COMPOUNDS FOR THE TREATMENT OF BATTEN DISEASE

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/221966, entitled “COMPOUNDS FOR THE TREATMENT OF BATTEN DISEASE”, filed on July 15, 2021; and of U.S. Provisional Application No. 63/134504, entitled “COMPOUNDS FOR THE TREATMENT OF BATTEN DISEASE”, filed on January 6, 2021; the contents of each of which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a sequence listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 5, 2022, is named A110770032WO00-SEQ-HCL and is 243,726 bytes in size.

BACKGROUND

Batten disease refers to a collective group of rare degenerative nervous system disorders called neuronal ceroid lipofuscinoses (NCLs). Juvenile-onset Batten disease (also known as CLN3 Batten disease) is the most common of these NCLs. A majority of CLN3 Batten disease patients harbor deletion of exons 7 and 8 from the neuronal ceroid- lipofuscinosis 3 lysosomal/endosomal transmembrane protein, battenin ( CLN3 ) gene. This loss-of-function mutation causes a frameshift, resulting in nonsense mediated decay of the CLN3 mRNA and a lack of CLN3 protein.

SUMMARY

Provided herein are compounds, methods and pharmaceutical compositions for the treatment or amelioration of symptoms and/or hallmarks of a neuronal ceroid lipofuscinosis (NCL) disease, such as juvenile NCL or CLN3 Batten disease. According to some aspects, the compounds, methods and pharmaceutical compositions provided herein affect the expression of splicing isoforms of CLN3 RNA, such as by increasing the expression of alternatively spliced isoforms. In certain embodiments, the compounds, methods and pharmaceutical compositions disclosed herein increase the amount of functional CLN3 protein in a cell or animal. According to some aspects, synthetic oligonucleotides and pharmaceutically acceptable salts thereof are provided herein. In some embodiments, a synthetic oligonucleotide, or a pharmaceutically acceptable salt thereof, comprises a nucleic acid sequence 10 to 30 nucleosides in length that is complementary to a region of at least 10 contiguous nucleotides of a CLN3 sequence as set forth in SEQ ID NO: 2, the synthetic oligonucleotide comprises at least one 2’-O-methoxyethyl (2’-MOE) or locked nucleic acid (LNA) modified nucleoside, and each cytidine residue of the synthetic oligonucleotide is a 5- methyl cytidine and each thymidine residue of the synthetic oligonucleotide is a 5-methyl uridine.

In some embodiments, the CLN3 sequence is a human CLN3 pre-mRNA sequence or a human CLN3 genomic sequence. In some embodiments, the region of at least 10 contiguous nucleotides is within intron 4, intron 4 and exon 5, exon 5, exon 5 and intron 5, intron 5, intron 5 and exon 6, exon 6, exon 6 and intron 6, intron 6, intron 8, intron 8 and exon 9, exon 9, exon 9 and intron 9, or intron 9 of a human CLN3 sequence.

In some embodiments, the nucleic acid sequence is complementary to a region of at least 18 contiguous oligonucleotides of the CLN3 sequence. In some embodiments, the nucleic acid sequence is complementary to a region of at least 20 contiguous oligonucleotides of the CLN3 sequence.

In some embodiments, the nucleic acid sequence is 15 to 22 nucleosides in length. In some embodiments, the nucleic acid sequence is 20 nucleosides in length.

In some embodiments, the synthetic oligonucleotide comprises a molecular species. In some embodiments, the molecular species is indirectly attached to a nucleotide of the synthetic oligonucleotide. In some embodiments, the molecular species is indirectly attached to the nucleotide at the 3’ end of the synthetic oligonucleotide. In some embodiments, the molecular species is indirectly attached to the nucleotide at the 5’ end of the synthetic oligonucleotide.

In some embodiments, the molecular species comprises or consists of a cholesterol or a tocopherol. In some embodiments, the molecular species comprises or consists of (N- cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-l-O-ph osphodiester (CholTEG).

In some embodiments, the synthetic oligonucleotide comprises a spacer. In some embodiments, the spacer comprises or consists of oligoethylene. In some embodiments, the oligoethylene is hexaethyleneglycol (HEG). In some embodiments, the spacer comprises or consists of two HEGs. In some embodiments, the spacer comprises or consists of HEG and triethyleneglycol (TEG). In some embodiments, the molecular species is indirectly attached to the nucleotide of the synthetic oligonucleotide through the spacer.

In some embodiments, the synthetic oligonucleotide comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the synthetic oligonucleotide comprises two or more phosphorothioate internucleoside linkages, or each intemucleoside linkage of the synthetic oligonucleotide is a phosphorothioate internucleoside linkage.

In some embodiments, the synthetic oligonucleotide comprises at least one phosphodiester intemucleoside linkage. In some embodiments, the synthetic oligonucleotide comprises two or more phosphodiester intemucleoside linkages, or each intemucleoside linkage of the synthetic oligonucleotide is a phosphodiester intemucleoside linkage.

In some embodiments, the synthetic oligonucleotide comprises at least two nucleotide modifications. In some embodiments, the at least two nucleotide modifications are selected from the group consisting of a 2’-O-methyl modification (2’-O-Me), a 2’-MOE modification, a 2’-O-methoxyethoxy-5-methyl (5-Me-MOE) modification, an LNA modification, a 5- methyl (5-Me) modification, a 7-deaza modification, and a 7-deaza-2’ -O-methyl (7deazaOM) modification.

In some embodiments, at least two nucleotides of the synthetic oligonucleotide comprise 2’-MOE modifications. In some embodiments, each nucleotide of the synthetic oligonucleotide comprises a 2’-MOE modification.

In some embodiments, the synthetic oligonucleotide comprises the nucleic acid sequence of any one of SEQ ID NO: 11-772.

According to some aspects, spherical nucleic acids (SNAs) are provided herein. In some embodiments, an SNA comprises a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises a synthetic oligonucleotide comprising a nucleic acid sequence 10 to 30 nucleotides in length that is complementary to a region of at least 10 contiguous nucleotides of a CLN3 sequence as set forth in SEQ ID NO: 1. In some embodiments, the synthetic oligonucleotide comprises at least one 2’-O-methoxyethyl (2’- MOE) or locked nucleic acid (LNA) modified nucleoside. In some embodiments, each cytidine residue of the synthetic oligonucleotide is a 5-methyl cytidine. In some embodiments, each thymidine residue of the synthetic oligonucleotide is a 5-methyl uridine.

In some embodiments, the synthetic oligonucleotide is a synthetic oligonucleotide disclosed herein.

In some embodiments, the synthetic oligonucleotide comprises a molecular species. In some embodiments, the molecular species is indirectly attached to a nucleotide of the synthetic oligonucleotide. In some embodiments, the molecular species is indirectly attached to the nucleotide at the 3’ end of the synthetic oligonucleotide. In some embodiments, the molecular species is indirectly attached to the nucleotide at the 5’ end of the synthetic oligonucleotide.

In some embodiments, the molecular species comprises or consists of a cholesterol or a tocopherol. In some embodiments, the molecular species comprises or consists of (N- cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-l-O-ph osphodiester (CholTEG).

In some embodiments, the synthetic oligonucleotide is anchored to the surface of the core through the molecular species.

In some embodiments, the core is a hollow core or solid core. In some embodiments, the hollow core is a liposome core. In some embodiments, the liposome core comprises or consists of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

In some embodiments, the oligonucleotide shell comprises or consists of 20 to 50 synthetic oligonucleotides. In some embodiments, the oligonucleotide shell comprises or consists of 25 to 35 synthetic oligonucleotides. In some embodiments, the oligonucleotide shell comprises or consists of 30 or about 30 synthetic oligonucleotides.

In some embodiments, the core is a liposome core comprising lipid molecules, and the synthetic oligonucleotides of the oligonucleotide shell are at a molar ratio of or about 50 to 1 of lipid molecules to synthetic oligonucleotides.

In some embodiments, the oligonucleotide shell comprises a second synthetic oligonucleotide comprising a second nucleic acid sequence 10 to 30 nucleotides in length that is complementary to a second region of at least 10 contiguous nucleotides of a CLN3 sequence as set forth in SEQ ID NO: 1. In some embodiments, the second synthetic oligonucleotide is a synthetic oligonucleotide disclosed herein.

According to some aspects, pharmaceutical compositions are provided herein. In some embodiments, a pharmaceutical composition comprises a synthetic oligonucleotide disclosed herein or pharmaceutically acceptable salt thereof. In some embodiments, a pharmaceutical composition comprises an SNA disclosed herein.

According to some aspects, methods of producing alternatively spliced CLN3 RNA in a cell are provided herein. In some embodiments, a method of producing alternatively spliced CLN3 RNA in a cell comprises contacting a cell comprising a CLN3 gene and/or a CLN3 gene product with a synthetic oligonucleotide disclosed herein, an SNA disclosed herein, or a pharmaceutical composition disclosed herein, to produce alternatively spliced CLN3 RNA in the cell. In some embodiments, the alternatively spliced CLN3 RNA lacks exon 5, exon 6, and/or exon 9. In some embodiments, the cell is a neuronal cell, a fibroblast, an astrocyte, a microglial cell, or a cell of the eye. In some embodiments, the cell of the eye is a retinal cell.

According to some aspects, methods of treating a disease or disorder in a subject are provided herein. In some embodiments, a method of treating a disease or disorder in a subject comprises administering to a subject an effective amount of a synthetic oligonucleotide disclosed herein, an SNA disclosed herein, or a pharmaceutical composition disclosed herein, in order to treat the disease or disorder in the subject.

In some embodiments, the disease or disorder is an inherited disease or disorder. In some embodiments, the disease or disorder is a neurodegenerative disease or disorder. In some embodiments, the disease or disorder is a neuronal ceroid lipofuscinosis (NCL). In some embodiments, the disease or disorder is Batten disease, juvenile NCL, or CLN3 Batten disease.

In some embodiments, administering the synthetic oligonucleotide, the SNA, or the pharmaceutical composition to the subject ameliorates or eliminates one or more symptoms or conditions associated with the disease or disorder in the subject. In some embodiments, the one or more symptoms or conditions are visual impairment, retinal degeneration, intellectual disability, impaired cognitive function, speech impairment, seizures, muscle rigidity or stiffness, hypokinesia, stooped posture, arrhythmia, and/or impaired motor function.

In some embodiments, the method comprises the administration of a second therapeutic agent.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety. Unless otherwise indicated, the following terms have the meanings indicated below.

As used herein, “2’-modified” or “2’-substituted” refers to a nucleoside comprising a sugar with a substituent group at the 2’ carbon position other than H or OH. 2’ -modified nucleosides, include, but are not limited to, bicyclic nucleosides and nucleosides with non- bridging 2’ substituents, such as allyl, amino, azido, thio, fluoro, O-allyl, O-C ₁ -C ₁₀ alkyl, - OCF3, O-(CH ₂ ) ₂ -O-CH ₃ , 2’-O(CH ₂ ) ₂ SCH ₃ , O-(CH ₂ ) ₂ -O-N(R _m )(R _n ), or O-CH ₂ -C(=0)- N(Rm)(Rn), where each R _m and R _n is, independently, H or substituted or unsubstituted C ₁ - C ₁₀ alkyl. Nucleosides with 2’ -modifications may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase. Nucleoside modifications are discussed in further detail below.

As used herein, “4’-2’ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2’ carbon atom and the 4’ carbon atom of the sugar ring. Nucleoside modifications are discussed in further detail below.

As used herein, “5-methyl cytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methyl cytosine is a modified nucleobase. Modified nucleobases are discussed in further detail below.

As used herein, “about” means within ±10% of a value. As a non-limiting example, statements in accordance with “about 70% inhibition” indicate that the referenced inhibition is within a range of 60% and 80%. Similarly, statements in accordance with “about 100” indicate values within a range of 90 to 110.

As used herein, “administration” or “administering” refers to routes of introducing a compound or composition provided herein to an individual to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion, intravitreal administration, intracistema magna administration, or intrathecal administration. Administration details are discussed in further detail below.

As used herein, “administered concomitantly” or “co-administration” means administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, by the same route of administration, or in parallel. Co-administration encompasses both parallel and sequential administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.

As used herein, “alternative splicing” refers to the use of different combinations of exons within gene products generated from a given transcript to generate multiple mRNA transcripts from a single gene. As a non-limiting example, alternative splicing may result in the production of one mRNA molecule comprising exons 1, 2, 3, 4, and 6 of a given gene and another mRNA molecule having exons 1, 2, 3, 5, and 6 of the gene.

As used herein, “ameliorate” in reference to a treatment means improvement or lessening in at least one indicator, sign or symptom of a disease, disorder or condition relative to the same indicator, sign or symptom in the absence of the treatment. In certain embodiments, amelioration is the reduction in the severity or frequency of an indicator, sign or symptom or the delayed onset or slowing of progression in the severity or frequency of a symptom. In certain embodiments, the symptom or hallmark includes progressive loss of motor function, seizures, vision loss, and loss of cognitive function. Symptoms and their amelioration are discussed in further detail below.

As used herein, “animal” means a human or non-human animal.

As used herein, an “antisense compound” refers to a compound comprising an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. Examples of antisense compounds include single-stranded and double- stranded compounds, such as, oligonucleotides, ribozymes, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

As used herein, “antisense inhibition” refers to reduction of one or more levels of a target nucleic acid facilitated by or corresponding to the presence of an antisense compound complementary to the target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

As used herein, “antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

As used herein, “antisense oligonucleotide” refers to an oligonucleotide having a nucleobase sequence that is complementary or sufficiently complementary to a target nucleic acid or region or segment thereof. In certain embodiments, an antisense oligonucleotide is specifically hybridizable to a target nucleic acid or region or segment thereof. Antisense oligonucleotides are discussed in further detail below.

As used herein, “bicyclic nucleoside” or “BNA” refers to a nucleoside comprising a bicyclic sugar moiety.

As used herein, “bicyclic sugar” and “bicyclic sugar moiety” refer to a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety. LNA is a bicyclic nucleoside. Bicyclic sugars and nucleosides are discussed in further detail below.

As used herein, “CLN3” or “a CLN3 sequence” or “CLN3 protein” refers to the ceroid-lipofuscinosis, neuronal 3 (CLN3) gene or a nucleic acid sequence or polypeptide/protein product thereof. CLN3 is also known as CLN3 lysosomal/endosomal transmembrane protein, Battenin, Batten Disease Protein, BTS, JNCL, and BTN1. The genomic sequence of human CLN3 is set forth in RefSeqGene (LRG_689) on chromosome 16, NCBI Reference Sequence: NG_008654.2 (SEQ ID NO: 1). The human CLN3 gene comprises nucleotides 5,001-30,650 of NCBI Reference Sequence: NG_008654.2 (SEQ ID NO: 1), and is provided as SEQ ID NO: 2. Additional human CLN3 sequences are provided as SEQ ID NO: 3-10 and the murine CLN3 gene sequence is provided as SEQ ID NO: 773. CLN3 and CLN3 sequences are discussed in further detail below.

As used herein, “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of the oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine and guanine (G), 5-methyl cytosine (5-Me-C) and guanine (G). Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, “fully complementary” or “100% complementary” in reference to oligonucleotides means that oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide. In certain embodiments, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target. One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the ability of the oligomeric compounds to remain in association. Therefore, described herein are antisense compounds that may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target). Preferably the antisense compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches. The remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art would recognize the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target nucleic acid. Sequence complementarity is discussed in further detail below.

As used herein, “contiguous” in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or intemucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” refers to nucleobases that are immediately adjacent to each other in a sequence.

As used herein, “differently modified” means chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, an MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising one 2’-OMe modified sugar and one unmodified adenine nucleobase and a nucleoside comprising one 2’-OMe modified sugar and one unmodified thymine nucleobase are not differently modified. Chemical modifications are discussed in further detail below.

As used herein, “dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous or intrathecal administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion (e.g., intravenous infusion) over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month. WO 2022/150369 - 1° ^' PCT/US2022/011291

As used herein, “dosing regimen” is a combination of doses designed to achieve one or more desired effects.

As used herein, “effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual’s medical condition, and other relevant factors.

As used herein, “efficacy” means the ability to produce a desired effect.

As used herein, “exon” refers to a region of a primary RNA transcript that remains in the mature RNA when it reaches the cytoplasm. Exons are “spliced” together to form the mature mRNA sequence. Intron-exon junctions are also referred to as “splice sites” with the 5' side of the junction often called the “5' splice site,” or “splice donor site” and the 3' side the “3' splice site” or “splice acceptor site.” “Cryptic” splice sites are those which are less often used but may be used when the “normal” splice site is blocked or unavailable. A splice modulator can be used to force a primary RNA transcript to use a cryptic splice site and generate an alternatively spliced RNA transcript.

As used herein, “expression” includes all the functions by which a gene’s coded information is converted into structures present and operating in a cell, tissue or animal. Such structures include, but are not limited to, the products of transcription and translation.

As used herein, “hybridization” means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

As used herein, the terms “internucleotide linkage” and “internucleoside linkage” refer to the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “modified internucleotide linkage” and “modified intemucleoside linkage” refer to any such linkage other than a phosphodiester (PO) linkage. “Phosphorothioate internucleotide linkage” or (PS) is a modified intemucleotide linkage in which one of the non-bridging oxygen atoms of a phosphodiester intemucleotide linkage is replaced with a sulfur atom. In some embodiments, the non-bridging oxygen atoms of a phosphodiester intemucleotide linkage is replaced with an organic or inorganic moiety selected from =S, =Se, =NR’, -SR’, - SeR’, -N(R’) ₂ , -B(R’) ₃ , -S-, -Se-, and -N(R’)-, wherein each R’ is independently as defined and described in the present disclosure. In some embodiments, an intemucleotide linkage is a phosphotriester linkage, or phosphorothioate triester linkage. In some embodiments, an intemucleotide linkage is one of, e.g., PNA (peptide nucleic acid) or PMO (phosphorodiamidate Morpholino oligomer) linkage. In some embodiments, a modified intemucleotide linkage is a non-negatively charged intemucleotide linkage. In some embodiments, a modified intemucleotide linkage is a neutral intemucleotide linkage. It is understood by a person of ordinary skill in the art that an intemucleotide linkage may exist as an anion or cation at a given pH due to the existence of acid or base moieties in the linkage. Internucleotide/intemucleoside linkages are discussed in further detail below.

As used herein, “intron” refers to a region of a primary RNA transcript that is not included in the mature mRNA. Introns are removed during RNA splicing and are retained in the nucleus.

As used herein, “linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

As used herein “linker”, or “linking moiety” and the like refer to any chemical moiety which connects one chemical moiety to another. As appreciated by those skilled in the art, a linker can be bivalent or trivalent or more, depending on the number of chemical moieties the linker connects. In some embodiments, a linker is a moiety which connects one oligonucleotide to another oligonucleotide in a multimer. In some embodiments, a linker is a moiety optionally positioned at the 3’ or 5’ end of the oligonucleotide. In some embodiments, in an oligonucleotide, a linker is a lipophilic moiety that facilitates the incorporation of an oligonucleotide into or onto the external surface of a core. In some embodiments, the linker comprises or consists of a cholesterol. In some embodiments, the linker comprises or consists of (N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-l-O -phosphodiester (CholTEG). Linkers are discussed in further detail below.

As used herein, “locked nucleic acid” or “LNA” or “LNA nucleoside” refers to a modified nucleoside which comprises a bridging moiety linking the 2’ and 4’ carbons of the ribose sugar ring (also referred to as a “2’-4’ bridge”), which restricts or locks the conformation of the ribose ring. LNA is an example of BNA. Non-limiting, exemplary LNA nucleosides are disclosed in WO 1999/014226, WO 2000/66604, WO 1998/039352, WO 2004/046160, WO 2000/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, and WO 2008/150729, the contents of each of which are incorporated herein by reference in their entireties. LNA modifications are discussed in further detail below.

As used herein, “mismatch” or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid molecule when the first and second oligonucleotide are aligned. For example, nucleobases including but not limited to a universal nucleobase, inosine, and hypoxanthine, are capable of hybridizing with at least one nucleobase but are still mismatched or non-complementary with respect to nucleobase to which it hybridized. As another example, a nucleobase of a first oligonucleotide that is not capable of hybridizing to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned is a mismatch or non-complementary nucleobase.

As used herein, “modulate” or “modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating CLN3 can mean to increase or decrease the level of CLN3 RNA and/or CLN3 protein in a cell, tissue, organ or organism. A “modulator” effects the change in the cell, tissue, organ or organism. For example, an SNA compound can be a modulator that increases the amount of alternatively spliced CLN3 RNA and/or functional CLN3 protein in a cell, tissue, organ or organism.

As used herein, “modulation of splicing” refers to altering the processing of a pre- mRNA transcript such that the spliced mRNA molecule contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion of one or more exons, or an additional sequence not normally found in the spliced mRNA (e.g., intron sequence). In the context of the present invention, modulation of splicing refers to altering splicing of CLN3 RNA under certain conditions or in response to certain events whereby a shortened version of CLN3 mRNA is produced in a cell tissue, organ or organism.

As used herein, a “morpholino oligomer” refers to a polymeric molecule having a backbone which supports bases capable of hydrogen bonding to typical oligonucleotides, wherein the polymer lacks a ribose sugar moiety, and more specifically a ribose sugar backbone linked by phosphodiester bonds which is typical of naturally occurring oligonucleotides and nucleosides, but instead contains a ring nitrogen with coupling through the ring nitrogen. Exemplary structures of morpholino oligonucleotides are described, for example, in Hudziak et ah, Antisense Nucleic Acid Drug Dev. 6: 267-272 (1996) and Summerton and Weller, Antisense Nucleic Acid Drug Dev. 7: 187-195 (1997), the contents of each of which are incorporated herein by reference in their entireties.

As used herein, “motif’ means the pattern of modified sugar moieties, nucleobases, and/or intemucleotide linkages, in an oligonucleotide.

As used herein, “neurodegenerative disease” refers to a condition marked by progressive loss of function or structure of components of the nervous system, including loss of motor function and death of neurons. In certain embodiments, the neurodegenerative disease is a neuronal ceroid lipofuscinosis (NCL). In certain embodiments, the neurodegenerative disease is CLN3 Batten disease. Neurodegenerative diseases are discussed in further detail below.

As used herein, “non-bicyclic modified sugar” or “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring. Sugar modifications are discussed in further detail below.

As used herein, “nucleic acid” refers to molecules composed of linked nucleosides. Nucleic acids include, but are not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single- stranded nucleic acids, and double- stranded nucleic acids. Nucleic acids are discussed in further detail below.

As used herein, “nucleobase” refers to an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase. Examples of modified nucleobases include but are not limited to 5-methyl cytosine, 7-deaza guanosine, and 7-deaza deoxyguanosine. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. Modified and unmodified nucleobases are discussed in further detail below.

As used herein, “nucleobase sequence” or “nucleic acid sequence” refers to the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or linkage modification(s).

As used herein, “nucleoside” refers to a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, “modified nucleoside” refers to a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase. “Linked nucleosides” are nucleosides that are connected in a contiguous sequence (i.e., no additional nucleosides are present between those that are linked). Nucleosides are discussed in further detail below.

As used herein, “oligomeric compound” refers to an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired. A “single- stranded oligomeric compound” is an unpaired oligomeric compound. The term “oligomeric duplex” means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a “duplexed oligomeric compound.” As disclosed herein, an “oligonucleotide” refers to a strand of nucleosides {i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to an exchangeable organic base, such as pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a purine (e.g., adenine (A) or guanine (G))) which are linked, having a length of typically between eight and 100 nucleobases. Each nucleoside and intemucleoside linkage of an oligonucleotide may be modified or unmodified, relative to a reference nucleoside sequence. As disclosed herein, a “modified oligonucleotide” refers to an oligonucleotide wherein at least one nucleoside or intemucleoside linkage is modified relative to a naturally-occurring strand of linked nucleosides. As used herein, an “unmodified oligonucleotide” refers to an oligonucleotide that does not comprise any nucleoside modifications or internucleotide linkage modifications. Oligonucleotides are discussed in further detail below.

As used herein, “parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intravitreal administration, intracranial administration, intrathecal administration, intracistema magna administration, and intracerebroventricular administration.

As used herein, “pharmaceutical agent” refers to a compound that provides a therapeutic benefit when administered to a subject. As used herein “pharmaceutical composition” refers to a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution (i.e., a pharmaceutically acceptable carrier). In certain embodiments, a pharmaceutical composition shows activity in free uptake assays in certain cell lines. Pharmaceutical agents and compositions are discussed in further detail below.

As used herein, “pharmaceutically acceptable carrier or diluent” refers to any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffer solution or sterile artificial cerebrospinal fluid.

As used herein, “phosphorothioate linkage” refers to a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate intemucleoside linkage is a modified intemucleoside linkage. Intemucleoside linkages are discussed in further detail below. As used herein, “prevent” refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely.

As used herein, a “region” of a nucleic acid sequence is a portion of the nucleic acid having at least one identifiable structure, function, or characteristic, including a particular sequence.

As used herein, “RNA” refers to an RNA transcript that encodes a protein and includes pre-mRNA and mature mRNA unless otherwise specified.

As used herein, “side effects” refers to physiological diseases and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function test abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality, increased bilirubin may indicate liver toxicity or liver function abnormality, and changes in glomerular filtration rate may represent renal toxicity or renal impairment.

As used herein, “single- stranded” in reference to a given nucleic acid compound means the compound has only one oligonucleotide or nucleic acid strand. A compound consisting of one oligonucleotide or nucleic acid molecule, wherein the oligonucleotide of the compound is self-complementary, is a single-stranded compound. A single- stranded compound may be capable of binding to a complementary compound to form a duplex.

As used herein, “spacer” refers to a group of atoms that connect two moieties, such as a linker and an oligonucleotide. In some embodiments, a spacer comprises or consists of an oligoethylene. In some embodiments, the oligoethylene is hexaethyleneglycol (HEG). In some embodiments, the spacer comprises or consists of two or more connected HEG molecules (e.g., two HEG molecules covalently bound via a phosphodiester bond). In some embodiments, the spacer comprises or consists of hexa(ethyleneglycol)phosphodiester- hexa(ethyleneglycol)phosphodiester (HEG-HEG). In some embodiments, two moieties (e.g., a linker and an oligonucleotide) are indirectly attached to one another through a spacer. In some embodiments, a spacer comprises or consists of one or more nucleosides, or does not comprise nucleosides. Spacers are discussed in further detail below.

As used herein, “spherical nucleic acid” or “SNA” refers to a three-dimensional arrangement of oligonucleotides comprising an oligonucleotide shell radially oriented around or on the exterior of a core. SNAs are discussed in further detail below. As used herein, “sugar moiety” refers to an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2’-OH(H) ribosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2’-H(H) deoxyribosyl sugar moiety, as found in DNA (an “unmodified DNA sugar moiety”). Unmodified sugar moieties have one hydrogen at each of the 1’, 3’, and 4’ positions, an oxygen at the 3’ position, and two hydrogens at the 5’ position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate. Sugar moieties are discussed in further detail below.

As used herein, “sugar surrogate” refers to a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an intemucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or target nucleic acids. Such modified nucleosides include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have bridge between the 2’ carbon and 4’ carbon on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the 2’ carbon and 3’ carbon (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non- sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

As used herein, “symptom” or “hallmark” refers to any physical feature or test result that indicates the existence or extent of a disease or disorder. In certain embodiments, a symptom is apparent to a subject or to a medical professional examining or testing said subject. In certain embodiments, a hallmark is apparent upon invasive diagnostic testing, including, but not limited to, post-mortem tests.

As used herein, “synergy” or “synergize” mean an effect of a combination of two or more components that is greater than the additive effects of each component alone at the same doses.

As used herein, “target gene” refers to a gene encoding a target molecule.

As used herein, “targeting” means the specific hybridization of a compound to a target nucleic acid in order to induce a desired effect. WO 2022/150369 - I ^{7 '} PCT/US2022/011291

As used herein, “target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” each refer to a nucleic acid capable of being targeted by compounds described herein.

As used herein, “target region” means a portion of a target nucleic acid to which one or more compounds is targeted or is capable of hybridizing.

As used herein, “target segment” means the sequence of nucleotides of a target nucleic acid to which a compound is targeted.

As used herein, “treat” refers to administering a compound or pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal.

As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutical agent or composition that provides a therapeutic benefit to a subject. For example, a therapeutically effective amount may improve a symptom of a disease. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition may be the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

Oligonucleotides

In some embodiments, an oligonucleotide disclosed herein is a synthetic oligonucleotide. As disclosed herein, a “synthetic oligonucleotide” refers to a non-naturally occurring oligonucleotide. A synthetic oligonucleotide, in some embodiments, refers to a synthetic DNA or synthetic RNA. In some embodiments, a synthetic oligonucleotide is produced through an in vitro transcription or a polymerization reaction (e.g., artificial (non- natural) chemical synthesis, solid phase nucleic acid synthesis, or another method known by one of ordinary skill in the art). In some embodiments, a synthetic oligonucleotide includes a modification at one or both ends of the nucleic acid sequence in the synthetic oligonucleotide. In some embodiments, the synthetic oligonucleotide is produced by nucleic acid synthesis (e.g., in vitro), chemical nucleic acid synthesis, and/or solid phase nucleic acid synthesis, or produced through other methods well known in the art. In some embodiments, one or more nucleosides of the oligonucleotide include a modification.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,

81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleosides (e.g., nucleobases) in length, or any range or combination thereof. In some embodiments, the nucleic acid sequence of a synthetic oligonucleotide disclosed herein is 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100 or more than 100 nucleosides (e.g., nucleobases) in length. In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is 10 to 30 nucleosides (e.g., nucleobases) in length. In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is 15 to 22 nucleosides (e.g., nucleobases) in length. In some embodiments, the oligonucleotide (e.g., synthetic oligonucleotide) is 20 nucleosides (e.g., nucleobases) in length. In some embodiments, the oligonucleotide (e.g., synthetic oligonucleotide) is 17 nucleosides (e.g., nucleobases) in length.

It is possible to increase or decrease the length of an oligonucleotide without eliminating activity. For example, in Woolf et al. ( Proc . Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotides were able to direct specific cleavage of the target RNA, albeit to a lesser extent than the oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.

In certain embodiments, oligonucleotides (including modified oligonucleotides) can have any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X<Y. For example, in certain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 2 1 to 22, 2 1 to 23, 2 1 to 24, 2 1 to 25, 2 1 to 26, 2 1 to 27, 2 1 to 28, 2 1 to 29, 2 1 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to

25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to

29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to

30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides.

In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides, e.g., synthetic oligonucleotides) disclosed herein are further described by their nucleobase sequence. In certain embodiments oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.

In some embodiments, the synthetic oligonucleotide comprises or consists of a nucleic acid sequence complementary to a region (e.g. a nucleic acid sequence segment) of a CLN3 sequence (e.g., a CLN3 sequence as set forth in any one of SEQ ID NO: 1-10 and 773). In some embodiments, a synthetic oligonucleotide disclosed herein comprises or consists of a nucleic acid sequence complementary to a region (e.g., a nucleic acid sequence segment) of at least 5 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or more) contiguous nucleotides of a CLN3 sequence (e.g., a CLN3 sequence as set forth in any one of SEQ ID NO: 1-10 and 773). In some embodiments, a synthetic oligonucleotide disclosed herein comprises or consists of a nucleic acid sequence complementary to a region of at least 15 contiguous nucleotides of a CLN3 sequence. In some embodiments, a synthetic oligonucleotide disclosed herein comprises or consists of a nucleic acid sequence complementary to a region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more contiguous nucleotides of a CLN3 sequence. In some embodiments, a synthetic oligonucleotide disclosed herein comprises or consists of a nucleic acid sequence complementary to a region of or about 20 contiguous nucleotides of a CLN3 sequence.

In some embodiments, the sequence of an oligonucleotide (e.g., a synthetic oligonucleotide) encompassed by the present disclosure is at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, or any range or combination thereof, identical to the sequence of an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein (e.g., any one of the oligonucleotides provided in Table 1, any one of SEQ ID NO: 11- 772).

In certain embodiments, modifications disclosed herein (e.g., sugar modifications, nucleobase modifications, and/or internucleotide linkage modifications) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each intemucleotide linkage of an oligonucleotide having a particular sugar modification motif may be modified or unmodified and may or may not follow the modification pattern of the sugar modifications. Unless otherwise indicated, all modifications are independent of nucleobase sequence.

Populations of modified oligonucleotides in which all of the modified oligonucleotides of the population have the same molecular formula can be stereorandom populations or chirally enriched populations. All of the chiral centers of all of the modified oligonucleotides are stereorandom in a stereorandom population. In a chirally enriched population, at least one particular chiral center is not stereorandom in the modified oligonucleotides of the population. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for beta-D ribosyl sugar moieties, and all of the phosphorothioate intemucleotide linkages are stereorandom. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for both beta-D ribosyl sugar moieties and at least one particular phosphorothioate intemucleotide linkage in a particular stereochemical configuration.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is any size useful for producing an effect (e.g., promoting or contributing to one or more beneficial phenotype(s) for the treatment, amelioration and/or elimination of one or more characteristics, conditions, and/or symptoms associated with Batten disease, as disclosed herein).

In some embodiments, the oligonucleotide (e.g., synthetic oligonucleotide) is single- stranded. In some embodiments, the oligonucleotide (e.g., synthetic oligonucleotide) is hybridized to a second oligonucleotide (e.g., synthetic oligonucleotide) and forms a double- stranded oligonucleotide. In some embodiments, the oligonucleotide (e.g., synthetic oligonucleotide) is not hybridized to a second oligonucleotide and does not form a double- stranded oligonucleotide. In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is a single-stranded oligonucleotide.

In certain embodiments, oligonucleotides (e.g., synthetic oligonucleotides) disclosed herein comprise an oligonucleotide having a nucleobase sequence complementary to that of a target nucleic acid. In certain embodiments, one oligonucleotide is paired with a second oligonucleotide to form a duplex. Such oligonucleotide duplexes comprise a first oligonucleotide having a region complementary to a target nucleic acid and a second oligonucleotide having a region complementary to the first oligonucleotide.

In certain embodiments, the first oligonucleotide of an oligonucleotide duplex disclosed herein comprises or consists of (1) a modified or unmodified oligonucleotide and optionally a spacer and/or a linker and (2) a second modified or unmodified oligonucleotide and optionally a spacer and/or a linker. Either or both oligomeric compounds of an oligomeric duplex may comprise a conjugate group. One or both oligonucleotides of an oligonucleotide duplex may include non-complementary overhanging nucleosides.

Oligonucleotide modifications

Oligonucleotide modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.) and 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, bases that base pair with an expanded repertoire of partners, and conjugated bases; (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar; as well as (d) intemucleoside linkage modifications, including modification or replacement of phosphodiester linkages. An oligonucleotide (e.g., a synthetic oligonucleotide disclosed herein) can comprise one or more of any of these modifications, or a combination thereof.

In some embodiments, the oligonucleotide modification is in one or more bases and/or sugars. For example, in some embodiments an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein includes nucleic acids having backbone sugars that are covalently attached to low molecular weight organic groups other than a hydroxyl group or hydrogen at the 2' position and other than a phosphate group or hydroxyl group at the 5' position. Thus, in some embodiments, a substituted or modified oligonucleotide includes a 2'- O-alkylated ribose group. In some embodiments, a modified oligonucleotide includes sugars such as hexose, 2’-F hexose, 2’-amino ribose, constrained ethyl (cEt), locked nucleic acid (LNA), bridged nucleic acid (BNA), arabinose or 2'-fluoroarabinose instead of ribose. Thus, in some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide-nucleic acids (which have an amino acid backbone with nucleic acid bases).

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein includes at least one LNA modification or modified nucleoside. An LNA modification or modified nucleoside is a modified RNA nucleoside in which the ribose moiety is modified with an additional linkage connecting the 2’ oxygen and the 4’ carbon.

Without wishing to be bound by theory, LNA modifications enhance base stacking and backbone organization, and significantly increase the hybridization properties of oligonucleotides. In some embodiments, the melting temperature of oligonucleotides comprising an LNA modification(s) can be increased relative to an unmodified oligonucleotide having the same nucleic acid sequence.

In some embodiments, the LNA modification is, comprises or consists of (2'-0, 4'-C methylene)-adenosine. In some embodiments, the LNA modification is, comprises or consists of 5 -methyl- (2 '-O, 4'-C methylene)-cytidine. In some embodiments, the LNA modification is, comprises or consists of (2'-0, 4'-C methylene)-cytidine. In some embodiments, the LNA modification is, comprises or consists of (2'-0, 4'-C methylene)-guanosine. In some embodiments, the LNA modification is, comprises or consists of 5-methyl-(2'-0, 4'-C methylene)-uridine. In some embodiments, the oligonucleotide includes two or more LNA modifications, each of which, in some embodiments, comprises, consists of, or consists essentially of an LNA modification disclosed herein. In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is DNA, RNA, PNA, cEt, LNA, ENA or hybrids including any chemical or natural modification thereof. Chemical and natural modifications are well known in the art and include but are not limited to those described above. Non-limiting examples of modifications include modifications designed to increase binding to a target strand (e.g., increase melting temperature of a hybridized pair of nucleic acid molecules, such as an oligonucleotide and a target nucleic acid), to assist in identification of the oligonucleotide or an oligonucleotide- target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides, to provide a mode of disruption (a terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein comprises at least one modification (e.g., a 2’-MOE modification, an LNA modification, or any other modification disclosed herein). In some embodiments, the oligonucleotide comprises at least two modifications. In some embodiments, the at least two modifications comprise an LNA modification and another modification disclosed herein. In some embodiments, the at least two modifications comprise a 2’-MOE modification and another modification disclosed herein. In some embodiments, the at least two modifications comprise a 2’-MOE modification and an LNA modification. In some embodiments, the oligonucleotide comprises two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 modifications (e.g., 2’-MOE modification(s), LNA modification(s), and/or any other modification(s) disclosed herein).

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein includes one or more 2’-O-methoxyethyl (2’-MOE) modifications. In some embodiments, the oligonucleotide includes one or more LNA modifications or modified nucleosides. In some embodiments, the oligonucleotide includes a 2’-MOE modification and an LNA modification.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein comprises an end modification. In some embodiments, 5’- and/or 3 ’-end modifications involve incorporation or addition of non-native or non-natural components to the 5’- and/or 3 ’-end of a nucleic acid or oligonucleotide. Such modifications may improve physicochemical properties, stability, resistance to nuclease degradation, etc. End modifications may include the addition of amino modifiers (e.g., 5’-DMS(0)MT-amino modifier C ₆ , 5’-amino modifier C3-TLA, 5’-amino modifier C12, 5’-amino modifier C ₆ - TLA, 5’-amino-dT, 5’-amino modifier-5, and 3’-amino modifier C7-CPG); thiol modifiers (e.g., 5 ’-thiol-modifier C ₆ S-S and 3 ’-thiol-modifier C ₆ S-S); 3’-glyceryl modification; binding modifiers (e.g., 5 ’-biotin, biotin-dT, biotin-TEG, 3’-biotin-TEG-CPG, digoxigenin, and 2,4-dinitrophenol-TEG); spacers (e.g., spacer 9, spacer 12, spacer 18, spacer C3, 3’- spacer-C3-CPG); nucleoside/nucleotide analogs (e.g., 3’-deoxynucleoside-CPG, 2’, 3’- dideoxycytidine, halogenated bases, 2’-deoxypseudouridine, 5,6-dihydro-dT, 5,6-dihydro-dU,

5-OH-dC, 5-OH-dU, 8-oxo-dA, 8-oxo-dG, thymidine glycol, dUracil, 2’-deoxynebularine, derivative K, derivative P, inosine, 5-nitroindole, 3-nitropyrrole, 2,6-diaminopurine, 5-Me- dC, 2-aminopurine, etheno-dA, N6-Me-dA, 06-Me-dG, 04-Me-dT, dSpacer, 5’-O-MedT, 7- deaza-dA, 7-deaza-dG, 7-deaza-dX, 7-deaza-8-aza-dA, and puromycin), intercalators (e.g., psoralen C2, and psoralen C ₆ ); cholesterol moieties (e.g., cholesteryl-TEG and 3’- cholesteryl-TEG-CPG) ; methyl RNA nucleotides (e.g., 2’-OMe-A, 2’-OMe-C, 2’-OMe-G and 2’-OMe-U); and/or thiophosphates to the 5’-end and/or the 3’-end of a nucleic acid or oligonucleotide.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein comprises a base modification. In some embodiments, a base modification involves replacement of a “natural” or “native” nucleobase of an oligonucleotide with a “non-natural” or “non-native” substituent, or involves chemical modification of a native nucleobase. Non- limiting examples of base modifications include methylation, hydroxymethylation, alkylation, methoxyethyl modifications, and substitutions with heterocyclic, stabilizing, destabilizing, promiscuous, or conjugated base moieties. “Natural” nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine and uracil. “Non-native” or “non-natural” substituents include 5-methyl-cytosine (5-Me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl (-CºC-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases,

6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8- thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5- bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and

7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Base modifications also include replacement of the native purine or pyrimidine base with other heterocycles, such as 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. Base modifications can improve various oligonucleotide properties, including stability (e.g., nuclease resistance, thermostability, chemical stability, biological stability, stability in various salt conditions, etc.), target hybridization, biocompatibility (e.g., reduced hepatotoxicity, reduced nephrotoxicity, reduced immune stimulation, etc.), mismatch discrimination, water solubility, etc.

In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleosides that do not comprise a nucleobase, referred to as an abasic nucleoside.

In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-prop yladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-CºC-CH3) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil),

4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7- methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine (7deazaG), 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N- isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine,

5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size- expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et ah, U.S. 3,687,808, those disclosed in Englisch et al. , Angewandte Chemie, International Edition, 1991, 30: 613, the contents of each of which are herein incorporated by reference in their entireties.

Publications that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, Manoharan et al., US2003/0158403; Manoharan et al., US2003/0175906; Dinh et al., U.S. 4,845,205; Spielvogel et al., U.S. 5,130,302; Rogers et al., U.S. 5,134,066; Bischofberger et al., U.S. 5,175,273; Urdea et al., U.S. 5,367,066; Benner et al., U.S. 5,432,272; Matteucci et al, U.S. 5,434,257; Gmeiner et al., U.S. 5,457,187; Cook et al., U.S. 5,459,255; Froehler et al., U.S. 5,484,908; Matteucci et al, U.S. 5,502,177; Hawkins et al., U.S. 5,525,711; Haralambidis et al, U.S. 5,552,540; Cook et al., U.S. 5,587,469; Froehler et al., U.S. 5,594,121; Switzer et al., U.S. 5,596,091; Cook et al., U.S. 5,614,617; Froehler et al., U.S. 5,645,985; Cook et al., U.S. 5,681,941; Cook et al, U.S. 5,81 1,534; Cook et al., U.S. 5,750,692; Cook et al., U.S. 5,948,903; Cook et al., U.S. 5,587,470; Cook et al., U.S. 5,457,191; Matteucci et al., U.S. 5,763,588; Froehler et al., U.S. 5,830,653; Cook et al., U.S. 5,808,027; Cook et al, 6,166,199; and Matteucci et al., U.S. 6,005,096, the contents of each of which are herein incorporated by reference in their entireties.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein comprises one or more (e.g., two or more, three or more, etc.) modified nucleosides (e.g., modified nucleosides with a base modification(s) and/or modified nucleosides with a sugar modification(s)). In some embodiments, the modification is a 2’-O-methyl (2’-O-Me) modification, a 2’-O-methoxyethyl (2’-MOE or MOE) modification, a 2’-O-methoxyethoxy- 5-methyl (5-Me-MOE) modification, an LNA modification, a 5-methyl (5-Me or iMe) modification (e.g., 5-methyl-cytidine or 5-methyl-uridine), a 5-methyl LNA modification, a 7-deaza modification, or a 7-deaza-2’-O-methyl (7deazaOM) modification.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein comprises an LNA modification and a 5-methyl modification. In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein comprises an LNA modification, a 5-methyl modification, and a 7-deaza modification. In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein comprises an MOE modification and a 5-methyl modification. In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein comprises an MOE modification, a 5-methyl modification, and a 7-deaza modification. In some embodiments, each nucleoside of an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein comprises an MOE modification. In some embodiments in which an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein comprises 3 or more linked guanosine nucleosides, a guanosine of the 3 or more linked guanosine nucleosides (e.g., an internal guanosine of the 3 or more linked guanosine nucleosides, such as the second of the 3 or more guanosine nucleosides) is 7-deaza modified. In some such embodiments, the oligonucleotide comprises two or more (e.g., 2, 3, 4, 5, or more) stretches of 3 or more linked guanosine nucleosides, in which case, in some embodiments, a guanosine of each of the two or more stretches of 3 or more linked guanosine nucleosides (e.g., an internal guanosine of each of the two or more stretches of 3 or more linked guanosine nucleosides, such as each second guanosine of the two or more stretches of 3 or more guanosine nucleosides) is 7-deaza modified. In some embodiments, the oligonucleotide further comprises one or more modified intemucleoside linkages, such as phosphorothioate intemucleoside linkage(s). As a non-limiting example, the oligonucleotide may comprise one or more LNA modified nucleosides, one or more 5-Me modified nucleosides, one or more 7-deaza modified nucleosides, one or more 2’-MOE modified nucleosides and/or one or more phosphorothioate intemucleoside linkages.

In some embodiments, a modified nucleoside is a 2’ -O-methyl adenosine (mA), a 2’- O-methyl cytidine (mC), a 2'-O-methyl guanosine (mG), a 2'-O-methyl uridine (mU), a deoxy adenosine (A, dA), a deoxycytidine (C, dC), a deoxyguanosine (G, dG), a deoxythymidine (T, dT), a 2'-O-methoxyethoxy adenosine (moeA, 2’-MOE-A), a 2'-O- methoxyethoxy-5-methyl cytidine (5-Me-MOE-C), a 2'-O-Methoxyethoxy Guanosine (moeG, 2’-MOE-G), a 2'-O-Methoxyethoxy-5-Methyl Uridine (moeT, 2’-MOE-T), an LNA Adenosine (1A), an LNA 5-Methyl Cytidine (5-Me-lC), an LNA Guanosine (1G), an LNA Thymidine (IT), a 5-Methyl deoxy Cytidine (iMe-dC), a 7-Deaza deoxy Guanosine (7deazaG), or a 7-deaza-2'-O-Methyl Guanosine (7deazaOMG).

In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, some or all of the guanine nucleobases in a modified oligonucleotide are 7-deaza guanine. In certain embodiments, all of the guanine nucleobases are 7-deaza guanines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methyl cytosines. In certain embodiments, all of the cytosine nucleobases are 5-methyl cytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases.

In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3 ’-end of the oligonucleotide.

In certain embodiments the block is within 3 nucleosides of the 3 ’-end of the oligonucleotide. In certain embodiments, the block is at the 5’-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 5 ’-end of the oligonucleotide. In certain embodiments, oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of said nucleoside is a 2’-deoxyribosyl sugar moiety. In certain embodiments, the modified nucleobase is selected from: a 2- thiopyrimidine and a 5-propynepyrimidine.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein comprises a sugar modification. In some embodiments, a sugar modification involves replacement of a “natural” or “native” sugar ring of a nucleoside of a nucleic acid sequence and/or oligonucleotide (e.g., a synthetic oligonucleotide) with a “non-natural” or “non- native” substituent, or involves chemical modification of a native sugar ring. Sugar ring substituent groups include OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N- alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C2 to C ₁ 0 alkenyl and alkynyl. These include O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ) _n OCH ₃ , O(CH ₂ ) _n NH ₂ , O(CH ₂ ) _n CH ₃ , O(CH ₂ ) ₂ ONH ₂ , and O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ ] ₂ , where n and m are from 1 to about 10. Other substituent groups include C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , S0 ₂ CH ₃ , ONO ₂ ,

N0 ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. In some embodiments, a sugar modification includes a 2'-O-methoxyethoxy (2'-O- CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2'-MOE), a 2'- dimethylaminooxyethoxy (i.e., a O(CH ₂ ) ₂ ON(CH ₃ ) ₂ group, also known as 2'-DMAOE), or 2'- dimethylaminoethoxyethoxy (also known as 2 '-O-dimethyl-amino-ethoxy-ethyl or 2'- DMAEOE; i.e., 2'-O-CH ₂ -O-CH ₂ -N(CH ₃ ) ₂ ). In some embodiments, a sugar modification includes an LNA modification, a 2’-O-Me modification, or a 2’-MOE modification.

In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties. In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more substituent groups none of which bridge two atoms of the furanosyl ring to form a bicyclic structure. Such non bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2’, 4’, and/or 5’ positions. In certain embodiments one or more non- bridging substituent of non-bicyclic modified sugar moieties is branched. Examples of 2’- substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2’-F, 2’-OCH ₃ (“OMe” or “O-methyl”), and 2’-O(CH ₂ )OCH ₃ (“MOE”). In certain embodiments, 2’ -substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF ₃ , OCF ₃ , O-C ₁ -C ₁₀ alkoxy, O- C ₁ - C ₁₀ substituted alkoxy, O-C ₁ -C ₁₀ alkyl, O-C ₁ - C ₁₀ substituted alkyl, S-alkyl, N(R _m )-alkyl, O-alkenyl, S-alkenyl, N(R _m )-alkenyl, O-alkynyl, S-alkynyl, N(R _m )-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O- aralkyl, O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ )ON(R _m )(R _n ) or OCH ₂ C(=0)-N(R _m )( R _n ), where each R _m and R _n is, independently, H, an amino protecting group, or substituted or unsubstituted C ₁ -C ₁₀ alkyl, and the 2’-substituent groups described in Cook et ah, U.S. 6,531,584; Cook et ah, U.S. 5,859,221; and Cook et ah, U.S. 6,005,087. Certain embodiments of these 2’ -substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N0 ₂ ), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4’ -substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et ah, WO 2015/106128. Examples of 5’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5-methyl (R or S), 5’-vinyl, and 5’-methoxy. In certain embodiments, non-bicyclic modified sugar moieties comprise more than one non-bridging sugar substituent, for example, 2’-F-5’-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et ah, WO 2008/101157 and Rajeev et ah, US2013/0203836.

In certain embodiments, a 2’ -substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’ -substituent group selected from: F, NF, N ₃ , OCF ₃ , OCH ₃ , O(CH ₂ ) ₃ NH ₂ , CH ₂ CH=CH ₂ , OCH ₂ CH=CH ₂ , OCH ₂ CH ₂ OCH ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ ON(CH ₃ ) ₂ , O(CH ₂ ) ₂ ON(R _m )(R _n ), O(CH ₂ ) ₂ O(CH ₂ ) ₂ N(CH ₃ ) ₂ , and N-substituted acetamide (OCH ₂ C(=O)-N(R _m )(R _n )), where each R _m and R _n is, independently, H, an amino protecting group, or substituted or unsubstituted C ₁ -C ₁₀ alkyl, e.g. OCH ₂ C(=0)-N(H)CH ₃ (“NMA”).

Certain modified sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4’ and the 2’ furanose ring atoms. Examples of such 4’ to 2’ bridging sugar substituents include but are not limited to: 4’-CH ₂ -2’, 4’-(CH ₂ ) ₂ -2' 4’-(CH ₂ ) ₃ -2' 4’-CH ₂ -O-2’ (“LNA”), 4’-CH ₂ -S-2' 4’- (CH ₂ ) ₂ -O-2’ (“ENA”), 4’-CH(CH ₃ )-O-2’ (“constrained ethyl” or “cEt”), 4’-CH ₂ -O-CH ₂ -2’, 4’-CH ₂ -N(R)-2’, 4’-CH(CH ₂ OCH ₃ )-O-2' (“constrained MOE” or “cMOE”) and analogs thereof {see, e.g., Seth et ah, U.S. 7,399,845, Bhat et al., U.S. 7,569,686, Swayze et ah, U.S. 7,741,457, and Swayze et al., U.S. 8,022,193, the contents of each of which are hereby incorporated by reference in their entireties), 4’-C(CH ₃ )(CH ₃ )-O-2’ and analogs thereof {see, e.g., Seth et al., U.S. 8,278,283, the contents of which are herein incorporated by reference in their entirety), 4’-CH ₂ -N(OCH ₃ )-2’ and analogs thereof {see, e.g., Prakash et al., U.S. 8,278,425, the contents of which are herein incorporated by reference in their entirety), 4’- CH ₂ -O-N(CH ₃ )-2’ {see, e.g., Allerson et al., U.S. 7,696,345 and Allerson et al., U.S. 8,124,745, the contents of each of which are hereby incorporated by reference in their entireties), 4’-CH ₂ -C(H)(CH ₃ )-2’ {see, e.g., Zhou, et al., /. Org. Chem., 2009, 74: 118-134), 4’-CH ₂ -C(=CH ₂ )-2’ and analogs thereof {see e.g., Seth et al., U.S. 8,278,426, the contents of which are hereby incorporated by reference in their entirety), 4’-C(R _a R _b )-N(R)-O-2’, 4’- C(R _a R _b )-O-N(R)-2' 4’-CH ₂ -O-N(R)-2’, and 4’-CH ₂ -N(R)-O-2' wherein each R, R _a , and R _b is, independently, H, a protecting group, or C ₁ -C ₁₂ alkyl {see, e.g. Imanishi et al., U.S.

7 ,· 427 ,672, the contents of which are hereby incorporated by reference in their entirety).

In certain embodiments, such 4’ to 2’ bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(R _a )(R _b )] _n -· -[C(R _a )(R _b )] _n -O-, -C(R _a )=C(R _b )-, -C(Ra)=N-, -C(=NRa)-, -C(=0)-, -C(=S)-, -O-, -Si(R _a ) ₂ -, -S(=0) _x -, and -N(R _a )-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R _a and R _b , is, independently, H, a protecting group, hydroxyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, Cs-C ₂ o aryl, substituted Cs-C ₂ o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, N JiJ ₂ , SJi, N ₃ , COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=0) ₂ -Ji), or sulfoxyl (S(=0)-Ji); and each Ji and J ₂ is, independently, H, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, Cs-C ₂ o aryl, substituted Cs-C ₂ o aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C ₁ -C ₁₂ aminoalkyl, substituted C ₁ -C ₁ 2 aminoalkyl, or a protecting group.

Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22): 4429-4443; Albaek et al., J. Org. Chem., 2006, 71:7731-7740; Singh et al., Chem. Commun., 1998, 4:455-456; Koshkin et al., Tetrahedron, 1998, 54:3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8:2219-2222; Singh et al., J. Org. Chem., 1998, 63:10035-10039; Chattopadhyaya U.S. 8,461,124; Wengel et al., U.S. 7,053,207; Imanishi et al., U.S. 6,268,490; Imanishi et al. U.S. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. 6,794,499; Wengel et al., U.S. 6,670,461; Wengel et al., U.S. 7,034,133; Wengel et al, U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582; and Ramasamy et al., U.S. 6,525,191; Torsten et al., WO 2004/106356; Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S. 8,088,746; Seth et al., U.S. 7,750,131; Seth et al., U.S. 8,030,467; Seth et al., U.S. 8,268,980; Seth et al., U.S. 8,546,556; Seth et al., U.S. 8,530,640; Migawa et al., U.S. 9,012,421; Seth et al., U.S. 8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawa et al., US2015/0191727, the contents of each of which are herein incorporated by reference in their entireties.

In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the a-L configuration or in the b-D configuration.

LNA (b-D-configuration) Alpha-L-LNA (a-L-configuration) Bridge = 4’-CH ₂ -O-2’ Bridge = 4’-CH ₂ -O-2’

Alpha-L-methyleneoxy (4’-CH ₂ -O-2’) or alpha-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids Research, 2003, 21: 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides ( e.g ., LNA or cEt) are identified in exemplified embodiments herein, they are in the b-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5 ’-substituted and 4’-2’ bridged sugars). In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4’ -sulfur atom and a substitution at the 2’-position (see, e.g., Bhat et al, U.S. 7,875,733 and Bhat et al., U.S. 7 ,939,677 , the contents of each of which are hereby incorporated by reference in their entireties) and/or the 5’ position.

In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), mannitol nucleic acid (“MNA”) (see, e.g., Leumann, CJ. Bioorg. &Med. Chem. 2002, 10: 841-854), fluoro HNA:

F-HNA

(“F-HNA”, see e.g. Swayze et al., U.S. 8,088,904; Swayze et al., U.S. 8,440,803; Swayze et al., U.S. 8,796,437; and Swayze et al., U.S. 9,005,906; F-HNA can also be referred to as a F-THP or 3'-fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula: wherein, independently, for each of said modified THP nucleoside:

Bx is a nucleobase moiety;

T3 and T4 are each, independently, an internucleotide linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an internucleotide linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a spacer, or a 5’ or 3 ’-linker; q ₁ , q ₂ , q ₃ , q ₄ q ₅ , q ₆ , and q ₇ are each, independently, H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, or substituted C ₂ -C ₆ alkynyl; and each of Ri and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJi, N3, OC(=X)Ji, OC(=X)NJIJ ₂ , NJ ₃ C(=X)NJIJ ₂ , and CN, wherein X is O, S or NJi, and each Ji, J ₂ , and J3 is, independently, H or C ₁ -C ₆ alkyl.

In certain embodiments, modified THP nucleosides are provided wherein q ₁ , q ₂ , q ₃ , q ₄ q ₅ , q ₆ , and q ₇ are each H. In certain embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ q ₅ , q ₆ , and q ₇ is other than H. In certain embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ q ₅ , q ₆ , and q ₇ is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of Ri and R2 is F. In certain embodiments, Ri is F and R2 is H, in certain embodiments, Ri is methoxy and R2 is H, and in certain embodiments, Ri is methoxyethoxy and R2 is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al, U.S. 5,166,315; Summerton et al, U.S. 5,185,444; and Summerton et al., U.S. 5,034,506, the contents of each of which are hereby incorporated by reference in their entireties). As used here, the term “morpholino” means a sugar surrogate having the following structure:

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

In certain embodiments, sugar surrogates comprise acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11: 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein comprises an internucleoside linkage modification. In some embodiments, an intemucleoside linkage modification involves replacement of a “natural” or “native” intemucleoside linkage of a nucleic acid molecule (e.g., an oligonucleotide) with a “non- natural” or “non-native” substituent, or involves chemical modification of a native intemucleoside linkage. In some embodiments, an intemucleoside linkage modification may comprise replacement of an oxygen of the phosphate group in a 3’,5’-phosphodiester bond with a substituent atom or a substituent group, or may comprise replacement of the 3 ’,5’- phosphodiester bond or both the 3’,5’-phosphodiester bond and the sugar moiety to facilitate linkage of one nucleobase of a nucleic acid molecule to the next. Non-limiting examples of modified intemucleoside linkages include phosphorothioate, phosphorodithioate, N3’ phosphoramidate, boranophosphate, 2’,5’-phosphodiester, phosphonoacetate (PACE), methylphosphonate, morpholino, amide, and peptide nucleic acid linkages. In some embodiments, an intemucleoside linkage modification comprised in an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is a phosphorothioate intemucleoside linkage modification.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein comprises a modified backbone. In some embodiments, the modified backbone comprises modified intemucleoside linkages. In some embodiments, the modified backbone comprises one or more phosphorothioate intemucleoside linkages. In some embodiments, all of the intemucleoside linkages of the oligonucleotide are phosphorothioate intemucleoside linkages. In some embodiments, modified intemucleoside linkages (e.g., linkages within a modified backbone) that do not include a phosphoms atom therein have intemucleoside linkages that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and Cth component parts.

Non-limiting examples of modified intemucleoside linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any internucleotide/internucleoside linkage. The two main classes of intemucleotide linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus -containing intemucleotide linkages include but are not limited to phosphates, which contain a phosphodiester bond (“P=0”) (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, phosphorothioates (“P=S”), and phosphorodithioates (“HS-P=S”). Representative non- phosphorus containing intemucleotide linking groups include but are not limited to methylenemethylimino (-CH ₂ -N(CH ₃ )-O-CH ₂ -), thiodiester, thionocarbamate (-O- C(=0)(NH)-S-); siloxane (-O-S1H2-O-); and N,N’-dimethylhydrazine (-CH2-N(CH3)- N(ϋ¾)-). Modified intemucleotide linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, intemucleotide linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Methods of preparation of phosphorous- containing and non-phosphorous-containing intemucleotide linkages are well-known to those skilled in the art.

Representative intemucleotide linkages having a chiral center include but are not limited to alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising intemucleotide linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom intemucleotide linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations. In certain embodiments, populations of modified oligonucleotides comprise phosphorothioate intemucleotide linkages wherein all of the phosphorothioate intemucleotide linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereochemical configuration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate intemucleotide linkages in a particular, independently selected stereochemical configuration. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et ah, JACS 125, 8307 (2003), Wan et al. Nuc. Add. Res. 42, 13456 (2014), and WO 2017/015555, the contents of each of which are herein incorporated by reference in their entireties. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration. In certain embodiments, modified oligonucleotides comprising (Rp) and/or (Sp) phosphorothioates comprise one or more of the following formulas, respectively, wherein “B” indicates a nucleobase:

Unless otherwise indicated, chiral internucleotide linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration.

Neutral intemucleotide linkages include, without limitation, phosphotriesters, methylpho sphonates , MMI (3’-CH ₂ -N(CH ₃ )-O-5’), amide-3 (3’-CH ₂ -C(=0)-N(H)-5’), amide-4 (3’-CH ₂ -N(H)-C(=0)-5’), formacetal (3’-O-CH ₂ -O-5’), methoxypropyl (MOP), and thioformacetal (3’-S-CH ₂ -O-5’)· Further neutral intemucleotide linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides. Further neutral internucleotide linkages include nonionic linkages comprising mixed N, O, S and CFh component parts.

In certain embodiments, oligonucleotides comprise modified and/or unmodified intemucleotide linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each intemucleotide linking group is a phosphodiester intemucleotide linkage (P=0). In certain embodiments, each intemucleotide linking group of a modified oligonucleotide is a phosphorothioate intemucleotide linkage (P=S). In certain embodiments, each intemucleotide linkage of a modified oligonucleotide is independently selected from a phosphorothioate intemucleotide linkage and phosphodiester intemucleotide linkage. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the intemucleotide linkages within the gap are all modified. In certain such embodiments, some or all of the intemucleotide linkages in the wings are unmodified phosphodiester intemucleotide linkages. In certain embodiments, the terminal intemucleotide linkages are modified. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer, and the intemucleotide linkage motif comprises at least one phosphodiester intemucleotide linkage in at least one wing, wherein the at least one phosphodiester linkage is not a terminal intemucleotide linkage, and the remaining intemucleotide linkages are phosphorothioate intemucleotide linkages.

In certain embodiments, each phosphorothioate intemucleotide linkage is independently selected from a stereorandom phosphorothioate, a (Sp) phosphorothioate, and a (Rp) phosphorothioate. In certain such embodiments, all of the phosphorothioate linkages are stereorandom. In certain embodiments, all of the phosphorothioate linkages in the wings are (Sp) phosphorothioates, and the gap comprises at least one Sp-Sp-Rp motif. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising such intemucleotide linkage motifs.

Substituted sugar moieties include, but are not limited to one of the following at the 2' position: H (deoxyribose); OH (ribose); F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl- O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein includes, for example, at least one nucleotide or nucleoside modified at the 2' position of the sugar. In some embodiments, the nucleoside modification is a 2'-O-alkyl, 2'-O-alkyl-O- alkyl or 2'-fluoro-modified nucleotide or an end cap. In some embodiments, nucleoside modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the oligonucleotide. In some embodiments, an oligonucleotide includes a single modified nucleoside. In some embodiments, an oligonucleotide includes at least two modified nucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more nucleosides, up to the entire length of the oligonucleotide (e.g., synthetic oligonucleotide). In some embodiments, each nucleoside of the oligonucleotide is a modified nucleoside.

Nucleosides or nucleobases include the natural purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleosides include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio) N6- (isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8- (alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl) adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6- (methyl)adenine, N6,N6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6- (alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8- (halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N- (methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3- (methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5- (methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6- (azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5- (methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4- (thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5- (methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5- (alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5- (guanidiniumalkyl)uracil, 5-(l,3-diazole-l-alkyl)uracil, 5-(cyanoalkyl)uracil, 5- (dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil- 5 -oxy acetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl- methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4- (thio)pseudouracil,2,4-(dithio)psuedouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5- (alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4- (dithio)pseudouracil, 1 -substituted pseudouracil, l-substituted-2(thio)-pseudouracil, 1- substituted-4-(thio)pseudouracil, l-substituted-2,4-(dithio)pseudouracil, 1- (aminocarbonylethylenyl)-pseudouracil, l-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1- (aminocarbonylethylenyl)-4-(thio)pseudouracil, l-aminocarbonylethylenyl)-2,4- (dithio)pseudouracil, l-(aminoalkylarninocarbonylethylenyl)-pseudouracil, 1- (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, l-(aminoalkylamino- carbonylethylenyl)-4-(thio)pseudouracil, l-(aminoalkylamino-carbonylethylenyl)-2,4- (dithio)pseudouracil, l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, l-(aza)-2-(thio)-3-(aza)- phenoxazin-l-yl, l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, l-(aza)-2-(thio)-3-(aza)-phenthiazin-

1-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, 7-substituted l-(aza)-2-(thio)-3- (aza)-phenoxazin-l-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7-substituted 1- (aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, 7-(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)- phenoxazin-l-yl, 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l- yl, 7- (aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7-(aminoalkylhydroxy)-l-(aza)-

2-(thio)-3-(aza)-phenthiazin-l-yl, 7- (guanidiniumalkylhydroxy)-l,3-(diaza)-2-(oxo)- phenoxazin-l-yl, 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxa zin-l-yl, 7- (guanidiniumalkyl-hydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin-l -yl, 7- (guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiaz in-l-yl, l,3,5-(triaza)-2,6- (dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3- (methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, diiluorotolyl, 4- (iluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5 nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino) purine, 5- substituted pyrimidines, N2-substituted purines, N6-substituted purines, 06-substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-

2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-substituted-6- phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-

3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-y l, ortho- (aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-(aminoalkylhydroxy)- 6-phenyl- pyrrolo-pyrimidin-2-on-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino- pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivatives thereof.

In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified intemucleotide linkages. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or intemucleotide linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and intemucleotide linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or intemucleotide linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is a chimeric oligonucleotide. Chimeric oligonucleotides may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, and/or oligonucleotide mimetics. Such compounds have also been referred to in the art as hybrids or mixed backbone or chimeric or gapmers. In particular a gapmer is an oligonucleotide that has at least three discrete portions, two of which are similar e.g., include one or more common backbone modifications or include one or more common nucleoside modifications, and surround a region that is distinct, e.g., does not include the common backbone modifications or does not include the common nucleoside modifications.

In some embodiments, the oligonucleotide has a gap segment. In some embodiments, the oligonucleotide does not have a gap segment. In some embodiments, the oligonucleotide comprises or consists of a 5 ’-wing segment, a 3 ’-wing segment, and a gap segment.

As disclosed herein, a “5 ’-wing segment” corresponds to two or more linked nucleosides positioned at the 5 ’-end of an oligonucleotide (e.g., a synthetic oligonucleotide) and corresponding to nucleosides positioned before the first nucleoside at the 5 ’-end of a gap segment. As disclosed herein, a “3 ’-wing segment” corresponds to two or more linked nucleosides positioned after the last nucleic acid at the 3’ end of the gap segment and including the last nucleic acid at the 3’ end of the oligonucleotide. In some embodiments, at least one nucleoside of the 5 ’-wing segment and/or at least one nucleoside of the 3 ’-wing segment comprises a modification.

In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides comprise or consist of a region having a gapmer motif, which is defined by two external regions or “wings” and a central or internal region or “gap.” The three regions of a gapmer motif (the 5’-wing, the gap, and the 3 ’-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3 ’-most nucleoside of the 5 ’-wing and the 5 ’-most nucleoside of the 3 ’-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5’-wing differs from the sugar motif of the 3’-wing (asymmetric gapmer).

As disclosed herein, a “gap segment” corresponds to two or more linked nucleosides in an oligonucleotide, which are positioned between the 5 ’-wing segment and the 3 ’-wing segment. In some embodiments, a gap segment refers to one or more linked nucleosides located at the center or near the center of an oligonucleotide, such as a synthetic oligonucleotide. In some embodiments, the gap segment consists of two to three, two to four, two to five, two to six, two to seven, two to eight, two to nine, two to 10, two to 20, two to 30, two to 40, two to 50, three to four, three to five, three to six, three to seven, three to eight, three to nine, three to 10, three to 20, three to 30, three to 40, three to 50, four to five, four to six, four to seven, four to eight, four to nine, four to 10, four to 20, four to 30, four to 40, four to 50, five to six, five to seven, five to eight, five to nine, five to 10, five to 20, five to 30, five to 40, five to 50, six to seven, six to eight, six to nine, six to 10, six to 20, six to 30, six to 40, six to 50, seven to eight, seven to nine, seven to 10, seven to 20, seven to 30, seven to 40, seven to 50, eight to nine, eight to 10, eight to 20, eight to 30, eight to 40, eight to 50, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 20 to 30, 20 to 40, 20 to 50, 30 to 40, 40 to 50, or 50 linked nucleosides, or any range or combination thereof.

In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides. In certain embodiments, each nucleoside of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least one nucleoside of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least two nucleosides of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least three nucleosides of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least four nucleosides of each wing of a gapmer comprises a modified sugar moiety.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer comprises a 2’-P-D-deoxyribosyl sugar moiety. In certain embodiments, at least one nucleoside of the gap of a gapmer comprises a modified sugar moiety.

In certain embodiments, the gapmer is a deoxy gapmer. In certain embodiments, the nucleosides on the gap side of each wing/gap junction comprise 2’-deoxyribosyl sugar moieties and the nucleosides on the wing sides of each wing/gap junction comprise modified sugar moieties. In certain embodiments, each nucleoside of the gap comprises a 2 ^? -b-ϋ- deoxyribosyl sugar moiety. In certain embodiments, each nucleoside of each wing of a gapmer comprises a modified sugar moiety.

In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified oligonucleotide comprises the same 2’-modification. In some embodiments, each nucleoside of an oligonucleotide disclosed herein comprises a 2’-MOE modification.

Herein, the lengths (number of nucleosides) of the three regions of a gapmer may be provided using the notation [number of nucleosides in the 5 ’-wing] - [number of nucleosides in the gap] - [number of nucleosides in the 3’-wing]. Thus, a 3-10-3 gapmer consists of 3 linked nucleosides in each wing and 10 linked nucleosides in the gap. Where such nomenclature is followed by a specific modification, that modification is the modification in each sugar moiety of each wing and the gap nucleosides comprise 2 ’ - b- D-dco x yri bo s y 1 sugar moieties. Thus, a 5-10-5 MOE gapmer consists of 5 linked 2’-MOE nucleosides in the 5’- wing, 10 linked 2’^-D-deoxynucleosides in the gap, and 5 linked 2’ -MOE nucleosides in the 3’-wing. A 3-10-3 LNA gapmer consists of 3 linked LNA nucleosides in the 5’-wing, 10 linked 2’-P-D-deoxynucleosides in the gap, and 3 linked LNA nucleosides in the 3 ’-wing.

In certain embodiments, modified oligonucleotides are 5-10-5 MOE gapmers. In certain embodiments, modified oligonucleotides are 3-10-3 BNA gapmers. In certain embodiments, modified oligonucleotides are 3-10-3 LNA gapmers. In certain embodiments, modified oligonucleotides are 3-10-3 cEt gapmers.

In some embodiments, the gap segment comprises a nucleoside having a 2’-O-Me modification. In some embodiments, the gap segment comprises two to 10 (i.e., 2, 3, 4, 5, 6,

7, 8, 9, or 10) nucleosides having 2’-O-Me modifications. In some embodiments, the gap segment does not comprise a nucleoside having a 2’-O-Me modification. In some embodiments, the gap segment comprises a phosphorothioate internucleoside linkage. In some embodiments, the gap segment comprises two to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10) phosphorothioate internucleoside linkages. In some embodiments, the gap segment comprises all phosphorothioate intemucleoside linkages (i.e., each internucleoside linkage within the gap segment is a phosphorothioate intemucleoside linkage). In some embodiments, the gap segment does not comprise phosphorothioate intemucleoside linkages.

In some embodiments, the gap segment comprises a phosphodiester intemucleoside linkage. In some embodiments, the gap segment comprises two to 10 phosphodiester intemucleoside linkages. In some embodiments, the gap segment comprises all phosphodiester intemucleoside linkages (i.e., each intemucleoside linkage within the gap segment is a phosphodiester intemucleoside linkage). In some embodiments, the gap segment does not comprise phosphodiester intemucleoside linkages.

In some embodiments, the modification is a 2’-O-Me modification. In some embodiments, the nucleosides in the synthetic oligonucleotide are modified with one or more other modifications disclosed herein. In some embodiments, the intemucleoside linkages within the gap segment and the linkages connecting the gap segment to the 3 ’-wing segment and/or the 5’-wing segment are all phosphorothioate linkages (*). In some embodiments, the intemucleoside linkages connecting the rest of the nucleosides of both the 5’ and 3 ’-wing segments are phosphodiester linkages. In some embodiments, the intemucleoside linkages connecting the rest of the nucleosides of both the 5’ and 3 ’-wing segments are phosphorothioate linkages (*). In some embodiments, all intemucleoside linkages connecting the nucleosides of the 5 ’-wing segment, the gap segment, and the 3 ’-wing segment are phosphorothioate linkages (*).

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein comprises a fully-phosphorothioate backbone (i.e., each intemucleoside linkage of the oligonucleotide is a phosphorothioate internucleoside linkage), and each nucleoside of the oligonucleotide comprises an MOE modification. In some such embodiments, one or more guanosine nucleosides is 7-deaza modified. In some embodiments, the oligonucleotide comprises a fully-phosphorothioate backbone, each nucleoside of the oligonucleotide comprises an MOE modification, one or more guanosine nucleosides of the oligonucleotide is 7-deaza modified, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or each) cytosine nucleoside of the oligonucleotide is 5-methyl modified.

Sequence complementarity

As disclosed herein, an oligonucleotide (e.g., a synthetic oligonucleotide) is “complementary” or “sufficiently complementary” to a nucleic acid (e.g. a region of a CLN3 sequence, such as a CLN3 sequence as set forth in any one of SEQ ID NO: 1-10 and 773) when hybridization can occur in an antiparallel configuration between the oligonucleotide and the nucleic acid. A double-stranded nucleic acid or oligonucleotide can be “complementary” or “sufficiently complementary” to another nucleic acid or oligonucleotide if hybridization can occur between one of the strands of the first nucleic acid or oligonucleotide and the second nucleic acid or oligonucleotide. Complementarity (e.g., the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base pairing (e.g., Watson-Crick base pairing) rules. An oligonucleotide (e.g., a synthetic oligonucleotide) may be complementary to or sufficiently complementary to a region of a nucleic acid even if the two base sequences are not 100% complementary, as long as the duplex structure formed between has the desired stability.

In some embodiments, two nucleic acids are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences are able to form base pairing interactions (e.g., Watson-Crick base pairing interactions) between at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% of their nucleotides, or any range or combination thereof.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions (e.g., Watson-Crick base pairing interactions) between at least or about 45% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 50% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 55% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 60% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 65% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 70% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 75% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 80% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 85% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 90% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 92.5% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 95% of their nucleotides. In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 96% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 97% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 98% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 99% of their nucleotides.

In some embodiments, two nucleic acid sequences are considered complementary to or sufficiently complementary to one another when their nucleic acid sequences form base pairing interactions between at least or about 100% of their nucleotides.

In a non-limiting example, two nucleic acid sequences that are each 20 nucleosides in length are considered 80% complementary if 16 of their respective 20 nucleosides are able to form base-pairing interactions. Two nucleic acid sequences that are of different lengths may also be complementary. For example, a nucleic acid sequence that is 20 nucleosides in length may be complementary to or sufficiently complementary to a nucleic acid sequence that is longer if their sequences are able to form base pairing interactions (e.g., Watson-Crick base pairing interactions) between a sufficient number of nucleotides of the 20 nucleoside length nucleic acid sequence and a sufficient number of nucleotides of the longer nucleic acid sequence. A nucleic acid sequence that is 20 nucleosides in length is considered 100% complementary to a longer nucleic acid sequence if all 20 of its nucleosides are able to form base-pairing interactions with 20 contiguous nucleosides of the longer nucleic acid sequence. Similarly, a nucleic acid sequence that is 20 nucleosides in length is considered 80% complementary to or sufficiently complementary to a longer nucleic acid sequence if 16 of its 20 nucleosides are able to form base-pairing interactions with 16 nucleosides of the longer nucleic acid sequence in a complementary sequence alignment.

It is possible to introduce mismatched bases without eliminating activity of an antisense oligonucleotide against its target(s). For example, Gautschi et al ( Journ . Natl. Cancer Inst. 93:463-471, 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

In certain embodiments, oligonucleotides are complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99%, 95%, 90%, 85%, or 80% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a region that is 100% or fully complementary to a target nucleic acid. In certain embodiments, the region of full complementarity is from 6 to 20, 10 to 18, or 18 to 20 nucleobases in length.

In certain embodiments, oligonucleotides comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain embodiments, antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, in certain embodiments selectivity of the oligonucleotide for its target is improved by the presence of a mismatch. In certain embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) comprises a mismatch at position 1, 2, 3, 4, 5, 6, 7, or 8 from its 5 ’-end. In certain embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) comprises a mismatch at position 1, 2, 3, 4, 5, 6, 7, or 8 from its 3’-end. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide having a gapmer motif. In certain embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5’-end of the gap region. In certain embodiments, the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3 ’-end of the gap region. In certain embodiments, the mismatch is at position 1, 2, 3, or 4 from the 5 ’-end of the wing region. In certain embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3 ’-end of the wing region.

Target nucleic acids

In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide (e.g., a synthetic oligonucleotide disclosed herein) comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is a mature mRNA or a pre- mRNA. In some embodiments, the target nucleic acid includes intronic, exonic and/or untranslated regions. In certain embodiments, the target RNA is a mature mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction (i.e., is partially within an intron and partially within an exon). In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is the RNA transcriptional product of a retrogene. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non- coding RNA is selected from: a long non-coding RNA (IncRNA), a short non-coding RNA, and an intronic RNA molecule.

In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide (e.g., a synthetic oligonucleotide disclosed herein) comprising a region that is complementary to a target nucleic acid. In some embodiments, the target nucleic acid is a CLN3 nucleic acid.

In certain embodiments, contacting a cell with an oligomeric compound comprising a nucleic acid sequence with complementarity to any one of SEQ ID NO: 1-10 and 773 reduces the amount of CLN3 RNA in the cell or reduces the amount of a particular CLN3 RNA isoform in the cell, and in certain embodiments modulates (e.g., reduces or increases) the amount of CLN3 protein or the amount of a particular CLN3 isoform in the cell. In certain embodiments, the oligomeric compound consists of a modified oligonucleotide (e.g., a synthetic oligonucleotide disclosed herein). In certain embodiments, contacting a cell with an oligomeric compound comprising a nucleic acid sequence with complementarity to any one of SEQ ID NO: 1-10 and 773 results in amelioration of symptoms of a disease or disorder (e.g., an NCL disease, such as CLN3 Batten disease). In certain embodiments, the oligomeric compound comprises or consists of a modified oligonucleotide (e.g., a synthetic oligonucleotide disclosed herein). In certain embodiments, the oligomeric compound comprises or consists of a modified oligonucleotide, at least one spacer moiety, and at least one linker moiety.

CLN3 sequences

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is complementary or sufficiently complementary to a region of a CLN3 sequence (e.g., a human CLN3 sequence, such as a CLN3 sequence provided in any one of SEQ ID NO: 1- 10). In some embodiments, the region is within exon la (nucleotides 5001-5283 of SEQ ID NO:l and nucleotides 1-283 of SEQ ID NO: 2); exon lb (nucleotides 5221-5589 of SEQ ID NO: 1 and nucleotides 221-589 of SEQ ID NO: 2); exon lc (nucleotides 5468-5589 of SEQ ID NO: 1 and nucleotides 468-589 of SEQ ID NO: 2); intron 1 (nucleotides 5590-5742 of SEQ ID NO:l and nucleotides 590-742 of SEQ ID NO: 2); exon 2 (nucleotides 5743-5821 of SEQ ID NO:l and nucleotides 743-821 of SEQ ID NO:2); intron 2 (nucleotides 5822-7916 of SEQ ID NO:l and nucleotides 822-916 of SEQ ID NO:2); exon 3 (nucleotides 7917-8013 of SEQ ID NO:l and nucleotides 2917-3013 of SEQ ID NO:2); intron 3 (nucleotides 8014-8640 of SEQ ID NO: 1 and nucleotides 3014-3640 of SEQ ID NO: 2); exon 4 (nucleotides 8641- 8712 of SEQ ID NO: 1 and nucleotides 3641-3712 of SEQ ID NO: 2); intron 4 (nucleotides 8713-9561 of SEQ ID NO: 1 and nucleotides 3713-4561 of SEQ ID NO: 2; SEQ ID NO: 3); exon 5 (nucleotides 9562-9641 of SEQ ID NO: 1 and nucleotides 4562-4641 of SEQ ID NO: 2; SEQ ID NO: 4); intron 5 (nucleotides 9642-9761 of SEQ ID NO: 1 and nucleotides 4642- 4761 of SEQ ID NO: 2; SEQ ID NO: 5); exon 6 (nucleotides 9762-9847 of SEQ ID NO: 1 and nucleotides 4762-4847 of SEQ ID NO: 2; SEQ ID NO: 6); intron 6 (nucleotides 9848- 10652 of SEQ ID NO: 1 and nucleotides 4848-5652 of SEQ ID NO: 2; SEQ ID NO: 7); exon 7 (nucleotides 10653-10725 of SEQ ID NO: 1 and nucleotides 5653-5725 of SEQ ID NO: 2); intron 7 (nucleotides 10726-10812 of SEQ ID NO: 1 and nucleotides 5726-5812 of SEQ ID NO: 2); exon 8 (nucleotides 10813-10956 of SEQ ID NO: 1 and nucleotides 5813-5956 of SEQ ID NO: 2); intron 8 (nucleotides 10957-13184 of SEQ ID NO: 1 and nucleotides 5957- 8184 of SEQ ID NO: 2; SEQ ID NO: 8); exon 9 (nucleotides 13185-13297 of SEQ ID NO: 1 and nucleotides 8185-8297 of SEQ ID NO: 2; SEQ ID NO: 9); intron 9 (nucleotides 13298- 14630 of SEQ ID NO: 1 and nucleotides 8298-9630 of SEQ ID NO: 2; SEQ ID NO: 10); exon 10 (nucleotides 14631-14677 of SEQ ID NO: 1 and nucleotides 9631-9677 of SEQ ID NO: 2); intron 10 (nucleotides 14678-14757 of SEQ ID NO: 1 and nucleotides 9678-9757 of SEQ ID NO: 2); exon 11 (nucleotides 14758-14826 of SEQ ID NO: 1 and nucleotides 9758- 9826 of SEQ ID NO: 2); intron 11 (nucleotides 14827-14920 of SEQ ID NO: 1 and nucleotides 9827-9920 of SEQ ID NO: 2); exon 12 (nucleotides 14921-14976 of SEQ ID NO: 1 and nucleotides 9921-9976 of SEQ ID NO: 2); intron 12 (nucleotides 14977-15104 of SEQ ID NO: 1 and nucleotides 9977-10104 of SEQ ID NO: 2); exon 13 (nucleotides 15105- 15198 of SEQ ID NO: 1 and nucleotides 10105-10198 of SEQ ID NO: 2); intron 13 (nucleotides 15199-19425 of SEQ ID NO: 1 and nucleotides 10199-14425 of SEQ ID NO:

2); exon 14 (nucleotides 19426-19566 of SEQ ID NO: 1 and nucleotides 14426-14566 of SEQ ID NO: 2); intron 14 (nucleotides 19567-19667 of SEQ ID NO: 1 and nucleotides 14567-14667 of SEQ ID NO: 2); or exon 15 (nucleotides 19668-20024 of SEQ ID NO: 1 and nucleotides 14668-15024 of SEQ ID NO: 2) of CLN3.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein is complementary or sufficiently complementary to a region of a murine CLN3 sequence (e.g., a murine CLN3 sequence provided in SEQ ID NO: 773).

In some embodiments, the region is within intron 4 of CLN3. In some embodiments, the region is within exon 5 of CLN3. In some embodiments, the region is within intron 5 of CLN3. In some embodiments, the region is within exon 6 of CLN3. In some embodiments, the region is within intron 6 of CLN3. In some embodiments, the region is within intron 8 of CLN3. In some embodiments, the region is within exon 9 of CLN3. In some embodiments, the region is within intron 9 of CLN3.

In some embodiments, the region overlaps a portion of an intron and a portion of an exon. For example, in some embodiments, the region is partially within intron 4 and partially within exon 5; partially within exon 5 and partially within intron 5; partially within intron 5 and partially within exon 6; partially within exon 6 and partially within intron 6; partially within intron 8 and partially within exon 9; or partially within exon 9 and partially within intron 9. In some embodiments, the region is between nucleotides 9,462 and 9,947 of SEQ ID NO: 1 (nucleotides 5462-5947 of SEQ ID NO: 2) or between nucleotides 13,185 and 13,397 of SEQ ID NO: 1 (nucleotides 8185-8397 of SEQ ID NO: 2).

Spherical nucleic acids (SNAs)

As disclosed herein, a “spherical nucleic acid” (SNA) refers to a three-dimensional arrangement of nucleic acids or oligonucleotides, such as synthetic oligonucleotides, forming an oligonucleotide shell, with oligonucleotides arranged radially around and/or on the exterior of a core (e.g., a nanoparticle core). In some embodiments, the core is a hollow core produced by a three-dimensional arrangement of molecules which form the outer boundary of the core. For instance, the molecules may be in the form of a lipid layer (e.g. lipid monolayer or lipid bilayer), which has a hollow center. Alternatively, the molecules may be in the form of lipids, such as amphipathic lipids (e.g., sterols), which are linked to or associated with, either directly or indirectly, an end of the oligonucleotide (e.g., synthetic oligonucleotide). In some embodiments, tocopherols or sterols, such as cholesterol, linked (e.g., indirectly attached) to an end of an oligonucleotide (e.g., a synthetic oligonucleotide) may associate with the outer surface of a core (e.g., a hollow core), such that the oligonucleotides radiate outward from the core.

In some embodiments, an SNA comprises an oligonucleotide shell comprising a synthetic oligonucleotide and a core. In some embodiments, a synthetic oligonucleotide is associated with the core (e.g., a nanoparticle core) through a covalent or non-covalent interaction. In some embodiments, a synthetic oligonucleotide is associated with the exterior surface of the core. In some embodiments, the synthetic oligonucleotides are associated with the core (e.g., exterior surface) through a molecular species via, for instance, a hydrophobic interaction. In some embodiments, the synthetic oligonucleotide is associated with a molecular species (e.g., a hydrophobic group), either directly or indirectly. In some embodiments, the synthetic oligonucleotide is indirectly associated with or indirectly attached to a molecular species (e.g., hydrophobic group) through a spacer. In some embodiments, the molecular species (e.g., hydrophobic group) is associated with the core. In some embodiments, the core is a liposome core. In some embodiments, the liposome core comprises a lipid bilayer. In some embodiments, the synthetic oligonucleotide is covalently attached to one or more lipids of the lipid layer. In some embodiments, the synthetic oligonucleotide is not covalently attached to one or more lipids of the lipid layer. In some embodiments, the synthetic oligonucleotide is covalently attached to a molecular species.

In some embodiments, an SNA disclosed herein comprises a single population of synthetic oligonucleotides (i.e., each synthetic oligonucleotide of the oligonucleotide shell is the same, having the same sequence). In some embodiments, an SNA disclosed herein may comprise two populations, three populations, four populations, five populations, six populations, seven populations, eight populations, nine populations, 10 populations, or more of distinct synthetic oligonucleotides (i.e., the synthetic oligonucleotides of the oligonucleotide shell represent two, three, four, etc. different synthetic oligonucleotides having different sequences).

In some embodiments, an SNA disclosed herein comprises a synthetic oligonucleotide comprising a molecular species indirectly attached to a nucleotide of the synthetic oligonucleotide. In some embodiments, the molecular species is linked to the core of the SNA. In some embodiments, the molecular species is indirectly attached to the nucleotide at the 3 ’-end of the synthetic oligonucleotide, such that the 3 ’-end of the synthetic oligonucleotide is linked to the core of the SNA and the 5 ’-end of the synthetic oligonucleotide is distal from the core (e.g., is oriented away from the core). In some embodiments, the molecular species is indirectly attached to the nucleotide at the 5 ’-end of the synthetic oligonucleotide, such that the 5 ’-end of the synthetic oligonucleotide is linked to the core of the SNA and the 3 ’-end of the synthetic oligonucleotide is distal from the core (e.g., is oriented away from the core).

In some embodiments, an SNA disclosed herein comprises a first synthetic oligonucleotide comprising a first nucleic acid sequence and a second synthetic oligonucleotide comprising a second nucleic acid sequence. In some embodiments, the oligonucleotide(s) of an SNA disclosed herein are indirectly attached to a molecular species. In some embodiments, a first molecular species is indirectly attached to a nucleotide of a first synthetic oligonucleotide and a second molecular species is indirectly attached to a nucleotide of a second synthetic oligonucleotide. In some embodiments, the first molecular species is indirectly attached to the nucleotide at the 3 ’-end of the first synthetic oligonucleotide and the second molecular species is indirectly attached to the nucleotide at the 5 ’-end of the second synthetic oligonucleotide. In some embodiments, the first molecular species is indirectly attached to the nucleotide at the 5 ’-end of the first synthetic oligonucleotide and the second molecular species is indirectly attached to the nucleotide at the 3 ’-end of the second synthetic oligonucleotide. In some embodiments, the first molecular species is indirectly attached to the nucleotide at the 5 ’-end of the first synthetic oligonucleotide and the second molecular species is indirectly attached to the nucleotide at the 5 ’-end of the second synthetic oligonucleotide. In some embodiments, the first molecular species is indirectly attached to the nucleotide at the 3 ’-end of the first synthetic oligonucleotide and the second molecular species is indirectly attached to the nucleotide at the 3 ’-end of the second synthetic oligonucleotide. In some embodiments, an SNA comprises additional synthetic oligonucleotides (e.g., a third synthetic oligonucleotide comprising a third nucleic acid sequence, a fourth synthetic oligonucleotide comprising a fourth nucleic acid sequence, etc.), each of which is indirectly attached via a nucleotide at its 3’ - or 5 ’-end to a molecular species. In some embodiments, the end of each synthetic oligonucleotide to which the molecular species is attached is linked to the core of the SNA and the opposite end of the synthetic oligonucleotide is distal from the core (e.g., is oriented away from the core).

As used herein, “conjugated,” “attached,” “linked,” or “directly attached” means two entities stably bound to one another by any physiochemical means. It is important that the nature of the attachment is such that it does not impair substantially the effectiveness of either entity. Keeping these parameters in mind, any covalent or non-covalent linkage known to those of ordinary skill in the art may be employed. In some embodiments, covalent linkage is preferred. Noncovalent conjugation includes hydrophobic interactions, ionic interactions, high affinity interactions such as biotin avidin and biotin streptavidin complexation and other affinity interactions. Such means and methods of attachment are well known to those of ordinary skill in the art.

In some embodiments, an SNA comprises an oligonucleotide shell. In some embodiments, an oligonucleotide shell includes one or more oligonucleotides (e.g., synthetic oligonucleotides) on and/or around the exterior of the core. In some embodiments, the oligonucleotide shell comprises or consists of one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about 10,000, about 15,000, about 20,000 or more synthetic oligonucleotides, or any range or combination thereof. In some embodiments, the oligonucleotide shell comprises or consists of 2-1000, 2-900, 2-800, 2-700, 2-600, 2-500, 2-400, 2-300, 2-200, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-45, 2-40, 2-35, 2- 30, 2-25, 2-20, 2-15 or 2-10 oligonucleotides (e.g., synthetic oligonucleotides). In some embodiments, the oligonucleotide shell comprises or consists of 5-100, 10-100, 15-100, 20- 100, 25-100, 50-100, 5-50, 10-50, 15-50, 20-50, 25-50, 5-40, 10-40, 15-40, 20-40, 25-40, 5- 30, 10-30, 15-30, 20-30, 25-30, 5-25, 10-25, 15-25 or 20-25 oligonucleotides (e.g., synthetic oligonucleotides). In some embodiments, the oligonucleotide shell comprises or consists of 30 or about 30 oligonucleotides (e.g., synthetic oligonucleotides).

In some embodiments, each synthetic oligonucleotide of the oligonucleotide shell comprises the same nucleic acid sequence. In some embodiments, the synthetic oligonucleotides of the oligonucleotide shell comprise different nucleic acid sequences (e.g., the synthetic oligonucleotides comprise two different sequences, three different sequences, four different sequences, five different sequences, six different sequences, seven different sequences, eight different sequences, or more). In embodiments in which the synthetic oligonucleotides of the oligonucleotide shell comprise more than one distinct nucleic acid sequence, each different nucleic acid sequence may be comprised within an equal proportion of the synthetic oligonucleotides, such that the proportion of synthetic oligonucleotides having a particular nucleic acid sequence within a shell comprising n distinct nucleic acid sequences is 1 In (e.g., two nucleic acid sequences are each comprised within 1/2 or 50% of the synthetic oligonucleotides, three nucleic acid sequences are each comprised within 1/3 or 33% of the synthetic oligonucleotides, four nucleic acid sequences are each comprised within 1/4 or 25% of the synthetic oligonucleotides, etc.). In embodiments in which the synthetic oligonucleotides of the oligonucleotide shell comprise more than one distinct nucleic acid sequence, each different nucleic acid sequence may be comprised within different proportions of the synthetic oligonucleotides (e.g., one nucleic acid sequence may be comprised within 75% of the synthetic oligonucleotides and a second nucleic acid sequence may be comprised within 25% of the synthetic oligonucleotides; one nucleic acid sequence may be comprised within 50% of the synthetic oligonucleotides, a second nucleic acid sequence may be comprised within 25% of the synthetic oligonucleotides and a third nucleic acid sequence may be comprised within 25% of the synthetic oligonucleotides, etc.).

In some embodiments, an SNA disclosed herein comprises or consists of lipids or lipid molecules and oligonucleotides (e.g., synthetic oligonucleotides) at a molar ratio between 100 to 1 and 10 to 1, between 100 to 1 and 20 to 1, between 100 to 1 and 30 to 1, between 100 to 1 and 40 to 1, between 100 to 1 and 50 to 1, between 100 to 1 and 60 to 1, between 100 to 1 and 70 to 1, between 100 to 1 and 80 to 1, between 100 to 1 and 90 to 1, between 90 to 1 and 10 to 1, between 90 to 1 and 20 to 1, between 90 to 1 and 30 to 1, between 90 to 1 and 40 to 1, between 90 to 1 and 50 to 1, between 90 to 1 and 60 to 1, between 90 to 1 and 70 to 1, between 90 to 1 and 80 to 1, between 80 to 1 and 10 to 1, between 80 to 1 and 20 to 1, between 80 to 1 and 30 to 1, between 80 to 1 and 40 to 1, between 80 to 1 and 50 to 1, between 80 to 1 and 60 to 1, between 80 to 1 and 70 to 1, between 70 to 1 and 10 to 1, between 70 to 1 and 20 to 1, between 70 to 1 and 30 to 1, between 70 to 1 and 40 to 1, between 70 to 1 and 50 to 1, between 70 to 1 and 60 to 1, between 60 to 1 and 10 to 1, between 60 to 1 and 20 to 1, between 60 to 1 and 30 to 1, between 60 to 1 and 40 to 1, between 60 to 1 and 50 to 1, between 50 to 1 and 10 to 1, between 50 to 1 and 20 to 1, between 50 to 1 and 30 to 1, between 50 to 1 and 40 to 1, between 40 to 1 and 10 to 1, between 40 to 1 and 20 to 1, between 40 to 1 and 30 to 1, between 30 to 1 and 10 to 1, between 30 to 1 and 20 to 1 of lipids or lipid molecules to oligonucleotide (e.g., synthetic oligonucleotide), or any range or combination thereof.

In some embodiments, an SNA disclosed herein comprises lipids or lipid molecules and oligonucleotides (e.g., synthetic oligonucleotides) at a molar ratio of at least or about 10 to 1, 20 to 1, 30 to 1, 40 to 1, 50 to 1, 60 to 1, 70 to 1, 80 to 1, 90 to 1, or 100 to 1 of lipids to oligonucleotide (e.g., synthetic oligonucleotide), or any range or combination thereof. In some embodiments, an SNA disclosed herein comprises lipids or lipid molecules and oligonucleotides (e.g., synthetic oligonucleotides) at a molar ratio of between 55 to 1 and 45 to 1 of lipid or lipid molecules to oligonucleotide. In some embodiments, an SNA disclosed herein comprises lipids or lipid molecules and oligonucleotides (e.g., synthetic oligonucleotides) at a molar ratio of or about 50 to 1 of lipid to oligonucleotide (e.g., synthetic oligonucleotide). In some embodiments, the oligonucleotide portion of the lipids or lipid molecules to oligonucleotide ratio is divided between more than one population of oligonucleotides (e.g., a first population of synthetic oligonucleotides, a second population of synthetic oligonucleotides, etc.), such that the ratio represents the total number of oligonucleotides of the SNA regardless of their nucleic acid sequences.

In some embodiments, an SNA comprising a first population of synthetic oligonucleotides and a second population of synthetic oligonucleotides may comprise lipids or lipid molecules at a molar ratio of 50 to 0.5 to 0.5 of lipids or lipid molecules to first population of synthetic oligonucleotides to second population of synthetic oligonucleotides.

In embodiments in which an SNA comprises more than two populations of synthetic oligonucleotides, the SNA may comprise lipids or lipid molecules at a molar ratio of 50 to 1, where the oligonucleotide portion is divided equally between the more than two populations of synthetic oligonucleotides. In some embodiments, an SNA comprising a first population of synthetic oligonucleotides and a second population of synthetic oligonucleotides may comprise different amounts of the first population of synthetic oligonucleotides and the second population of synthetic oligonucleotides, such that the molar ratio of lipids or lipid molecules to the first population of synthetic oligonucleotides to the second population of synthetic oligonucleotides is, for example, 50 to 0.1 to 0.9, 50 to 0.2 to 0.8, 50 to 0.3 to 0.7,

50 to 0.4 to 0.6, 50 to 0.6 to 0.4, 50 to 0.7 to 0.3, 50 to 0.8 to 0.2, or 50 to 0.9 to 0.1, or any range or combination thereof, of lipid molecules to first population of synthetic oligonucleotides to second population of synthetic oligonucleotides. In embodiments in which an SNA comprises more than two populations of synthetic oligonucleotides, the SNA may comprise lipids or lipid molecules at a molar ratio of 50 to 1, where the oligonucleotide portion is divided unequally between the more than two populations of synthetic oligonucleotides (e.g., the total oligonucleotide portion sums to 1, but each individual population does not represent an equal proportion of the total).

In some embodiments, the oligonucleotides (e.g., synthetic oligonucleotides) are on the exterior surface of the core (e.g., liposome core). In some embodiments, at least one oligonucleotide (e.g., a synthetic oligonucleotide) has its 5 ’-terminus exposed on the exterior surface away from the core (e.g., the 5 ’-terminus is located at the end of the oligonucleotide distal from the core). In some embodiments, all of the oligonucleotides (e.g., synthetic oligonucleotides) in an SNA have their 5 ’-termini exposed on the exterior surface away from the core (e.g., the 5’-termini are located at the ends of the oligonucleotides distal from the core). In some embodiments, at least one oligonucleotide (e.g., a synthetic oligonucleotide) has its 3 ’-terminus exposed on the exterior surface away from the core (e.g., the 3 ’-terminus is located at the end of the oligonucleotide distal from the core). In some embodiments, all of the oligonucleotides (e.g., synthetic oligonucleotides) in an SNA have their 3 ’-termini exposed on the exterior surface away from the core (e.g., the 3 ’-termini are located at the ends of the oligonucleotides distal from the core). In some embodiments, the SNA does not include an oligonucleotide (e.g., a synthetic oligonucleotide) inside the core (e.g., liposome core).

In some embodiments, an SNA comprises a first synthetic oligonucleotide and a second synthetic oligonucleotide. In some embodiments, the 5’-terminus of the first synthetic oligonucleotide is exposed on the exterior surface of the SNA away from the core, and the 3’- terminus of the second synthetic oligonucleotide is exposed on the surface of the SNA away from the core. In some embodiments, the 3 ’-terminus of the first synthetic oligonucleotide is exposed on the exterior surface of the SNA away from the core, and the 5 ’-terminus of the second synthetic oligonucleotide is exposed on the surface of the SNA away from the core. In some embodiments, the 3 ’-terminus of the first synthetic oligonucleotide is exposed on the exterior surface of the SNA away from the core, and the 3 ’-terminus of the second synthetic oligonucleotide is exposed on the surface of the SNA away from the core. In some embodiments, the 5 ’-terminus of the first synthetic oligonucleotide is exposed on the exterior surface of the SNA away from the core, and the 5 ’-terminus of the second synthetic oligonucleotide is exposed on the surface of the SNA away from the core.

In some embodiments, two or more of the oligonucleotides (e.g., synthetic oligonucleotides) in an SNA are crosslinked to one another. In some embodiments, all of the oligonucleotides (e.g., synthetic oligonucleotides) in an SNA are crosslinked to one or more additional oligonucleotides. In some embodiments, the oligonucleotides (e.g., synthetic oligonucleotides) in an SNA are not crosslinked.

In some embodiments, an SNA disclosed herein has a diameter of, or a population or plurality of SNAs disclosed herein has a mean diameter of about 10 to about 150 nm. In some embodiments, the mean diameter of the population of SNAs is from or about 15 nm to about 100 nm, about 20 nm to about 100 nm, about 25 nm to about 100 nm, about 15 nm to about 50 nm, about 20 nm to about 50 nm, about 10 nm to about 70 nm, about 15 nm to about 70 nm about 20 nm to about 70 nm, about 10 nm to about 30 nm, about 15 nm to about 30 nm, about 20 nm to about 30 nm, about 10 nm to about 40 nm, about 15 nm to about 40 nm, about 20 nm to about 40 nm, about 10 nm to about 80 nm, about 15 nm to about 80 nm, or about 20 nm to about 80 nm. In some embodiments, the mean diameter of the population of SNAs is from about 15 nm to about 45 nm. In some embodiments, an SNA disclosed herein has a diameter, or a population of SNAs disclosed herein has a mean diameter of or about 10 nm, about 15 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In some embodiments, an SNA disclosed herein has a diameter, or a population or a plurality of SNAs disclosed herein have a mean diameter of 30 nm or about 30 nm.

The SNAs disclosed herein may be stable self-assembling nanostructures. For instance, the SNA may comprise an oligonucleotide (e.g., a synthetic oligonucleotide) of 18- 21 nucleosides in length having a sequence disclosed herein, wherein a hydrophobic group at the 3’ or 5’ terminus of the oligonucleotide self-associates to form the core or associates with the core of the nanostructure in water or other suitable solvents. A hydrophobic group as used herein may include cholesterol, a cholesteryl or modified cholesteryl residue, adamantine, dihydrotesterone, long chain alkyl, long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins, such as vitamin E, fatty acids either saturated or unsaturated, fatty acid esters, such as triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen.

Nanoparticle cores

In certain embodiments, an oligonucleotide described herein is attached to the external surface of a core to form a corona or shell of oligonucleotides. In certain embodiments, an oligonucleotide described herein is comprised in a spherical nucleic acid (SNA). Those skilled in the art would recognize that a variety of materials can be used as the core of an SNA. In certain embodiments, the core is a liposome, an inorganic nanoparticle, a self-organizing oligonucleotide-based structure, a dendrimer, or a protein.

In some embodiments, the nanoparticle core of an SNA disclosed herein is a solid core or a hollow core.

In some embodiments, a solid core is a spherical- shaped material with or without a hollow center. As disclosed herein, a “spherical shape” refers to a general shape and does not imply or is not limited to a perfect sphere or round shape. In some embodiments, a spherical shape includes imperfections. In some embodiments, the core comprises or consists of a metal core (e.g., any metal). Non-limiting examples of metals include gold, silver, platinum, aluminum, palladium, copper, cobalt, indium, nickel and mixtures thereof. In some embodiments, the core comprises gold or is a gold core. In some embodiments, the core can be a lattice structure including degradable gold. In some embodiments, the core may comprise semiconductor and/or magnetic materials. Solid cores can be constructed from a wide variety of materials known to those skilled in the art including but not limited to: noble metals (gold, silver), transition metals (iron, cobalt) and metal oxides (silica). A solid core may be inert, paramagnetic, or superparamagnetic. The solid cores can be constructed from either pure compositions of described materials, or in combinations of mixtures of any number of materials, or in layered compositions of materials. In some embodiments, a solid core can be composed of a polymeric core such as amphiphilic block copolymers, hydrophobic polymers such as polystyrene, poly(lactic acid), poly(lactic co-glycolic acid), poly(glycolic acid), poly(caprolactone) and other biocompatible polymers known to those skilled in the art. In some embodiments, the core comprises or consists of a solid core or a semi-solid core.

In some embodiments, the core of an SNA disclosed herein is a hollow core. In some embodiments, a hollow core has at least some space in the center region of a shell material. In some embodiments, a hollow core is a liposome core. A liposome core as used herein refers to a centrally located core compartment formed by a component of the lipids or phospholipids that form a lipid bilayer. “Liposomes” are artificial, self-closed vesicular structures of various sizes and shapes, where one or several membranes encapsulate an aqueous core. Most typically liposome membranes are formed from lipid bilayer membranes, where the hydrophilic head groups are oriented towards aqueous environments, and the lipid chains are oriented away from aqueous environments. Liposomes can be formed as well from other amphiphilic monomeric and polymeric molecules, such as polymers, like block copolymers, or polypeptides. Unilamellar vesicles are liposomes defined by a single membrane enclosing an aqueous space. In contrast, oligo- or multilamellar vesicles are built up of several membranes. Typically, the membranes are roughly 4 nm thick and are composed of amphiphilic lipids, such as phospholipids, of natural or synthetic origin. Optionally, the membrane properties can be modified by the incorporation of other lipids such as sterols or cholic acid derivatives.

The lipid bilayer is composed of two layers of lipids or lipid molecules. Each lipid or lipid molecule in a layer is oriented substantially parallel to adjacent lipids or lipid molecules, and two layers of lipids or lipid molecules that form a bilayer have the polar ends of their molecules exposed to the aqueous phase and the non-polar ends adjacent to each other. The two lipid layers that form the lipid bilayer are substantially parallel to one another. The central aqueous region of the liposomal core may be empty or filled fully or partially with water, an aqueous emulsion, oligonucleotides, or other therapeutic or diagnostic agents or aqueous solutions thereof.

A lipid bilayer or liposome core can be constructed from one or more lipids known to those in the art including but not limited to: l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), l-palmitoyl-2-oleoyl-sn- phosphatidylcholine (POPC), l,2-distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DSPG), l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DOPG), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l,2-di-(9Z- octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine (DPPE), sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phosphosphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives, phospholipids such as phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, lysophosphatidylglycerols, phosphatidylserines, lysophosphatidylserines, phosphatidylinositols, inositol phosphates, LPI, cardiolipins, lysocardiolipins, bis(monoacylglycero) phosphates, (diacylglycero) phosphates, ether lipids, diphytanyl ether lipids, and plasmalogens of various lengths, saturation states, and their derivatives, sterols such as cholesterol, desmosterol, stigmasterol, lanosterol, lathosterol, diosgenin, sitosterol, zymosterol, zymostenol, 14-demethyl-lanosterol, cholesterol sulfate, DHEA, DHEA sulfate, 14-demethyl- 14-dehydrlanosterol, sitostanol, campesterol, ether anionic lipids, ether cationic lipids, lanthanide chelating lipids, A-ring substituted oxysterols, B-ring substituted oxysterols, D-ring substituted oxysterols, side-chain substituted oxysterols, double substituted oxysterols, cholestanoic acid derivatives, fluorinated sterols, fluorescent sterols, sulfonated sterols, phosphorylated sterols, and polyunsaturated sterols of different lengths, saturation states, and their derivatives. In some embodiments, the liposome core comprises or consists of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

As disclosed herein, a “lipid” refers to its conventional sense as a generic term encompassing fats, lipids, and alcohol-ether-soluble constituents of protoplasm, which are insoluble in or immiscible with water. Lipids usually consist of a hydrophilic and a hydrophobic moiety. In water, lipids can self-organize to form bilayer membranes, where the hydrophilic moieties (head groups) are oriented towards the aqueous phase, and the lipophilic moieties (acyl chains) are embedded in the hydrophobic region between the two hydrophilic layers comprised within the bilayer. Lipids can also comprise two hydrophilic moieties (bola amphiphiles). In that case, membranes may be formed from a single lipid layer, and not a bilayer. Typical non-limiting examples of lipids are fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, glycolipids, sulpholipids, aminolipids, chromolipids, and fatty acids. The term encompasses both naturally occurring and synthetic lipids.

In some embodiments, the lipids are steroids, sterols (e.g., cholesterol), phospholipids, including phosphatidyl, phosphatidylcholines and phosphatidylethanolamines and sphingomyelins. Where there are fatty acids, they could be about 12-24 carbon chains in length, containing up to 6 double bonds. The fatty acids are linked to a backbone, which may be derived from glycerol. The fatty acids within one lipid can be different (asymmetric), or there may be only 1 type of fatty acid chain present, e.g. lysolecithins. Mixed formulations are also possible, particularly when the non-cationic lipids are derived from natural sources, such as lecithins (phosphatidylcholines) purified from egg yolk, bovine heart, brain, liver or soybean. In some embodiments, the SNA includes a neutral lipid. The neutral lipid may be, for example, l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn- phosphatidylcholine (DMPC), l-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2- distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DSPG), l,2-dioleoyl-sn-glycero-3- phospho-(l'-rac-glycerol) (DOPG), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), and l,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), any related phosphatidylcholine or neutral lipids available from commercial vendors. In some embodiments, the SNA includes l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

In some embodiments, the core is a niosome core. A noisome is a vesicle formed from non-ionic surfactant oriented in a bilayer. Niosomes commonly have cholesterol added as an excipient, but other lipid-based and non-lipid-based constituents can also be included. Methods for preparation of niosomes are known in the art. In some embodiments polyethylene glycol (PEG) is included during or following niosome preparation. Niosome vesicles are structurally and functionally analogous to liposomes, but are based on non-ionic surfactant rather than lipid as the primary constituent. Common non-ionic surfactants used include sorbitans (spans) or polysorbates (tween); however, a wide variety of non-ionic surfactants can be used to prepare niosomes.

In some embodiments, the core comprises or consists of a protein. In some embodiments, the protein is an antibody, a glycoprotein, an enzyme, a receptor, a repeat containing protein, or an engineered alternative scaffold. In some embodiments, the core is a protein selected from any of asialoglycoprotein, albumin, fibronectin, transferrin, alpha- galactosidase, b-glucosidase, b-cerebrosidase, a-glucosidase, a-mannosidase, b- glucuronidase, b-hexosamininidase A, acid lipase, lipase, hydrogenase, protease, oxygenase, non-naturally occurring alternative scaffold proteins such as a Centyrin (see Diem et al, Protein Engineering, Design and Selection , 2014, page 419-419; Dudkin et al, WO 2019/118818), a DARPin (see Stumpp et al, Biodrugs, 2020, page 423-433).

Those skilled in the art would understand that nanostructures (e.g., a nanoparticle core of an SNA) can be made entirely of oligonucleotides. In some embodiments, the core is a self-assembled three-dimensional structure made entirely from oligonucleotides. In some embodiments the core is a dendrimer made from oligonucleotides (see Getts et al, WO 2017/143171, Kadushin et al, WO 2010/017544, Getts et al, WO 2016/168578, Nilsen et al, US 5,484,904, Nilsen et al, US 6,274,723). In some embodiments, the core is a dendrimer made from materials other than oligonucleotides. In some embodiments, the core comprises dendrimers comprising poly(amidoamine) (PAMAM). Methods for making PAMAM dendrimers are known to those of skill in the art and generally involve a twostep iterative reaction sequence that produces concentric shells (generations) of dendritic b-alanine units around a central initiator core.

This PAMAM core-shell architecture grows linearly in diameter as a function of added shells (generations). Meanwhile, the surface groups amplify exponentially at each generation according to dendritic-branching mathematics. They are available in generations GO-10 with 5 different core types and 10 functional surface groups. The dendrimer-branched polymer may consist of PAMAM, polyester, polyether, poly-lysine, or polyethylene glycol (PEG), polypeptide dendrimers.

In some embodiments, the core (e.g., liposome core) of an SNA disclosed herein has a diameter of, or the cores (e.g., liposome cores) of a population of SNAs disclosed herein has a mean diameter of about 10 to about 150 nm. In some embodiments, the diameter of the core or the mean diameter of the population of cores is from about 15 nm to about 100 nm, about 20 nm to about 100 nm, about 25 nm to about 100 nm, about 15 nm to about 50 nm, about 20 nm to about 50 nm, about 10 nm to about 70 nm, about 15 nm to about 70 nm, about 20 nm to about 70 nm, about 10 nm to about 30 nm, about 15 nm to about 30 nm, about 20 nm to about 30 nm, about 10 nm to about 40 nm, about 15 nm to about 40 nm, about 20 nm to about 40 nm, about 10 nm to about 80 nm, about 15 nm to about 80 nm, or about 20 nm to about 80 nm. In some embodiments, the core (e.g., liposome core) of an SNA disclosed herein has a diameter of, or the cores (e.g., liposome cores) of a population of SNAs disclosed herein has a mean diameter of about 15 nm to about 45 nm. In some embodiments, the core (e.g., liposome core) of an SNA disclosed herein has a diameter of, or the cores (e.g., liposome cores) of a population of SNAs disclosed herein has a mean diameter of about 10 nm, about 15 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In some embodiments, the core (e.g., liposome core) of an SNA disclosed herein has a diameter of, or the cores (e.g., liposome cores) of a population of SNAs disclosed herein has a mean diameter of about 30 nm.

In some embodiments, the core (e.g., liposome core) of an SNA disclosed herein has a diameter, or the cores (e.g., liposome cores) of a population of SNAs disclosed herein has a mean diameter of about or less than about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, and/or 40 nm, or any range or combination thereof. In some embodiments, the core (e.g., liposome core) of an SNA disclosed herein has a diameter, or the cores (e.g., liposome cores) of a population of SNAs disclosed herein has a mean diameter of about or less than 40 nm. In some embodiments, the core (e.g., liposome core) of an SNA disclosed herein has a diameter, or the cores (e.g., liposome cores) of a population of SNAs disclosed herein has a mean diameter of or about 20 nm.

Molecular species and spacers

In certain embodiments, provided herein are oligomeric compounds, which consist of an oligonucleotide (modified or unmodified) and optionally one or more spacers and/or linkers. In some embodiments, spacers comprise or consist of one or more moiety which connects the linker moiety to the oligonucleotide. In some embodiments, a linker moiety may be attached to either or both ends of an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein, or a linker moiety may be attached to an intermediate position of an oligonucleotide. In certain embodiments, linker moieties attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, the linker is attached at the 3’ and/or 5’-end of the oligonucleotide. In some embodiments, the linker is attached at or near the 3 ’-end of the oligonucleotide. In certain embodiments, the linker is attached at or near the 5 ’-end of the oligonucleotide.

In some embodiments, a synthetic oligonucleotide comprises a molecular species. In some embodiments, the molecular species anchors the synthetic oligonucleotide to the core (e.g., liposome core) of an SNA disclosed herein. In certain embodiments, the molecular species is a linker. A molecular species may be attached at various positions of a synthetic oligonucleotide (e.g., at the 3 ’-end or 5 ’-end of a nucleotide sequence of a synthetic oligonucleotide disclosed herein). In some embodiments, a molecular species is attached to the nucleotide at the 3 ’-end of a synthetic oligonucleotide disclosed herein. In some embodiments, a molecular species is attached to the nucleotide at the 5 ’-end of a synthetic oligonucleotide disclosed herein. In some embodiments, a molecular species is attached to a nucleotide not at a terminus of a synthetic oligonucleotide disclosed herein (e.g., an internal nucleotide one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more positions away from the 3’- or 5’-end).

In some embodiments, the molecular species enhances the stability of the synthetic oligonucleotide against 3’ - or 5 ’-exonucleases. In some embodiments, a molecular species is attached to or associated with an internal nucleotide or a nucleotide on a branch of a synthetic oligonucleotide. In some embodiments, a molecular species is attached to a 2’-position of a nucleoside of a synthetic oligonucleotide. In some embodiments, a molecular species is linked to the heterocyclic base of a nucleoside of a synthetic oligonucleotide. In some embodiments, the molecular species is modified. In some embodiments, the molecular species comprises multiple moieties. In some embodiments, the molecular species is, comprises or consists of a lipophilic (i.e., hydrophobic) moiety. In some embodiments, the molecular species is, comprises or consists of a lipophilic (i.e., hydrophobic) moiety and a hydrophilic moiety. In some embodiments, the molecular species comprises multiple lipophilic (i.e., hydrophobic) moieties and/or multiple hydrophilic moieties. In some embodiments, the molecular species is, comprises or consists of a hydrophobic moiety, such as a cholesterol or a cholesteryl ester.

In some embodiments, the molecular species is, comprises or consists of a cholesterol and an additional moiety. In some embodiments, the molecular species is, comprises or consists of a cholesterol and a hydrophilic moiety. In some embodiments, the molecular species comprises a cholesterol and a triethylene glycol (TEG). In some embodiments, the molecular species comprises one or more cholesterols and one or more TEGs. In some embodiments, the molecular species comprises or consists of a cholesterol linked (e.g., covalently linked) to a TEG. In some embodiments, the molecular species is a cholesteryl ester. In some embodiments, the molecular species is a cholesteryl ester linked (e.g., covalently linked) to a TEG. In some embodiments, the molecular species is, comprises or consists of N-cholesteryl-3-aminopropyl-triethyleneglycol-glyceryl- 1-O-phosphodiester (3CholTEG).

In some embodiments, the molecular species is a lipid, a sterol, lipid moieties such as a cholesterol moiety, cholic acid, a thioether (e.g., hexyl- S-tritylthiol), a thiocholesterol, an aliphatic chain (e.g., dodecandiol or undecyl residues), a phospholipid (e.g., di-hexadecyl- rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, stearyl, C ₁ 6 alkyl chain, bile acids, cholic acid, taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins, such as vitamin E, saturated fatty acids, unsaturated fatty acids, fatty acid esters, such as triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl, t- butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy576), Hoechst 33258 dye, psoralen, or ibuprofen.

In some embodiments, oligonucleotides (e.g., synthetic oligonucleotides) disclosed herein are anchored to the surface of a core through a molecular species. In some embodiments, the molecular species is selected from tocopherols, sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phosphosphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives, phospholipids such as phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, lysophosphatidylglycerols, phosphatidylserines, lysophosphatidylserines, phosphatidylinositols, inositol phosphates, LPI, cardiolipins, lysocardiolipins, bis(monoacylglycero) phosphates, (diacylglycero) phosphates, ether lipids, diphytanyl ether lipids, and plasmalogens of various lengths, saturation states, and their derivatives, sterols such as cholesterol, desmosterol, stigmasterol, lanosterol, lathosterol, diosgenin, sitosterol, zymosterol, zymostenol, 14-demethyl-lanosterol, cholesterol sulfate, DHEA, DHEA sulfate, 14-demethyl- 14-dehydrlanosterol, sitostanol, campesterol, ether anionic lipids, ether cationic lipids, lanthanide chelating lipids, A-ring substituted oxysterols, B-ring substituted oxysterols, D-ring substituted oxysterols, side-chain substituted oxysterols, double substituted oxysterols, cholestanoic acid derivatives, fluorinated sterols, fluorescent sterols, sulfonated sterols, phosphorylated sterols, and polyunsaturated sterols of different lengths, saturation states, and their derivatives.

In some embodiments, a molecular species is connected to an oligonucleotide (e.g., a synthetic oligonucleotide) disclosed herein through a spacer (e.g., a non-nucleotidic spacer). Non-limiting examples of spacers include abasic residues (dSpacer), oligoethyleneglycol, such as triethyleneglycol (spacer 9 or iSp9; TEG) or hexaethylenegylcol (spacer 18 or iSp 18; HEG), alkane-diol (e.g., butanediol), C ₂₂ alkyl, C ₂₀ alkyl, C½ alkyl, C ₁₀ alkyl, C ₂₁ alkyl, C ₁₉ alkyl, C ₁₈ alkyl, C ₁ 5 alkyl, Cu alkyl, C ₁ 3 alkyl, C ₁ 2 alkyl, C ₁ 1 alkyl, C ₉ alkyl, C ₈ alkyl, C ₇ alkyl, Ce alkyl, C ₅ alkyl, C ₂₂ alkenyl, C ₂₀ alkenyl, C _\ e alkenyl, C ₁₀ alkenyl, C ₂₁ alkenyl, C ₁₉ alkenyl, C ₁ s alkenyl, C ₁₅ alkenyl, C ₁₄ alkenyl, C ₁₃ alkenyl, C ₁₂ alkenyl, C ₁₁ alkenyl, C ₉ alkenyl, Cs alkenyl, C ₇ alkenyl, Ce alkenyl, or C ₅ alkenyl. In some embodiments, the spacer comprises or consists of one or more nucleotides. In some embodiments, the spacer does not comprise or consist of an oligonucleotide.

In some embodiments, the synthetic oligonucleotide is attached to the spacer through a covalent bond (e.g., phosphodiester, phosphorodithioate or phosphorothioate bond). In some embodiments, the spacer does not comprise or consist of an oligonucleotide spacer. The spacer in some embodiments appears just once in the molecule or in some embodiments is incorporated several times (e.g., via phosphodiester, phosphorothioate, methylphosphonate, or amide linkages). In embodiments in which multiple spacer moieties are incorporated, the individual spacer moieties may be attached to one another and/or to the synthetic oligonucleotide via phosphodiester, phosphorothioate, methylphosphonate, or amide linkages. In some embodiments, the spacer comprises a TEG and/or a HEG. In some embodiments, the spacer comprises or consists of hexa(ethyleneglycol)phosphodiester- hexa(ethyleneglycol)phosphodiester (HEG-HEG). In some embodiments, the molecular species connected to the spacer comprises or consists of hexa(ethyleneglycol)phosphodiester- hexa(ethyleneglycol)phosphodiester-3-O-(N-cholesteryl-3-amin opropyl)-triethyleneglycol- glyceryl- 1-O-phosphodiester (HEG-HEG-CholTEG or CholTEG-HEG-HEG) . The HEG- HEG-CholTEG or CholTEG-HEG-HEG may be connected (e.g., directly or indirectly) to the 5’-end and/or the 3’-end of a nucleic acid or oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein.

In some embodiments, the spacer comprises or consists of the structure

HEG-HEG spacer), wherein a and b label attachment sites, e.g., for covalent linkage to an oligonucleotide and/or a molecular species. In some embodiments, the molecular comprises or consists of the structure

CholTEG linker), wherein c labels an attachment site, e.g., for covalent linkage to a spacer and/or an oligonucleotide. In some embodiments, the molecular species connected to the spacer comprises or consists of the structure

(III; HEG-HEG-CholTEG), wherein a labels an attachment site, e.g., for covalent linkage to an oligonucleotide. In some embodiments, a synthetic oligonucleotide disclosed herein comprises, consists essentially of, or consists of the structure

(IV). In some embodiments, the oligonucleotide in Formula IV has the nucleic acid sequence of an Antisense Sequence listed in Table 1. In some embodiments, a synthetic oligonucleotide comprising, consisting essentially of, or consisting of the structure of Formula IV has the structure of an ASO Structure listed in Table 1.

In certain embodiments, it is desirable for the spacer and/or the linker to be cleaved from the oligonucleotide. For example, in certain circumstances oligonucleotide compounds comprising a particular spacer and/or linker are better taken up by a particular cell type, but once the compounds have been taken up, it is desirable that the spacer and/or linker be cleaved to release the oligonucleotide. Thus, certain spacers and linkers may comprise one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide bond. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a spacer moiety or a linker moiety.

In certain embodiments, a cleavable moiety comprises or consists of one or more nucleosides. In certain such embodiments, the one or more nucleosides are linked to one another and/or to the remainder of the oligonucleotide through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2'-deoxynucleoside that is attached to either the 3' or 5'- terminal nucleoside of an oligonucleotide by a phosphate internucleotide linkage and covalently attached to the remainder of the spacer or linker moiety by a phosphate or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is 2'- deoxy adenosine.

Modulation of CLN3 splice variant and protein levels

In some embodiments, administration of an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) to a subject increases the amount of a particular splicing isoform of CLN3 in a cell or subject. In some embodiments, the level is increased in the cell or subject by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about 21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about 25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about 29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about 33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about 37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about 41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about 45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about 49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about two-fold, at least or about three-fold, at least or about four-fold, at least or about five- fold, at least or about six-fold, at least or about seven-fold, at least or about eight-fold, at least or about nine-fold, at least or about 10-fold or more, or any range or combination thereof, relative to a reference level.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) is contacted with a cell to increase the amount of a particular splicing isoform of CLN3 in the cell. In some embodiments, the level in the cell is increased by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about 21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about 25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about 29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about 33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about 37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about 41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about 45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about 49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about two-fold, at least or about three-fold, at least or about four-fold, at least or about five-fold, at least or about six- fold, at least or about seven-fold, at least or about eight-fold, at least or about nine-fold, at least or about 10-fold or more, relative to a reference level.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) is administered to a subject at a dose which results in an increased amount of a particular splicing isoform of CLN3 in the subject by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about 21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about 25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about 29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about 33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about 37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about 41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about 45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about 49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about two-fold, at least or about three-fold, at least or about four-fold, at least or about five-fold, at least or about six-fold, at least or about seven-fold, at least or about eight-fold, at least or about nine-fold, at least or about 10-fold or more, relative to a reference level. In some embodiments, the CLN3 splicing isoform levels are increased in a cell or cells of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are increased in a tissue or tissues of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are increased in an organ or organs of a subject with a disease or disorder (e.g., Batten disease).

In some embodiments, the CLN3 splicing isoform levels are increased in the nervous system of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are increased in the eye of a subject with a disease or disorder (e.g. Batten disease). In some embodiments, the CLN3 splicing isoform levels are increased in the cortex, cerebellum, hippocampus, midbrain, thalamus, and/or occipital lobe of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are increased in a neuron, a microglial cell, and/or an astrocyte of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are increased in a cell of the eye (e.g., a retinal cell) of a subject with a disease or disorder (e.g., Batten disease).

In some embodiments, administration of an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) to a subject decreases the amount of a particular splicing isoform of CLN3 in a cell or subject. In some embodiments, the level is decreased in the cell or subject by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about

21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about

25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about

29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about

33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about

37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about

41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about

45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about

49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about

65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about

85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about

97%, at least or about 98%, at least or about 99%, or about 100%, or any range or combination thereof, relative to a reference level.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) is contacted with a cell to decrease the amount of a particular splicing isoform of CLN3 in the cell. In some embodiments, the level in the cell is decreased by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about 21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about 25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about 29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about 33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about 37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about 41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about 45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about 49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% relative to a reference level.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) is administered to a subject at a dose which results in a decreased amount of a particular splicing isoform of CLN3 in the subject by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about 21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about 25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about 29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about 33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about 37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about 41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about 45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about 49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% relative to a reference level.

In some embodiments, the CLN3 splicing isoform levels are decreased in a cell or cells of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are decreased in a tissue or tissues of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are decreased in an organ or organs of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are decreased in the nervous system of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are decreased in the eye of a subject with a disease or disorder (e.g. Batten disease). In some embodiments, the CLN3 splicing isoform levels are decreased in the cortex, cerebellum, hippocampus, midbrain, thalamus, and/or occipital lobe of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are decreased in a neuron, a microglial cell, and/or an astrocyte of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 splicing isoform levels are decreased in a cell of the eye (e.g., a retinal cell) of a subject with a disease or disorder (e.g., Batten disease).

In some embodiments, administration of an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) to a cell or subject increases the ratio of one CLN3 splicing isoform (e.g., a CLN3 mRNA splicing isoform) to another. In some embodiments, a CLN3 splicing isoform lacking exons 5, 7 and 8 is increased relative to a CLN3 splicing isoform lacking only exons 7 and 8, or not lacking exon 5. In some embodiments, a CLN3 splicing isoform lacking exons 6, 7 and 8 is increased relative to a CLN3 splicing isoform lacking only exons 7 and 8, or not lacking exon 6. In some embodiments, a CLN3 splicing isoform lacking exons 7, 8 and 9 is increased relative to a CLN3 splicing isoform lacking only exons 7 and 8, or not lacking exon 9. In some embodiments, one CLN3 splicing isoform (e.g., a CLN3 splicing isoform lacking exons 5, 7 and 8; a CLN3 splicing isoform lacking exons 6, 7 and 8; or a CLN3 splicing isoform lacking exons 7, 8 and 9) is increased relative to another CLN3 splicing isoform (e.g.,. a CLN3 splicing isoform lacking exons 7 and 8) such that the shorter isoform (e.g., lacking three exons) is 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13- fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more higher than the longer isoform (e.g., lacking two exons). In some preferred embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) increases one CLN3 isoform to a level at least 1.3-fold relative to a second CLN3 isoform.

In some embodiments, administration of an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) to a subject increases the amount of CLN3 protein in a cell or subject. In some embodiments, the level is increased in the cell or subject by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about 21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about 25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about 29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about 33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about 37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about 41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about 45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about 49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about two-fold, at least or about three-fold, at least or about four-fold, at least or about five- fold, at least or about six-fold, at least or about seven-fold, at least or about eight-fold, at least or about nine-fold, at least or about 10-fold or more, or any range or combination thereof, relative to a reference level.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) is contacted with a cell to increase the amount of CLN3 protein in the cell. In some embodiments, the level in the cell is increased by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about 21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about 25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about 29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about 33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about 37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about 41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about 45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about 49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about two-fold, at least or about three-fold, at least or about four-fold, at least or about five-fold, at least or about six-fold, at least or about seven-fold, at least or about eight-fold, at least or about nine-fold, at least or about 10-fold or more, relative to a reference level.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) is administered to a subject at a dose which results in an increased amount of CLN3 protein in the subject by at least or about 5%, at least or about 10%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%, at least or about

21%, at least or about 22%, at least or about 23%, at least or about 24%, at least or about

25%, at least or about 26%, at least or about 27%, at least or about 28%, at least or about

29%, at least or about 30%, at least or about 31%, at least or about 32%, at least or about

33%, at least or about 34%, at least or about 35%, at least or about 36%, at least or about

37%, at least or about 38%, at least or about 39%, at least or about 40%, at least or about

41%, at least or about 42%, at least or about 43%, at least or about 44%, at least or about

45%, at least or about 46%, at least or about 47%, at least or about 48%, at least or about

49%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about

65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about

85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about

97%, at least or about 98%, at least or about 99%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about two-fold, at least or about three-fold, at least or about four-fold, at least or about five-fold, at least or about six-fold, at least or about seven-fold, at least or about eight-fold, at least or about nine-fold, at least or about 10-fold or more, relative to a reference level.

In some embodiments, the CLN3 protein levels are increased in a cell or cells of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 protein levels are increased in a tissue or tissues of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 protein levels are increased in an organ or organs of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 protein levels are increased in the nervous system of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 protein levels are increased in the eye of a subject with a disease or disorder (e.g. Batten disease). In some embodiments, the CLN3 protein levels are increased in the cortex, cerebellum, hippocampus, midbrain, thalamus, and/or occipital lobe of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 protein levels are increased in a neuron, a microglial cell, and/or an astrocyte of a subject with a disease or disorder (e.g., Batten disease). In some embodiments, the CLN3 protein levels are increased in a cell of the eye (e.g., a retinal cell) of a subject with a disease or disorder (e.g., Batten disease).

The level of a CLN3 nucleic acid, including a particular splicing isoform of a CLN3 RNA, can be measured according to methods known in the art. In some embodiments, measuring the level of a CLN3 nucleic acid may refer to using laboratory methods known to those of ordinary skill in the art to quantify or qualitatively evaluate the amount of CLN3 RNA (e.g., CLN3 pre-mRNA and/or CLN3 mRNA) in a given sample, using methods including but not limited to quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), Northern blotting, in situ hybridization, or spectrophotometric analysis.

The level of a CLN3 protein, including a particular splicing isoform of a CLN3 protein, can be measured according to methods known in the art. In some embodiments, measuring CLN3 protein levels (e.g., a CLN3 splicing isoform) may refer to using laboratory methods known to those of ordinary skill in the art to quantify or qualitatively evaluate the amount of CLN3 protein in a given sample, using methods including but not limited to Western blotting, enzyme-linked immunosorbent assays (ELISA), immunohistochemical staining, immunofluorescent staining, mass spectrometry, spectrophotometric analysis, or colorimetric analysis such as bicinchinonic acid (BCA) assays.

As disclosed herein, a “reference level,” refers to a corresponding level in a subject with a disease or disorder (e.g., a subject with Batten disease) who has been administered a linear oligonucleotide (e.g., a synthetic oligonucleotide disclosed herein) which is not in an SNA format or pharmaceutical composition thereof; to a corresponding level in a subject with a disease or disorder (e.g., Batten disease) who has not been administered an SNA (e.g., an SNA disclosed herein) or pharmaceutical composition thereof; to a corresponding level in a subject with a disease or disorder (e.g., Batten disease) who has not been administered a linear oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein; to a corresponding level in a subject with the disease or disorder (e.g., Batten disease) prior to administration of an oligonucleotide (e.g., synthetic oligonucleotide), SNA or composition thereof (e.g., pharmaceutical composition thereof) disclosed herein to the subject with the disease or disorder (e.g., Batten disease); to a corresponding level in a subject with a disease or disorder (e.g., Batten disease) who has been administered a treatment other than a synthetic oligonucleotide, SNA, or composition thereof (e.g., pharmaceutical composition thereof) disclosed herein; to a corresponding level in a subject without Batten disease; or to a corresponding level in a subject without a disease or disorder, such as a neurodegenerative disease or disorder.

As disclosed herein, “a value that is less than” and equivalent phrases refer to values which are decreased or quantified to be smaller than a value or values to which they are compared (e.g., a reference value or reference values). As disclosed herein, “a value that is greater than” and equivalent phrases refer to values which are increased or quantified to be larger than a value or values to which they are compared (e.g., a reference value or reference values).

Methods of Use/Treatment

As disclosed herein, a disease or disorder refers to a disease or disorder in a subject.

In some embodiments, the disease or disorder is a neurodegenerative disease or disorder. In some embodiments, the disease or disorder is associated with or caused by genetic mutations, such as in the gene CLN3. In some embodiments, the disease or disorder is associated with mutations in the CLN3 gene causing production of an abnormally short protein, which may be broken down or may interfere with normal cellular processes. In some embodiments, the disease or disorder is associated with the accumulation of proteins and other cellular products in lysosomes, which may occur in cells throughout the body. In some embodiments, the accumulation of proteins and other cellular products in lysosomes results in damage to cells of the nervous system, including neurons. In some embodiments, the disease or disorder is associated with or causative of one or more symptoms, including visual impairment, retinal degeneration, intellectual disability, impaired cognitive function, speech impairment, seizures, muscle rigidity or stiffness, hypokinesia, stooped posture, arrhythmia, and impaired motor function. In some embodiments, the disease or disorder is a neuronal ceroid lipofuscinosis (NCL). In some embodiments, the disease or disorder is Batten disease. In some embodiments, the disease or disorder is CLN3 Batten disease.

As disclosed herein, a “cell” refers to a cell obtained from a subject or existing within a subject. In some embodiments, the cell is a cell which produces CLN3 transcripts (e.g., pre- mRNA molecules or mRNA molecules) and/or CLN3 protein. The cell may be a cell of the nervous system (e.g., a neuron), or the cell may be a non-neuronal cell (e.g., a skin cell, such as a fibroblast). In some embodiments, the cell is from a subject having a disease or disorder, such as a neurodegenerative disease or disorder disclosed herein. In some embodiments, the cell is from a subject having CLN3 Batten disease.

In some embodiments, the cell is contacted with an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein at a concentration of at least 0.001 nM, at least 0.01 nM, at least 0.1 nM, at least 1 nM, at least 10 nM, at least 100 nM, at least 1000 nM, at least 10 mM, at least 100 mM, at least 1000 mM, or above 1000 mM. In some embodiments, the cell is contacted with oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition (e.g., pharmaceutical composition) disclosed herein at a concentration range of 0.001 nM to 0.01 nM, 0.01 nM to 0.1 nM, 0.1 nM to 1 nM, 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1000 nM, 1000 nM to 10 mM, 10 mM to 100 mM, or 100 mM to 1000 mM. In some embodiments, the cell is contacted with oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition (e.g., pharmaceutical composition) disclosed herein at a concentration of 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10 mM, 100 mM, 1000 mM or above 1000 mM. In some embodiments, the concentration refers to the total weight or total mass of an oligonucleotide (e.g., synthetic oligonucleotide) disclosed herein in a volume of solution. In a non-limiting example, a concentration of 0.001 nM refers to 0.001 nmoles of oligonucleotide (e.g., synthetic oligonucleotide) per liter of solution (e.g., pharmaceutically acceptable carrier). In some embodiments, the concentration refers to the total weight or total mass of an SNA disclosed herein in a volume of solution. In a non-limiting example, a concentration of 0.001 nM refers to 0.001 nmoles of SNA per liter of solution (e.g., pharmaceutically acceptable carrier).

As disclosed herein, to “ameliorate” or “eliminate” a disease or a symptom of a disease refers to decreasing the severity of the disease or the symptom or removing evidence of the disease or the symptom in a subject. In some instances, ameliorating or eliminating a disease or symptom may refer to a change in a metric associated with the disease or symptom as evaluated by a medical professional or as measured by a laboratory test. In some instances, ameliorating or eliminating a disease, disorder or symptom may refer to a change in a metric associated with the disease, disorder or symptom as reported by the subject having the disease or symptom. In some instances, ameliorating or eliminating a disease or symptom may refer to a decrease or elimination of one or more neurodegenerative symptoms associated with the disease, disorder or symptom.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), SNA or pharmaceutically acceptable salt thereof disclosed herein is delivered to the central and/or peripheral nervous system.

As disclosed herein, a “second therapeutic agent” refers to a therapeutic agent other than an oligonucleotide (e.g., synthetic oligonucleotide), SNA or composition disclosed herein. The second therapeutic agent may refer to any composition, compound, or device that is administered to or utilized on a subject having a disease or disorder, which may enhance the therapeutic effect of the synthetic oligonucleotide or SNA disclosed herein, or which may ameliorate one or more symptoms of the disease or disorder. Second therapeutic agents include, but are not limited to antidepressants or other psychiatric medications and anticonvulsants.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is a human. In some embodiments, the mammal is a vertebrate animal including, but not limited to, a mouse, rat, dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, monkey, fish (e.g., aquaculture species, salmon, etc.). Thus, the disclosure herein can be used to treat diseases or disorders, such as a neurodegenerative disease (e.g., a neuronal ceroid lipofuscinosis, such as CLN3 Batten disease) in human or non-human subjects.

The disclosure herein can be used to treat a neurodegenerative disease in a human or non-human subject. The disclosure herein can be used to treat neuronal ceroid lipofuscinoses in a human or non-human subject. The disclosure herein can be used to treat Batten disease in a human or non-human subject. The disclosure herein can be used to treat CLN3 Batten disease in a human or non-human subject.

In some embodiments, the oligonucleotide (e.g., synthetic oligonucleotide) and/or SNA is administered in a pharmaceutical composition. Routes of administration include but are not limited to cutaneous, subcutaneous, nodal, systemic, intravenous, intrathecal, intravitreal, intracisterna magna, intracranial, oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal. Administration may be by any acceptable method, including but not limited to injection, topical administration, ingestion, or inhalation.

As disclosed herein, the dose (e.g., an effective amount) as it relates to administration of an oligonucleotide (e.g., synthetic oligonucleotide) and/or an SNA disclosed herein (e.g., without limitation the phrases “an effective amount of a synthetic oligonucleotide,” “an effective amount of a pharmaceutical composition,” “an effective amount of a spherical nucleic acid”, etc.) refers to the total weight or total mass of active agent (e.g., total weight or total mass of the synthetic oligonucleotides) in the SNA administered to the subject (e.g., subject with a disease or disorder). As disclosed herein, an “effective amount” refers to an amount that is capable of improving one or more symptoms of a disease or disorder or an amount that is capable of improving a metric associated with a disease or disorder.

In some embodiments, a dose disclosed herein is considered a fixed dose or a discrete dose. In some embodiments, a dose disclosed herein can be adjusted to depend on body weight or be made dependent on body weight. A non-limiting example includes a dose of 2 mg of an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein, that can also be administered as 2 mg/kg body weight, which depends on kg of body weight. In some embodiments, a dose disclosed herein is independent of body weight. In some embodiments, a dose disclosed herein can be adjusted to depend on body surface area (e.g., total body surface area, skin surface area to be treated, etc.) or be made dependent on body surface area. A non-limiting example includes a dose of 2 mg of an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein, that can also be administered as 2 mg/m ² body surface area, which depends on m ² of body surface area (e.g., total body surface area). In some embodiments, a dose disclosed herein is independent of body surface area.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is administered once a day, once every three days, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nice weeks, once every 10 weeks, once every 12 weeks, once every 18 weeks, once every 24 weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every 10 months, once every 11 months, once a year, once every two years, once every three years, once every four years.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is administered to a subject once a week, twice a week or three times per week, for four weeks, six weeks, eight weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 24 weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, one year, two years, three years, four years, five years, six years.

In some embodiments an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is administered to a subject every three weeks for four weeks, six weeks, eight weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 24 weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, one year, two years, three years, four years, five years, six years, seven years, eight years, nine years, or 10 years. In some embodiments, the SNA is administered every three weeks. In some embodiments, the SNA is administered about or at least about every four weeks, five weeks, six weeks, 2 months, three months, six months, nine months, one year, 1.5 years, two years, 2.5 years, three years, 3.5 years, four years, 4.5 years, five years, 5.5 years, or six years.

In some embodiments, the duration of the method for treating a disease or disorder (e.g., CLN3 Batten disease) with an oligonucleotide (e.g., synthetic oligonucleotide), a SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is for about or at least 3 months, for about or at least six months, for about or at least nine months, for about or at least one year, for about or at least 1.5 years, for about or at least two years, for about or at least 2.5 years, for about or at least 3 years, for about or at least 3.5 years, for about or at least 4 years, for about or at least 4.5 years, for about or at least 5 years, for about or at least 5.5 years, for about or at least 6 years, for about or at least 6.5 years, for about or at least 7 years, for about or at least 7.5 years, for about or at least 8 years, for about or at least 8.5 years, for about or at least 9 years, for about or at least 9.5 years, for about or at least 10 years, for about or at least 15 years, for about or at least 20 years or more than 20 years, or for the lifetime of the subject.

In some embodiments, an effective amount is from about 0.1 pg to 10,000 mg per dose, at least about 1 pg to 8,000 mg per dose, or 10 pg to 100 pg per dose. In some embodiments the dose administered is about or at least about 1 microgram, about or at least about 5 microgram, about or at least about 10 microgram, about or at least about 50 microgram, about or at least about 100 micro gram, about or at least about 200 microgram, about or at least about 350 microgram, about or at least about 500 microgram, about or at least about 1 milligram, about or at least about 5 milligram, about or at least about 10 milligram, about or at least about 50 milligram, about or at least about 100 milligram, about or at least about 200 milligram, about or at least about 350 milligram, about or at least about 500 milligram, about or at least about 1000 mg or more per dose, and any range or combination thereof. In non-limiting examples of a derivable range from the doses disclosed herein, a range of about 5 mg to about 100 mg, about 5 microgram to about 500 milligram, etc., can be administered based on the doses disclosed herein.

Stated in terms of subject body weight, in some embodiments the dose administered is about or at least about 1 microgram/kg of body weight, about or at least about 5 microgram/kg of body weight, about or at least about 10 microgram/kg of body weight, about or at least about 50 microgram/kg of body weight, about or at least about 100 microgram/kg of body weight, about or at least about 200 microgram/kg of body weight, about or at least about 350 microgram/kg of body weight, about or at least about 500 microgram/kg of body weight, about or at least about 1 milligram/kg of body weight, about or at least about 5 milligram/kg of body weight, about or at least about 10 milligram/kg of body weight, about or at least about 50 milligram/kg of body weight, about or at least about 100 milligram/kg of body weight, about or at least about 200 milligram/kg of body weight, about or at least about 350 milligram/kg of body weight, about or at least about 500 milligram/kg of body weight, to about or at least about 1000 mg/kg of body weight or more per administration, and any range or combination thereof. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg of body weight to about 100 mg/kg of body weight, about 5 microgram/kg of body weight to about 500 milligram/kg of body weight, etc., can be administered, based on the numbers disclosed above.

The absolute amount (e.g., a discrete dose) will depend upon a variety of factors including the concurrent treatment, the number of doses and the individual patient parameters including age, physical condition, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide), an SNA, or composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is administered at a dose between 0.1 mg and 10 mg, between 0.2 mg and 10 mg, between 0.3 mg and 10 mg, between 0.4 mg and 10 mg, between 0.5 mg and 10 mg, between 0.6 mg and 10 mg, between 0.7 mg and 10 mg, between 0.8 mg and 10 mg, between 0.9 mg and 10 mg, between 1 mg and 10 mg, between 1 mg and 1,000 mg, between 1 mg and 900 mg, between 1 mg and 800 mg, between 1 mg and 700 mg, between 1 mg and 600 mg, between 1 mg and 500 mg, between 1 mg and 450 mg, between 1 mg and 400 mg, between 1 mg and 350 mg, between 1 mg and 300 mg, between 1 mg and 250 mg, between 1 mg and 200 mg, between 1 mg and 150 mg, between 1 mg and 100 mg, between 1 mg and 90 mg, between 1 mg and 80 mg, between 1 mg and 70 mg, between 1 mg and 60 mg, between 1 mg and 60 mg, between 1 mg and 50 mg, between 1 mg and 49 mg, between 1 mg and 48 mg, between 1 mg and 47 mg, between 1 mg and 46 mg, between 1 mg and 45 mg, between 1 mg and 44 mg, between 1 mg and 43 mg, between 1 mg and 42 mg, between 1 mg and 41 mg, between 1 mg and 40 mg, between 1 mg and 39 mg, between 1 mg and 38 mg, between 1 mg and 37 mg, between 1 mg and 36 mg, between 1 mg and 35 mg, between 1 mg and 34 mg, between 1 mg and 33 mg, between 1 mg and 32 mg, between 1 mg and 31 mg, between 1 mg and 30 mg, between 1 mg and 29 mg, between 1 mg in 28 mg, between 1 mg and 27 mg, between 1 mg and 26 mg, between 1 mg and 25 mg, between 1 mg and 24 mg, between 1 mg and 23 mg, between 1 mg and 22 mg, between 1 mg and 21 mg, between 1 mg and 20 mg, between 1 mg and 19 mg, between 1 mg and 18 mg, between 1 mg and 17 mg, between 1 mg and 16 mg, between 1 mg and 15 mg, between 1 mg and 14 mg, between 1 mg and 13 mg, between 1 mg and 12 mg, between 1 mg and 11 mg, between 1 mg and 10 mg, between 1 mg and 9 mg, between 1 mg and 8 mg, between 1 mg and 7 mg, between 1 mg and 6 mg, between 1 mg and 5 mg, between 1 mg and 4 mg, between 1 mg and 2 mg, between 1 mg and 1.5 mg, between 1 mg and 3 mg, between 3 mg and 5 mg, between 5 mg and 7 mg, between 7 mg and 9 mg, between 9 mg and 14 mg, between 15 mg and 17 mg, between 18 mg and 31 mg, between 31 mg and 33 mg, between 0.5 mg and 2 mg, between 2 mg and 4 mg, between 11 mg and 13 mg, between 23 mg and 25 mg, between 2 mg and 31 mg, between 2 mg and 30 mg, between 2 mg and 29 mg, between 2 mg and 28 mg, between 2 mg and 27 mg, between 2 mg and 26 mg, between 2 mg and 25 mg, between 2 mg and 24 mg, between 2 mg and 23 mg, between 2 mg and 22 mg, between 2 mg and 21 mg, between 2 mg and 20 mg, between 2 mg and 19 mg, between 2 mg and 18 mg, between 2 mg and 17 mg, between 2 mg and 16 mg, between 2 mg and 15 mg, between 2 mg and 14 mg, between 2 mg and 13 mg, between 2 mg and 12 mg, between 2 mg and 11 mg, between 2 mg and 10 mg, between 2 mg and 9 mg, between 2 mg and 8 mg, between 2 mg and 7 mg, between 2 mg and 6 mg, between 2 mg and 5 mg, between 2 mg and 3 mg.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide), an SNA, or composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is administered at a dose of or about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 30 mg, 13.5 mg, 40 mg, 14.5 mg, 50 mg, 15.5 mg, 60 mg, 16.5 mg, 70 mg, 70.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg, 25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5 mg, 28 mg, 28.5 mg, 29 mg, 29.5 mg, 30 mg, 30.5 mg, 31 mg, 31.5 mg, 32 mg, 32.5 mg, 33 mg, 33.5 mg, 34 mg, 34.5 mg, 35 mg, 35.5 mg, 36 mg, 36.5 mg, 37 mg, 37.5 mg, 38 mg, 38.5 mg, 39 mg, 39.5 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 50 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, six and 50 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg, or any range or combination thereof.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide), an SNA, or composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is administered at a dose of at least or at least about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg,

1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 30 mg, 13.5 mg, 40 mg, 14.5 mg, 50 mg, 15.5 mg, 60 mg,

16.5 mg, 70 mg, 70.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg, 25.5 mg, 26 mg, 26.5 mg, 27 mg,

27.5 mg, 28 mg, 28.5 mg, 29 mg, 29.5 mg, 30 mg, 30.5 mg, 31 mg, 31.5 mg, 32 mg, 32.5 mg, 33 mg, 33.5 mg, 34 mg, 34.5 mg, 35 mg, 35.5 mg, 36 mg, 36.5 mg, 37 mg, 37.5 mg, 38 mg,

38.5 mg, 39 mg, 39.5 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg,

49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 50 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg, or any range or combination thereof.

In some embodiments, an oligonucleotide (e.g., a synthetic oligonucleotide), an SNA, or composition thereof (e.g., pharmaceutical composition) disclosed herein is administered at a dose greater than or greater than about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg,

1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.5 mg, 5 mg,

5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg,

11.5 mg, 12 mg, 12.5 mg, 30 mg, 13.5 mg, 40 mg, 14.5 mg, 50 mg, 15.5 mg, 60 mg, 16.5 mg, 70 mg, 70.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22 mg,

22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg, 25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5 mg, 28 mg, 28.5 mg, 29 mg, 29.5 mg, 30 mg, 30.5 mg, 31 mg, 31.5 mg, 32 mg, 32.5 mg, 33 mg,

33.5 mg, 34 mg, 34.5 mg, 35 mg, 35.5 mg, 36 mg, 36.5 mg, 37 mg, 37.5 mg, 38 mg, 38.5 mg, 39 mg, 39.5 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg,

50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 50 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg, or any range or combination thereof.

As disclosed herein, “pharmaceutically acceptable salts” are physiologically and pharmaceutically acceptable salts of the nucleic acids (e.g., salts that retain the desired biological activity of the compound of interest and do not impart undesired toxicological effects thereto). Pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

Pharmaceutical compositions of the present disclosure comprise an effective amount of one or more agents, dissolved or dispersed in a pharmaceutically acceptable carrier.

Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

In some embodiments, a “pharmaceutical or pharmacologically acceptable” composition refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. The compounds are generally suitable for administration to humans. This term requires that a compound or composition be nontoxic and sufficiently pure so that no further manipulation of the compound or composition is needed prior to administration to humans.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.000001% (w/w) of an active agent (e.g., an oligonucleotide, such as a synthetic oligonucleotide, or an SNA disclosed herein). In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit (w/w), or between about 25% to about 60%, for example, and any range or combination thereof. In some embodiments, the active agent (e.g., an oligonucleotide, such as a synthetic oligonucleotide, or an SNA disclosed herein) disclosed herein comprises between 0.000001% and 0.00001%, between 0.00001% and 0.0001%, between 0.0001% and 0.001%, between 0.001% and 0.01%, between 0.01% and 0.1%, between 0.1% and 1%, between 1% and 5%, between 5% and 10%, between 10% and 15%, between 15% and 20%, between 20% and 25%, between 25% and 30%, between 30% and 40%, between 40% and 50% (w/w), and any range or combination thereof. In some embodiments, the active agent (e.g., oligonucleotide, such as a synthetic oligonucleotide or an SNA disclosed herein) comprises 0.00007%, 0.007%, 0.01%, 0.1%, 1% (w/w).

In certain embodiments, disclosed herein are pharmaceutical compositions comprising one or more oligonucleotide (e.g., a synthetic oligonucleotide disclosed herein). In certain embodiments, disclosed herein are pharmaceutical compositions comprising one or more SNA. In certain embodiments, the oligonucleotides comprised within the one or more SNA each consist of a modified oligonucleotide. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises or consists of a sterile saline solution and one or more SNA. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises or consists of one or more SNA and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises or consists of one or more SNA and phosphate-buffered saline (PBS). In certain embodiments, the sterile PBS is pharmaceutical grade PBS.

In certain embodiments, a pharmaceutical composition comprises a modified SNA and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists of an SNA and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists essentially of an SNA and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade. In some embodiments, artificial cerebrospinal fluid contains sodium dihydrogen phosphate dihydrate, sodium phosphate dibasic anhydrous, sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, a suitable acid and base to balance pH, and water. In certain embodiments, each 1 mL of artificial cerebrospinal fluid contains:

Ingredient Quantity

Sodium dihydrogen phosphate dihydrate 0.050 mg Sodium phosphate dibasic anhydrous 0.097 mg

Sodium chloride 8.766 mg

Potassium chloride 0.224 mg

Calcium chloride dihydrate 0.206 mg

Magnesium chloride hexahydrate 0.163 mg

Suitable acid and base to balance pH As needed

Water To 1 mL

In certain embodiments, pharmaceutical compositions comprise one or more SNA and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, dextrose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, SNA may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In certain embodiments, pharmaceutical compositions comprising an SNA encompass any pharmaceutically acceptable salts of the oligonucleotide, esters of the oligonucleotide, or salts of such esters. In certain embodiments, modified oligonucleotides or SNA are in aqueous solution with sodium. In certain embodiments, modified oligonucleotides or SNA are in aqueous solution with potassium. In certain embodiments, modified oligonucleotides or SNA are in PBS. In certain embodiments, modified oligonucleotides or SNA are in water. In certain such embodiments, the pH of the solution is adjusted with NaOH and/or HC ₁ to achieve a desired pH.

In certain embodiments, pharmaceutical compositions comprise one or more tissue- specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include a tissue-specific antibody.

In certain embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid, gel, cream, or aerosol form, and whether it need to be sterile for such routes of administration as injection.

An oligonucleotide (e.g., synthetic oligonucleotide), SNA, or compositions thereof (e.g., pharmaceutical composition thereof) disclosed herein can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intrathecally, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravascularly, mucosally, intrapericardially, intraumbilically, intraocularly (e.g., intravitreally), via intracisterna magna injection, via eyedrops, orally, topically (e.g., cutaneously), locally, via inhalation (e.g., aerosol inhalation), via injection, via infusion, via continuous infusion, via localized perfusion bathing target cells directly, via a catheter, or via a lavage. In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), SNA, or compositions thereof (e.g., pharmaceutical composition thereof) disclosed herein can be administered intradermally.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), SNA, or compositions thereof (e.g., pharmaceutical composition thereof) disclosed herein can be administered in creams, in gels, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art.

In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is administered into the central nervous system, such as by intrathecal injection or intracisterna magna injection. In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed herein is administered to the eye, such as by intravitreal injection. In some embodiments, an oligonucleotide (e.g., synthetic oligonucleotide), an SNA, or a composition thereof (e.g., pharmaceutical composition thereof) disclosed here may be administered both into the central nervous system (e.g., by intrathecal administration or intracisterna magna administration) and to the eye (e.g., by intravitreal injection). In embodiments in which an oligonucleotide, SNA, or composition thereof is administered both into the central nervous system and to the eye, such administrations may be substantially at the same time (e.g., within the same hour or on the same day, etc.), or may be temporally separated (e.g., a day or more apart, a week or more apart, etc.).

In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), intracisterna magna (ICM), intravitreal, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

In any case, the composition may comprise various antioxidants to retard oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The agent, which may be an oligonucleotide (e.g., synthetic oligonucleotide), SNA, or compositions thereof (e.g., pharmaceutical composition thereof) as disclosed, may be formulated into a composition in a free base, neutral or salt form.

An oligonucleotide (e.g., synthetic oligonucleotide), SNA, or compositions thereof (e.g., pharmaceutical composition thereof) disclosed herein may be administered directly to a tissue. Direct tissue administration may be achieved by direct injection, topical application, or local application. The compounds may be administered once, or alternatively they may be administered in a plurality of administrations. If administered multiple times, the compounds may be administered via different routes. For example, the first (or the first few) administrations may be made directly into the affected tissue while later administrations may be systemic.

The formulations are administered in pharmaceutically acceptable compositions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, excipients, and optionally other therapeutic ingredients. A composition disclosed herein (e.g., synthetic oligonucleotide,

SNA, etc.) may be administered in a pharmaceutical composition. In general, a pharmaceutical composition comprises the composition and a pharmaceutically-acceptable carrier. As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Pharmaceutically acceptable carriers include, without limitation, diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the disclosure. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

A composition disclosed herein (e.g., oligonucleotide, synthetic oligonucleotide,

SNA, etc.) may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections, and usual ways for oral, parenteral or surgical administration. The disclosure also embraces pharmaceutical compositions which are formulated for local administration, such as by implants.

A composition disclosed herein (e.g., synthetic oligonucleotide, SNA, etc.), when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

In some embodiments, a delivery vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compound, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.

Equivalents

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the Detailed Description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one,

B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention disclosed herein. Such equivalents are intended to be encompassed by the following claims.

All references, including patent documents, disclosed herein are incorporated by reference in their entirety.

EXAMPLES

The examples below illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high- affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated. Where a particular lipid core is provided, alternative cores that yield similar SNA structure are considered suitable.

Example 1. SNAs for modulation of CLN3 splicing

Spherical nucleic acids (SNAs) bearing oligonucleotides complementary to select regions of the pre-mRNA expressed from human CLN3 gene (NCBI RefSeq NG_008654.2, SEQ ID NO: 1) were tested. The synthetic oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. The oligonucleotides listed in Table 1 were synthesized on an automated DNA synthesizer using commercially available reagents and the appropriate phosphor amidite chemistry. The oligonucleotides were deprotected and isolated to desired purity and used to prepare SNA compounds. The oligonucleotides provided in Table 1 were uniformly 2’-MOE modified, with each internucleoside linkage being a phosphorothioate linkage. Each cytosine base was a 5-methyl cytosine, and select guanosine residues were replaced with 7-Deaza deoxyguanosine. The oligonucleotides contained the lipophilic linker cholesterol- triethyleneglycol (CholTEG) at the 3’ end. The linker was attached to the nucleobase portion of the synthetic oligonucleotide via a spacer consisting of two hexaethylene glycols (HEG, iSp 18) linked via phosphodiester linkages.

Liposomes synthesized from DOPC and having a mean diameter of about 20 nanometers were used as cores to prepare SNAs. SNAs were prepared by mixing one population of synthetic oligonucleotides with the DOPC liposomes at lipid to oligonucleotide molar ratio of about 50 to 1. Oligonucleotides were anchored via their 3’ ends to the exterior surface of the liposome core by the lipophilic linker to produce the SNA structure. The synthetic oligonucleotides of each SNA target the pre-mRNA expressed from human CLN3 gene. The start sites listed in Table 1 associated with each oligonucleotide are the 5’ positions of the portion of SEQ ID NO: 1 to which each oligonucleotide is complementary.

To test the ability of the prepared SNAs to modulate splicing of human CLN3 pre- mRNA, a CLN3 ^A78/A78 patient fibroblast cell line (BAT_055, National Institutes of Health) was used. This cell line is homozygous for the most common 1.02 kb deletion in exons 7 and 8 of human CLN3 mRNA. Cells were seeded at 10,000 cells per well in 96 well plates. Approximately 24 hours after plating, SNAs diluted in sterile PBS were added directly to wells to achieve a final treatment concentration of 2000 nM of oligonucleotide. Forty-eight hours after treatment, cells were lysed and RNA was extracted using the Macherey-Nagel NucleoSpin 96-well plate kit. cDNA was synthesized from each RNA sample using equal amounts of RNA, and was then combined with Green GoTaq® buffer and primers spanning CLN3 exons 4 to 9 (for SNAs targeting splicing out of Exons 5 and/or 6) or spanning exons 4 to 10 (for SNAs targeting splicing out of Exon 9). PCR products were resolved on TapeStation dlOOO tapes (Agilent), and the optical density of the spliced product (A “targeted-exon” + D78) was divided by the optical density of the unmodified product (D78) for each sample, to generate an edit ratio. For example, for a sample from cells treated with an exon 5-targeting SNA, the fold-change was calculated between PCR products lacking exons 5, 7 and 8 (“D578”) and PCR products lacking only exons 7 and 8 (“D78”). These resulting fold-change splicing modulation values are shown in Table 1.

Example 2. Efficacy of CLN3-targeting SNAs in mouse model of Batten disease

To test the in vivo efficacy of CLN3 -targeting ASOs formulated into SNAs as described above, a mouse model of Batten disease was used. The mice express a CLN3 locus in which exons 7 and 8 have been deleted (CLN3Aexon7/8), resulting in non-functional CLN3. CLN3Aexon7/8 mice were treated with CLN3 -targeting SNAs or vehicle via intravitreal injection. CLN3 mRNA levels were measured in the retinae of the SNA-treated CLN3Aexon7/8 mice and compared with vehicle treated CLN3Aexon7/8 mice, untreated wild-type mice (i.e., mice with wild-type CLN3 alleles, not the CLN3Aexon7/8 allele), and untreated heterozygous mutant mice (i.e., mice with one copy of the CLN3Aexon7/8 allele). Oligonucleotide sequence moeA*moeT*moeG*moeA*moeG*moeA*moeA*moeA*moeA*moeG*moeG*moeC* moeA *moeA*moeC*moeC*moeA*moeG*moeG*moeA/iSpl8//iSpl8//3CholTEG/ (SEQ ID NO: 355) was used to form the SNA where moeA = 2’-methoxyethoxy adenosine; moeC = 2’- methoxyethoxy cytidine; moeG = 2’-methoxyethoxy guanosine; moeT = 2’-methoxyethosy- 5-methyl uridine; 3CholTEG = cholesterol-triethyleneglycol (3’ end); iSp 18 = hexaethyleneglycol; * = phosphorothioate intemucleoside linkage.

The results show that treatment with CLN3 -targeting SNA restore CLN3 mRNA levels to near wild-type and heterozygote levels in retinae from CLN3Aexon7/8 mice.

Example 3. CLN3-targeted SNAs modulate CLN3 splicing in vivo after injection in mouse eyes

Spherical nucleic acids (SNAs) bearing oligonucleotides complementary to the pre- mRNA expressed from the murine CLN3 gene (SEQ ID NO: 773) were tested. The synthetic oligonucleotide used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. The modified oligonucleotide listed in Table 2 were synthesized on an automated DNA synthesizer using commercially available reagents and the appropriate phosphoramidite chemistry. The oligonucleotide was deprotected and isolated to desired purity and used to prepare the SNA compound. Each cytosine base was a 5-methyl cytosine. The oligonucleotide contained the lipophilic linker cholesterol-triethyleneglycol (CholTEG) at the 3’ end. The linker was attached to the nucleobase portion of the synthetic oligonucleotide via a spacer consisting of two hexaethylene glycols (HEG, iSp 18) linked via phosphodiester linkages. Liposomes synthesized from DOPC and having a mean diameter of about 20 nanometers were used as cores to prepare the SNA. The SNA was prepared by mixing one population of synthetic oligonucleotides with the DOPC liposomes at lipid to oligonucleotide molar ratio of about 50 to 1. Oligonucleotides were anchored via their 3’ ends to the exterior surface of the liposome core by the lipophilic linker to produce the SNA structure. The start site listed in Table 2 is the 5’-position of the portion of SEQ ID NO: 773 to which each oligonucleotide is complementary.

The effect of SNA treatment on CLN3 splicing in the eye was investigated in homozygous CLN3Aexon7/8 transgenic mice, which carry the same mutation found in a majority of Batten Disease patients. Vehicle only (phosphate-buffered saline [PBS]) or CLN3-targeted SNA (4.7 pg in 1 pL) was administered bilaterally by intravitreal injection (IVI) in eight mice per treatment group, 4 male and 4 female. Mice were euthanized at 15 +/- 1 days post-treatment and the retina and retinal pigmented epithelium (RPE; the RPE dissection also contained choroid) was dissected from the right eye of each animal. Total RNA was isolated from each retina or RPE sample, then CLN3 splice switching was assessed by endpoint PCR and stable CLN3 transcript levels were assessed by qRT-PCR.

The results, as shown in Table 2, demonstrate that a single IVI dose of SNA produced CLN3 splice modulation and increased the level of stable CLN3 mRNA in both the retina and RPE of CLN3Aex7/8 transgenic mice.

Table 2. CLN3-targeted SNA activity in mouse eyes Abbreviations used in Table 2: 5-Me-moeC = 2’-methoxyethoxy-5-methyl cytidine; moeA = 2’-methoxyethoxy adenosine; moeG = 2’-methoxyethoxy guanosine; moeT = 2’- methoxyethoxy-5-methyl uridine; moe = 2’-methoxyethoxy; 3CholTEG = cholesterol- triethyleneglycol (3’ end); iSp 18 = hexaethyleneglycol; * = phosphorothioate internucleoside linkage; ASO = antisense oligonucleotide.

ADDITIONAL EMBODIMENTS

Embodiment 1. A synthetic oligonucleotide comprising a nucleic acid sequence of 12 to 50 linked nucleosides, wherein the nucleic acid sequence is at least 70% complementary to an equal length portion of a CLN3 RNA, and wherein the synthetic oligonucleotide comprises at least one modification selected from a modified sugar, a sugar surrogate, a modified internucleoside linkage, a spacer and a linker.

Embodiment 2. A synthetic oligonucleotide comprising a nucleic acid sequence of 12 to 50 linked nucleosides having a nucleobase sequence comprising at least 12, 13, 14, 15, or 16 consecutive nucleobases of any one of SEQ ID NO: 11-772.

Embodiment 3. A synthetic oligonucleotide comprising a nucleic acid sequence of 12 to 50 linked nucleosides having a nucleobase sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases of an equal length portion of nucleobases 9,462-9,947 of SEQ ID NO: 1 or an equal length portion of nucleobases 13,185-13,397 of SEQ ID NO: 1.

Embodiment 4. The synthetic oligonucleotide of any one of Embodiments 1-3, wherein the synthetic oligonucleotide comprises at least one modified nucleoside.

Embodiment 5. The synthetic oligonucleotide of any one of Embodiments 1-4, wherein the synthetic oligonucleotide comprises at least one modified sugar moiety.

Embodiment 6. The synthetic oligonucleotide of Embodiment 5, wherein the modified sugar moiety is a 2’ -modified sugar moiety.

Embodiment 7. The synthetic oligonucleotide of Embodiment 6, wherein the 2’-modified sugar moiety is a 2’-O-methoxyethyl (2’-MOE) modified sugar moiety.

Embodiment 8. The synthetic oligonucleotide of any one of Embodiments 1-7, wherein the synthetic oligonucleotide comprises at least one modified nucleoside comprising a bicyclic sugar moiety having a 2’-4’ bridge, wherein the 2’-4’ bridge is selected from -O- CH2- and -O-CH(CH3)-. Embodiment 9. The synthetic oligonucleotide of any one of Embodiments 1-8, wherein the synthetic oligonucleotide comprises at least one modified nucleoside comprising a non-bicyclic modified sugar moiety.

Embodiment 10. The synthetic oligonucleotide of any one of embodiments 1-9, wherein the synthetic oligonucleotide comprises at least one modified nucleoside comprising a sugar surrogate.

Embodiment 11. The synthetic oligonucleotide of embodiment 10, wherein the sugar surrogate is a morpholino or a peptide nucleic acid (PNA).

Embodiment 12. A synthetic oligonucleotide comprising an antisense oligonucleotide listed in Table 1, or a pharmaceutically acceptable salt thereof.

Embodiment 13. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof.

Embodiment 14. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 17, or a pharmaceutically acceptable salt thereof.

Embodiment 15. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt thereof.

Embodiment 16. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt thereof.

Embodiment 17. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 31, or a pharmaceutically acceptable salt thereof.

Embodiment 18. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 34, or a pharmaceutically acceptable salt thereof.

Embodiment 19. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 35, or a pharmaceutically acceptable salt thereof.

Embodiment 20. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 41, or a pharmaceutically acceptable salt thereof.

Embodiment 21. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 44, or a pharmaceutically acceptable salt thereof.

Embodiment 22. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt thereof.

Embodiment 23. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt thereof.

Embodiment 24. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt thereof. Embodiment 25. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt thereof.

Embodiment 26. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 55, or a pharmaceutically acceptable salt thereof.

Embodiment 27. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 56, or a pharmaceutically acceptable salt thereof.

Embodiment 28. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 62, or a pharmaceutically acceptable salt thereof.

Embodiment 29. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 66, or a pharmaceutically acceptable salt thereof.

Embodiment 30. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 67, or a pharmaceutically acceptable salt thereof.

Embodiment 31. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 70, or a pharmaceutically acceptable salt thereof.

Embodiment 32. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 71, or a pharmaceutically acceptable salt thereof.

Embodiment 33. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 72, or a pharmaceutically acceptable salt thereof.

Embodiment 34. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 75, or a pharmaceutically acceptable salt thereof.

Embodiment 35. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 76, or a pharmaceutically acceptable salt thereof.

Embodiment 36. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 77, or a pharmaceutically acceptable salt thereof.

Embodiment 37. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.

Embodiment 38. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 80, or a pharmaceutically acceptable salt thereof.

Embodiment 39. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 81, or a pharmaceutically acceptable salt thereof.

Embodiment 40. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 82, or a pharmaceutically acceptable salt thereof.

Embodiment 41. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 83, or a pharmaceutically acceptable salt thereof. Embodiment 42. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 84, or a pharmaceutically acceptable salt thereof.

Embodiment 43. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 85, or a pharmaceutically acceptable salt thereof.

Embodiment 44. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 86, or a pharmaceutically acceptable salt thereof.

Embodiment 45. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 87, or a pharmaceutically acceptable salt thereof.

Embodiment 46. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 88, or a pharmaceutically acceptable salt thereof.

Embodiment 47. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 89, or a pharmaceutically acceptable salt thereof.

Embodiment 48. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 90, or a pharmaceutically acceptable salt thereof.

Embodiment 49. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 91, or a pharmaceutically acceptable salt thereof.

Embodiment 50. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 92, or a pharmaceutically acceptable salt thereof.

Embodiment 51. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 94, or a pharmaceutically acceptable salt thereof.

Embodiment 52. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 95, or a pharmaceutically acceptable salt thereof.

Embodiment 53. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 96, or a pharmaceutically acceptable salt thereof.

Embodiment 54. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 97, or a pharmaceutically acceptable salt thereof.

Embodiment 55. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 98, or a pharmaceutically acceptable salt thereof.

Embodiment 56. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 99, or a pharmaceutically acceptable salt thereof.

Embodiment 57. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 103, or a pharmaceutically acceptable salt thereof.

Embodiment 58. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 105, or a pharmaceutically acceptable salt thereof. WO 2022/150369 - 1°° ^' PCT/US2022/011291

Embodiment 59. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 106, or a pharmaceutically acceptable salt thereof.

Embodiment 60. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 108, or a pharmaceutically acceptable salt thereof.

Embodiment 61. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 111, or a pharmaceutically acceptable salt thereof.

Embodiment 62. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 112, or a pharmaceutically acceptable salt thereof.

Embodiment 63. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 114, or a pharmaceutically acceptable salt thereof.

Embodiment 64. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof.

Embodiment 65. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 116, or a pharmaceutically acceptable salt thereof.

Embodiment 66. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 117, or a pharmaceutically acceptable salt thereof.

Embodiment 67. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 118, or a pharmaceutically acceptable salt thereof.

Embodiment 68. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 120, or a pharmaceutically acceptable salt thereof.

Embodiment 69. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 121, or a pharmaceutically acceptable salt thereof.

Embodiment 70. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 122, or a pharmaceutically acceptable salt thereof.

Embodiment 71. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 124, or a pharmaceutically acceptable salt thereof.

Embodiment 72. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 126, or a pharmaceutically acceptable salt thereof.

Embodiment 73. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 127, or a pharmaceutically acceptable salt thereof.

Embodiment 74. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 128, or a pharmaceutically acceptable salt thereof.

Embodiment 75. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 129, or a pharmaceutically acceptable salt thereof. Embodiment 76. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 130, or a pharmaceutically acceptable salt thereof.

Embodiment 77. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 131, or a pharmaceutically acceptable salt thereof.

Embodiment 78. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 132, or a pharmaceutically acceptable salt thereof.

Embodiment 79. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 133, or a pharmaceutically acceptable salt thereof.

Embodiment 80. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 134, or a pharmaceutically acceptable salt thereof.

Embodiment 81. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 135, or a pharmaceutically acceptable salt thereof.

Embodiment 82. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 136, or a pharmaceutically acceptable salt thereof.

Embodiment 83. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 137, or a pharmaceutically acceptable salt thereof.

Embodiment 84. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 138, or a pharmaceutically acceptable salt thereof.

Embodiment 85. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 139, or a pharmaceutically acceptable salt thereof.

Embodiment 86. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 140, or a pharmaceutically acceptable salt thereof.

Embodiment 87. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 141, or a pharmaceutically acceptable salt thereof.

Embodiment 88. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 143, or a pharmaceutically acceptable salt thereof.

Embodiment 89. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 144, or a pharmaceutically acceptable salt thereof.

Embodiment 90. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 145, or a pharmaceutically acceptable salt thereof.

Embodiment 91. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 146, or a pharmaceutically acceptable salt thereof.

Embodiment 92. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 147, or a pharmaceutically acceptable salt thereof. WO 2022/150369 - 1° ^{2 '} PCT/US2022/011291

Embodiment 93. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 149, or a pharmaceutically acceptable salt thereof.

Embodiment 94. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 150, or a pharmaceutically acceptable salt thereof.

Embodiment 95. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 151, or a pharmaceutically acceptable salt thereof.

Embodiment 96. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 153, or a pharmaceutically acceptable salt thereof.

Embodiment 97. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 154, or a pharmaceutically acceptable salt thereof.

Embodiment 98. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 155, or a pharmaceutically acceptable salt thereof.

Embodiment 99. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 156, or a pharmaceutically acceptable salt thereof.

Embodiment 100. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 157, or a pharmaceutically acceptable salt thereof.

Embodiment 101. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 159, or a pharmaceutically acceptable salt thereof.

Embodiment 102. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 160, or a pharmaceutically acceptable salt thereof.

Embodiment 103. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 161, or a pharmaceutically acceptable salt thereof.

Embodiment 104. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 162, or a pharmaceutically acceptable salt thereof.

Embodiment 105. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 163, or a pharmaceutically acceptable salt thereof.

Embodiment 106. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 164, or a pharmaceutically acceptable salt thereof.

Embodiment 107. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 166, or a pharmaceutically acceptable salt thereof.

Embodiment 108. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 167, or a pharmaceutically acceptable salt thereof.

Embodiment 109. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 168, or a pharmaceutically acceptable salt thereof. Embodiment 110. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 169, or a pharmaceutically acceptable salt thereof.

Embodiment 11E A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 170, or a pharmaceutically acceptable salt thereof.

Embodiment 112. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 171, or a pharmaceutically acceptable salt thereof.

Embodiment 113. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 172, or a pharmaceutically acceptable salt thereof.

Embodiment 114. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 173, or a pharmaceutically acceptable salt thereof.

Embodiment 115. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 174, or a pharmaceutically acceptable salt thereof.

Embodiment 116. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 175, or a pharmaceutically acceptable salt thereof.

Embodiment 117. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 176, or a pharmaceutically acceptable salt thereof.

Embodiment 118. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 178, or a pharmaceutically acceptable salt thereof.

Embodiment 119. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 180, or a pharmaceutically acceptable salt thereof.

Embodiment 120. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 181, or a pharmaceutically acceptable salt thereof.

Embodiment 121. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 182, or a pharmaceutically acceptable salt thereof.

Embodiment 122. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 187, or a pharmaceutically acceptable salt thereof.

Embodiment 123. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 188, or a pharmaceutically acceptable salt thereof.

Embodiment 124. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 190, or a pharmaceutically acceptable salt thereof.

Embodiment 125. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 195, or a pharmaceutically acceptable salt thereof.

Embodiment 126. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 198, or a pharmaceutically acceptable salt thereof. Embodiment 127. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 199, or a pharmaceutically acceptable salt thereof.

Embodiment 128. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 224, or a pharmaceutically acceptable salt thereof.

Embodiment 129. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 227, or a pharmaceutically acceptable salt thereof.

Embodiment 130. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 228, or a pharmaceutically acceptable salt thereof.

Embodiment 131. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 229, or a pharmaceutically acceptable salt thereof.

Embodiment 132. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 233, or a pharmaceutically acceptable salt thereof.

Embodiment 133. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 238, or a pharmaceutically acceptable salt thereof.

Embodiment 134. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 239, or a pharmaceutically acceptable salt thereof.

Embodiment 135. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 240, or a pharmaceutically acceptable salt thereof.

Embodiment 136. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 241, or a pharmaceutically acceptable salt thereof.

Embodiment 137. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 242, or a pharmaceutically acceptable salt thereof.

Embodiment 138. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 244, or a pharmaceutically acceptable salt thereof.

Embodiment 139. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 250, or a pharmaceutically acceptable salt thereof.

Embodiment 140. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 270, or a pharmaceutically acceptable salt thereof.

Embodiment 141. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 273, or a pharmaceutically acceptable salt thereof.

Embodiment 142. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 275, or a pharmaceutically acceptable salt thereof.

Embodiment 143. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 276, or a pharmaceutically acceptable salt thereof. WO 2022/150369 - 1° ^{5 '} PCT/US2022/011291

Embodiment 144. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 278, or a pharmaceutically acceptable salt thereof.

Embodiment 145. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 279, or a pharmaceutically acceptable salt thereof.

Embodiment 146. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 282, or a pharmaceutically acceptable salt thereof.

Embodiment 147. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 283, or a pharmaceutically acceptable salt thereof.

Embodiment 148. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 286, or a pharmaceutically acceptable salt thereof.

Embodiment 149. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 287, or a pharmaceutically acceptable salt thereof.

Embodiment 150. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 288, or a pharmaceutically acceptable salt thereof.

Embodiment 151. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 289, or a pharmaceutically acceptable salt thereof.

Embodiment 152. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 291, or a pharmaceutically acceptable salt thereof.

Embodiment 153. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 293, or a pharmaceutically acceptable salt thereof.

Embodiment 154. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 294, or a pharmaceutically acceptable salt thereof.

Embodiment 155. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 295, or a pharmaceutically acceptable salt thereof.

Embodiment 156. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 297, or a pharmaceutically acceptable salt thereof.

Embodiment 157. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 298, or a pharmaceutically acceptable salt thereof.

Embodiment 158. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 299, or a pharmaceutically acceptable salt thereof.

Embodiment 159. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 304, or a pharmaceutically acceptable salt thereof.

Embodiment 160. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 305, or a pharmaceutically acceptable salt thereof. WO 2022/150369 - 1° ⁶ - PCT/US2022/011291

Embodiment 161. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 306, or a pharmaceutically acceptable salt thereof.

Embodiment 162. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 307, or a pharmaceutically acceptable salt thereof.

Embodiment 163. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 308, or a pharmaceutically acceptable salt thereof.

Embodiment 164. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 310, or a pharmaceutically acceptable salt thereof.

Embodiment 165. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 313, or a pharmaceutically acceptable salt thereof.

Embodiment 166. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 316, or a pharmaceutically acceptable salt thereof.

Embodiment 167. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 322, or a pharmaceutically acceptable salt thereof.

Embodiment 168. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 323, or a pharmaceutically acceptable salt thereof.

Embodiment 169. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 335, or a pharmaceutically acceptable salt thereof.

Embodiment 170. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 338, or a pharmaceutically acceptable salt thereof.

Embodiment 171. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 339, or a pharmaceutically acceptable salt thereof.

Embodiment 172. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 344, or a pharmaceutically acceptable salt thereof.

Embodiment 173. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 347, or a pharmaceutically acceptable salt thereof.

Embodiment 174. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 354, or a pharmaceutically acceptable salt thereof.

Embodiment 175. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 335, or a pharmaceutically acceptable salt thereof.

Embodiment 176. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 357, or a pharmaceutically acceptable salt thereof.

Embodiment 177. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 358, or a pharmaceutically acceptable salt thereof. WO 2022/150369 - 1° ^{7 '} PCT/US2022/011291

Embodiment 178. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 360, or a pharmaceutically acceptable salt thereof.

Embodiment 179. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 363, or a pharmaceutically acceptable salt thereof.

Embodiment 180. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 366, or a pharmaceutically acceptable salt thereof.

Embodiment 181. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 367, or a pharmaceutically acceptable salt thereof.

Embodiment 182. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 370, or a pharmaceutically acceptable salt thereof.

Embodiment 183. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 372, or a pharmaceutically acceptable salt thereof.

Embodiment 184. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 383, or a pharmaceutically acceptable salt thereof.

Embodiment 185. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 387, or a pharmaceutically acceptable salt thereof.

Embodiment 186. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 389, or a pharmaceutically acceptable salt thereof.

Embodiment 187. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 390, or a pharmaceutically acceptable salt thereof.

Embodiment 188. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 392, or a pharmaceutically acceptable salt thereof.

Embodiment 189. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 395, or a pharmaceutically acceptable salt thereof.

Embodiment 190. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 396, or a pharmaceutically acceptable salt thereof.

Embodiment 191. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 397, or a pharmaceutically acceptable salt thereof.

Embodiment 192. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 398, or a pharmaceutically acceptable salt thereof.

Embodiment 193. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 405, or a pharmaceutically acceptable salt thereof.

Embodiment 194. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 407, or a pharmaceutically acceptable salt thereof. WO 2022/150369 - 1° ^{8 '} PCT/US2022/011291

Embodiment 195. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 425, or a pharmaceutically acceptable salt thereof.

Embodiment 196. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 426, or a pharmaceutically acceptable salt thereof.

Embodiment 197. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 431, or a pharmaceutically acceptable salt thereof.

Embodiment 198. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 452, or a pharmaceutically acceptable salt thereof.

Embodiment 199. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 453, or a pharmaceutically acceptable salt thereof.

Embodiment 200. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 459, or a pharmaceutically acceptable salt thereof.

Embodiment 201. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 464, or a pharmaceutically acceptable salt thereof.

Embodiment 202. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 465, or a pharmaceutically acceptable salt thereof.

Embodiment 203. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 483, or a pharmaceutically acceptable salt thereof.

Embodiment 204. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 486, or a pharmaceutically acceptable salt thereof.

Embodiment 205. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 513, or a pharmaceutically acceptable salt thereof.

Embodiment 206. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 519 or a pharmaceutically acceptable salt thereof.

Embodiment 207. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 522, or a pharmaceutically acceptable salt thereof.

Embodiment 208. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 549, or a pharmaceutically acceptable salt thereof.

Embodiment 209. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 555, or a pharmaceutically acceptable salt thereof.

Embodiment 210. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 561, or a pharmaceutically acceptable salt thereof.

Embodiment 211. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 579, or a pharmaceutically acceptable salt thereof. Embodiment 212. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 609, or a pharmaceutically acceptable salt thereof.

Embodiment 213. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 627, or a pharmaceutically acceptable salt thereof.

Embodiment 214. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 630, or a pharmaceutically acceptable salt thereof.

Embodiment 215. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 636, or a pharmaceutically acceptable salt thereof.

Embodiment 216. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 660, or a pharmaceutically acceptable salt thereof.

Embodiment 217. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 663, or a pharmaceutically acceptable salt thereof.

Embodiment 218. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 666, or a pharmaceutically acceptable salt thereof.

Embodiment 219. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 675, or a pharmaceutically acceptable salt thereof.

Embodiment 220. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 678, or a pharmaceutically acceptable salt thereof.

Embodiment 221. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 681, or a pharmaceutically acceptable salt thereof.

Embodiment 222. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 683, or a pharmaceutically acceptable salt thereof.

Embodiment 223. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 686, or a pharmaceutically acceptable salt thereof.

Embodiment 224. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 692, or a pharmaceutically acceptable salt thereof.

Embodiment 225. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 696, or a pharmaceutically acceptable salt thereof.

Embodiment 226. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 697, or a pharmaceutically acceptable salt thereof.

Embodiment 227. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 699, or a pharmaceutically acceptable salt thereof.

Embodiment 228. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 738, or a pharmaceutically acceptable salt thereof. Embodiment 229. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 740, or a pharmaceutically acceptable salt thereof.

Embodiment 230. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 741, or a pharmaceutically acceptable salt thereof.

Embodiment 231. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 743, or a pharmaceutically acceptable salt thereof.

Embodiment 232. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 746, or a pharmaceutically acceptable salt thereof.

Embodiment 233. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 747, or a pharmaceutically acceptable salt thereof.

Embodiment 234. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 750, or a pharmaceutically acceptable salt thereof.

Embodiment 235. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 760, or a pharmaceutically acceptable salt thereof.

Embodiment 236. A synthetic oligonucleotide comprising the sequence of SEQ ID NO: 764, or a pharmaceutically acceptable salt thereof.

Embodiment 237. A spherical nucleic acid (SNA) comprising a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises the synthetic oligonucleotide of any one of embodiments 1-236 and 279-280.

Embodiment 238. A pharmaceutical composition comprising a synthetic oligonucleotide of any one of embodiments 1-236 and 279-280 or a spherical nucleic acid of embodiment 237.

Embodiment 239. A method of producing alternatively spliced CLN3 RNA in a cell, the method comprising contacting a cell comprising a CLN3 gene and/or a CLN3 gene product with a synthetic oligonucleotide of any one of embodiments 13-236 and 279-280, a spherical nucleic acid of embodiment 237, or a pharmaceutical composition of embodiment 238, to produce alternatively spliced CLN3 RNA in the cell.

Embodiment 240. A method of producing a CLN3 RNA lacking exon 5 in a cell, the method comprising contacting a cell comprising a CLN3 and/or a CLN3 gene product with a synthetic oligonucleotide comprising the sequence of any one of SEQ ID NO: 11-190, a spherical nucleic acid comprising the synthetic oligonucleotide and a core, or a pharmaceutical composition thereof, to produce CLN3 RNA lacking exon 5 in the cell.

Embodiment 241. The method of embodiment 240, wherein the synthetic oligonucleotide is a synthetic oligonucleotide of any one of embodiments 13-124. Embodiment 242. A method of producing a CLN3 RNA lacking exon 6 in a cell, the method comprising contacting a cell comprising a CLN3 and/or a CLN3 gene product with a synthetic oligonucleotide comprising the sequence of any one of SEQ ID NO: 191-477 and 772, a spherical nucleic acid comprising the synthetic oligonucleotide and a core, or a pharmaceutical composition thereof, to produce CLN3 RNA lacking exon 6 in the cell.

Embodiment 243. The method of embodiment 242, wherein the synthetic oligonucleotide is a synthetic oligonucleotide of any one of embodiments 125-202 and 279- 280.

Embodiment 244. A method of producing a CLN3 RNA lacking exon 9 in a cell, the method comprising contacting a cell comprising a CLN3 and/or a CLN3 gene product with a synthetic oligonucleotide comprising the sequence of any one of SEQ ID NO: 478- 771, a spherical nucleic acid comprising the synthetic oligonucleotide and a core, or a pharmaceutical composition thereof, to produce CLN3 RNA lacking exon 9 in the cell.

Embodiment 245. The method of embodiment 244, wherein the synthetic oligonucleotide is a synthetic oligonucleotide of any one of embodiments 203-236.

Embodiment 246. A method of ameliorating a symptom of CLN3 Batten disease, wherein the method comprises administering to a subject in need thereof an effective amount of a synthetic oligonucleotide disclosed herein, a spherical nucleic acid (SNA) disclosed herein, or a pharmaceutical composition disclosed herein, in order to ameliorate the symptom of CLN3 Batten disease in the subject.

Embodiment 247. The method of embodiment 246, wherein the synthetic oligonucleotide is a synthetic oligonucleotide of any one of embodiments 1-236 and 279-280, the SNA is an SNA of embodiment 237, or the pharmaceutical composition is a pharmaceutical composition of embodiment 238.

Embodiment 248. The method of embodiment 246 or 247, wherein the symptom of CLN3 Batten disease is visual impairment, retinal degeneration, intellectual disability, impaired cognitive function, speech impairment, seizures, muscle rigidity or stiffness, hypokinesia, stooped posture, arrhythmia, and/or impaired motor function.

Embodiment 249. A method of administering a synthetic oligonucleotide, a spherical nucleic acid (SNA), or a pharmaceutical composition to a subject, wherein the synthetic oligonucleotide is a synthetic oligonucleotide disclosed herein, the SNA is an SNA disclosed herein, or the pharmaceutical composition is a pharmaceutical composition disclosed herein, wherein the method comprises administering the synthetic oligonucleotide, the SNA, or the pharmaceutical composition to the subject. Embodiment 250. The method of embodiment 249, wherein the synthetic oligonucleotide comprises the sequence of any one of SEQ ID NO: 11-772.

Embodiment 251. The method of embodiment 249, wherein the synthetic oligonucleotide is a synthetic oligonucleotide of any one of embodiments 1-236 and 279-280.

Embodiment 252. The method of embodiment 249, wherein the SNA is an SNA comprising a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises a synthetic oligonucleotide comprising the sequence of any one of SEQ ID NO: 11-772.

Embodiment 253. The method of embodiment 249, wherein the SNA is an SNA of embodiment 237.

Embodiment 254. The method of embodiment 249, wherein the pharmaceutical composition is a pharmaceutical composition of embodiment 238.

Embodiment 255. The method of any one of embodiments 249-254, wherein the subject is an animal.

Embodiment 256. The method of any one of embodiments 249-255, wherein the subject is a human.

Embodiment 257. The method of embodiment 256, wherein the human is a patient having or suspected of having CLN3 Batten disease.

Embodiment 258. The method of any one of embodiments 249-257, wherein the synthetic oligonucleotide, the SNA, or the pharmaceutical composition is administered to the subject via intrathecal injection, intracistema magna injection, or intravitreal injection.

Embodiment 259. The method of any one of embodiments 249-258, wherein the synthetic oligonucleotide, the SNA, or the pharmaceutical composition is administered to the central nervous system of the subject.

Embodiment 260. The method of embodiment 259, wherein the synthetic oligonucleotide, the SNA, or the pharmaceutical composition is administered to one or more regions of the brain of the subject.

Embodiment 261. The method of embodiment 260, wherein the one or more regions of the brain are the cortex, the cerebellum, the hippocampus, the midbrain, the thalamus, and/or the occipital lobe.

Embodiment 262. The method of any one of embodiments 249-258, wherein the synthetic oligonucleotide, the SNA, or the pharmaceutical composition is administered to one or more cells of the eye of the subject.

Embodiment 263. The method of embodiment 262, wherein the one or more cells of the eye are retinal cells. Embodiment 264. A method of administering a synthetic oligonucleotide, a spherical nucleic acid (SNA), or a pharmaceutical composition to a subject, wherein the synthetic oligonucleotide is a synthetic oligonucleotide disclosed herein, the SNA is an SNA disclosed herein, or the pharmaceutical composition is a pharmaceutical composition disclosed herein, and wherein the synthetic oligonucleotide, SNA, or pharmaceutical composition is administered via intrathecal or intravitreal injection.

Embodiment 265. Use of a spherical nucleic acid (SNA) in a method of treating a disease or disorder, the SNA comprising a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises a synthetic oligonucleotide disclosed herein.

Embodiment 266. Use of a spherical nucleic acid (SNA) in a method of treating a disease or disorder, the SNA comprising a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises the synthetic oligonucleotide of any one of embodiments 1- 236 and 279-280.

Embodiment 267. A synthetic oligonucleotide for use as a medicament, wherein the synthetic oligonucleotide is a synthetic oligonucleotide disclosed herein.

Embodiment 268. A synthetic oligonucleotide for use as a medicament, wherein the synthetic oligonucleotide is a synthetic oligonucleotide of any one of embodiments 1-236 and 279-280.

Embodiment 269. A spherical nucleic acid (SNA) for use as a medicament, wherein the SNA comprises a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises a synthetic oligonucleotide disclosed herein.

Embodiment 270. A spherical nucleic acid (SNA) for use as a medicament, wherein the SNA comprises a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises a synthetic oligonucleotide of any one of embodiments 1-236 and 279-280.

Embodiment 271. A synthetic oligonucleotide for use in treating a disease or disorder, wherein the synthetic oligonucleotide is a synthetic oligonucleotide disclosed herein.

Embodiment 272. A synthetic oligonucleotide for use in treating a disease or disorder, wherein the synthetic oligonucleotide is a synthetic oligonucleotide of any one of embodiments 1-236 and 279-280.

Embodiment 273. The synthetic oligonucleotide of embodiment 271 or 272 for use in treating a neurodegenerative disease or disorder.

Embodiment 274. The synthetic oligonucleotide of embodiment 273, wherein the neurodegenerative disease or disorder is CLN3 Batten disease. Embodiment 275. A spherical nucleic acid (SNA) for use in treating a disease or disorder, wherein the SNA comprises a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises a synthetic oligonucleotide disclosed herein.

Embodiment 276. A spherical nucleic acid (SNA) for use in treating a disease or disorder, wherein the SNA comprises a core and an oligonucleotide shell, wherein the oligonucleotide shell comprises a synthetic oligonucleotide of any one of embodiments 1-236 and 279-280.

Embodiment 277. The SNA of embodiment 275 or 276 for use in treating a neurodegenerative disease or disorder.

Embodiment 278. The SNA of embodiment 277, wherein the neurodegenerative disease or disorder is CLN3 Batten disease.

Embodiment 279. A synthetic oligonucleotide comprising the structure moeA*moeT*moeG*moeA*moeG*moeA*moeA*moeA*moeA*moeG*moeG*moeC* moeA *moeA*moeC*moeC*moeA*moeG*moeG*moeA/iSpl8//iSpl8//3CholTEG/ (Seq ID No: 355), or a pharmaceutically acceptable salt thereof.

Embodiment 280. A synthetic oligonucleotide comprising the structure /5-Me- moeC/*moeT* moeG*moeA*moeG*moeA*moeG*moeA*moeA* moeG*moeG*/5-Me- moeC/*moeA*moeA* moeC*/5-Me-moeC/* moeA*moeG*moeA*moeA/iSp 18//iSp 18/ /3CholTEG/ (Seq ID No: 772), or a pharmaceutically acceptable salt thereof.

SEQUENCES

Although the sequences disclosed herein are shown as either “RNA” or “DNA” (and indicated as such in the accompanying sequence listing, as required), in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will understand that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2’ -OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2’ -OH in place of one 2’-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of an uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in this section and in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligonucleotide having the nucleobase sequence “ATCGATCG” encompasses any oligonucleotide having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” (e.g., wherein each T nucleotide is optionally substituted with a U, and vice versa) and those having other modified nucleobases, such as “AT-moeC-GAUCG,” wherein moeC indicates a cytosine base comprising a sugar moiety with 2’-MOE modification (e.g., wherein each individual nucleotide is optionally chemically modified).

Certain compounds disclosed herein (e.g., synthetic oligonucleotides) have one or more asymmetric center and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (Rp) or (Sp), as alpha or beta such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms, unless specified otherwise. Likewise, tautomeric forms of the compounds herein are also included unless otherwise indicated.

The compounds disclosed herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the Ή hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to: ¾ or ³ H in place of 'H, ¹³ C or ¹⁴ C in place of ¹² C, ¹⁵ N in place of ¹⁴ N, ¹⁷ 0 or ¹⁸ 0 in place of ¹⁶ 0, and ³³ S, ³⁴ S, ³⁵ S, or ³⁶ S in place of ³² S. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.

Abbreviations: Unless otherwise noted, 5-Me-moeC = 2’-methoxyethoxy-5-methyl cytidine; moeA = 2’-methoxyethoxy adenosine; moeG = 2’-methoxyethoxy guanosine; moeT = 2’-methoxyethosy-5-methyl uridine; 7deazaG = 7-deaza deoxyguanosine; moe = 2’- methoxyethoxy; A = deoxyadenosine; C = deoxycytidine; G = deoxyguanosine; T = deoxythymidine; 3CholTEG = cholesterol-triethyleneglycol (3’ end); iSp 18 = hexaethyleneglycol; * = phosphorothioate intemucleoside linkage; NR = not reported; ASO = antisense oligonucleotide.

Table 1. CLN3 splicing modulation by SNAs ^† Sequence start site indicates the position of the 5’-most nucleotide within SEQ ID NO: 1 to which each oligonucleotide is complementary.

Genomic locus: Homo sapiens CLN3 lysosomal/endosomal transmembrane protein (CLN3)

(RefSeqGene (LRG_689) on chromosome 16; NCBI Reference Sequence: NG_008654.2; SEQ ID NO: 1)

(32641 nucleotides in length)

TCCTCACGGTCCGGGAGGCCCACAGCCACCGCTTGCAGCCTCCAGCCTCTTCTGGAT GTTCTGTCAGCCTCCGCC

TCCTCATCCTCAGTTTCTCTGGCATAGGCCTCTCCCAGTGACGGGCAAGGCCCTGCG TCTGCCCCTGTGCTTCCG

TCCAGCTCCTGGTTCTCTGAGACAGATGCCTCTCCCTCCTCAGTTCCACATCCCGCG TCCTGGGTTGTCAGCCCC

TCCCCGCCTGCCTCTGGGACTTCTGATAGTTCAGACTCTCGGTCTCCTTCAGCCTCA GCCGCCAGGGTCTGGCCT

CCCGCAGTCTCCTCCTCCTCCCGCTGCTCGCCATCGGCCGCATCCTCCTGAGTATTT CCTCTCTCCATGTCCTTT

CTCGTGCGGTTTTCCCAGGCTCCTGGGAAGGCCTCGGAGGCCTCAGGCTTTACAGAC CCCGCGTGCCTGGGACCT

GCCTCCAGGCTCCTCTCCTGCTCCTCTTTGCTCAGGGCCAGAACTCCCATGAGCTCC ACCTCCCCTGCCTCCCTT

TCCTCGGGTTGCTTAGTTGGGGTCTGGGCGCCCCCCATCAGCTCAGGTCCTTGGCCT TTGGTGACGCTGTGTGTG

AGGCCACCCCATTCCACGCCCAGTAGGCCACAGCTAGTCTGGGCCGCCTCACTCTCC TCCCCTGTGAGCTCCTCC

TCAGGCCTGGCCTCTGGAGTTGCCTCCAAGTCGGCAGTGCCTTCAGCCTCTCCATCT GAGGGCAGCTCAGCCACT

GGATCCCTGCTGCTCCCTCTCTCCTCCTGGGCCTCCTCTGCCTGTACCAGCTCTTCC ATCCTGGCCCGGTCGGCC

CTGATCCCCAAGTCCTGCCTCATCTCAGCCTCCTTACCACGCAGGTCTACCTGGCCC TCAAAGCTCTCCCCTGCC

TCCTGGCCTCCCACAGCTTCCCTCCCTGCGGTCTGGCTCTCAGCAGCCTCTTCTGTT CTTTCAGTGCCCAGGACC

TGGGGCTGTTTGGGGAAAGGGCTCACCTCAGCCTCTCTCTCCTCATCCCCCTCCTCC TCTAGGACCCAGACTTTT

CCAGTAGCCTCTAGGAGCCTTTCTCCTGGGACTGCTCCATATTCTGTCTGTCTGACT CCCAGCAGGTCAGCCTCC

TCTTTGCCTGAGGTTGTCCAGGCCTCGTCCCCTCCTGAGGCTGTCCCGGCCTCCTCC CCTCCTGAGGCTGTCCCG

GCCTCCTCCCCTCCTGAGGCTGTCCCAGCCTCCCCGCCTGAGGCTATCCCGGCCTCC TCCCCTCCCGAGGCTGTC

CCGGCCTCCTCCCCGCCTGAGGCTGTTTCAGCCTCCTCCCCGCCTGAGGCTGTCTCA GCCTCCTCCCCGCCTGAG

ATTGTCCTGGCTTCCTCCCCATCTAAAATTGCCCTGGCCTCTTCCCTACCTGGGGTT GTCCTGGCCTCCTCTCTG

CCTAAGATTCCCCAGTCCTCAGGCTCTGCCCCTGGGCCCCACGTCCCCCATGCCCTA GTGCTTTCACAGGCCTTC

TCCACCACTACCACCTCTTCTCCTTTCCCAGCCCCTTCGGCCCCAGGCTCCTCAGTT TCCCCTGCATCTGCCTCC

CTGACCCCCTGCTCCATCTCCCGGTTGTCCCCTGCCGCCTTTGGCCCAACAGCACCA GCCTTCCCCTCCGTCTCC

CCATGCCAGGTCCACTCTGACTCCGCCCCTCTGGCCATCCCTGGCTCTCTTGCCCTG ACCTCTTCCTCCTCCTCC

TCCTGTTCCCAGCTTCTCAGCCTCTCTTCTCTGTTCACTTCCTGCTCATGGGACTCC TGCCTCTCCTGGGCTTGG

CCGCTCCTGTCTTGGCAAGCCCCAGACCCTGCCTTGGATTTCTTTCTGGCCTCCAAG TGGGCGCTTGGCTCCTGG CACCTGGCAGCCTTGGCTGTCTCCCCAGCCCCACTGTCCTGCCTCTCTGCTTGGGACCCA TGGGAGCTGCCATCT

CCCCAGCCCCAGGTCTGTTCTACAGCTGAGCTCCCCACTTCATGTCTTCTGTCATCT CCAGGCCCTCTCAGCCTT

CCAGCCCCCTCGTTTTGGCTGCCTCTAAGGCCCTCCAGGTCCTCCTGGGCTTCCTCC TCTACAATCTTCCCTGTC

TTTCCCACCGCCACCACCCCCAGTTCCTCAGCCGCCTGCGCCTCCCGCTCTACAGTG GGGACTGCATCTCCCAGG

AGGTAGGAGACAAAGGTGCCGAGGGAATCCTAGGAAAAAAGAGAGAGAGAGGGGAGA GTCATTGAGGAGAGTCCC

AGCCCTTTCCCTGGGGGCCCTGGCTGGAGGAGCCCAGGTTAAGGAAGAGGGTACTGG GATCTGAAAGGAGAAGAA

GAGGATTTGCAGAAGGAGCCAGAAGGGGCATCGATTAGGGGTACGAGGACGCATGGT TGGAGGTGGGGACTGAAA

TTCAGAAAATATTAGGCCAGGAGTCTGCCTGAACCTGAGAAAGCAATCTCCAGTTTG AGGAGATCAGAAGGAGGA

GGGGAAAGGTGAGGCCCCAGGGGAGGTGCCCGGAAGTGAGGCCCCAGGGAATGGGGT GCAAGTATCTGGCAGCTG

TCTGCCCTTCTCACCAGTGCCCCCCTCAAGGCCTGGTGCAGCCCAGGGAGGTATAGC CGGAGGAAGTCCATCCTG

TCTGTCTCTGTCTGTGTGTCCAGAAAGCTGATAATGACCGTCCCCACCCACCAAGAC AAGCCAACCTTCCCCAGG

ACCAAAGGCACTTCCTCTTCCCCTGTCTCCCTGATGCGTCCCCACCCCAGGCCCTGC CCACGGCCCCTGATGCCC

ACAGTAGCCCCCACTGCAGCCCGGGAAATGGGTGTACATCACACAGGGGACTCAGGT GCACATTTGGAAATAAAG

GACAAGCAAGAGGCCAGAAAGGACAAAGTGGGAGCAGGAGACCTTAGAACTGGGGAG GGGGAGAGGGAGGGGACA

TGTGGCAACAGCAGCTCAAGTGACTCAGGTCTCGTATCCGCACCTCAGCCATTTCTG CCTCCCCTACAAGCCACA

GCCACTCTGCAAGAGGAAGCTGGGGTTTGTGGTTCCTGTTTTGCCTGGGAAGAAAGA GTAGCAGTGAAGAGGGGG

CTGGTGGGAGCTCTGACACATTTCGGCGTCATTAATGCTTCCCCCAGGGGCGGAAGG GGCCGGATTGGTGGCTCT

GGGGTGCATCCCCTCACCTATGATTAGGCTTGCAAAAAAGAACTTGGCTAAACTTTA TATTGTGGATATCTGTGT

CTGCCTCCCACCTTCCTGTGACTTCCTTGATGGCAGATGCCATGTATGAAACGCTGC TGAATTTCTAGAGCCCTG

CACGTTAGTAGGATTCCATTTTCCATCAGCTGAGTGACCTGGGAGGCACACTAGAGA GGGCACATTTCTTTTCTT

TTCTTTTCTTTTTTTTTTTGAGACAGTCTCACTCTGTCACCCAGGCTGGAGTGTAGT GGCGCAGTCTCAGCTCAC

TGCAACCTCTGCCTCCTGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCGAGTAGCT GGGATTACAGGTGCGTGC

CACCACACCTGGTTAATTTTTGTATCTTTAGTAGAGACAGGGTTTCACCATGTTAGC CAGGCTGGTTTCGAACTC

CTGACCTCAGGTGATCTGCCCACCTTGGCCTCCCAAAGTGCTGGGATGACAGGCATG AGCCACCACACCCAGACG

AGAGGGCACATTTCCAACTGCATTCTGAAGGCCTGGTGGTGAGAAAGAGCATATAGA GGTGGCACAGCCCATAGC

AGAGATGAGGCCAAGGACAAGAAGAGAATGCAGGATGCCGCTTTCCACCTAGAAGAA GGTAAGGGTATGATTCCT

TATTTGGAACTACATAAAGTGACCTTGCTCTACTTCTCTTCCCCATACCCTCCTTCT CCCCTCCCCAGCCCCACT

TTCCTATTCCAATTAGGGCAATTCTATGAAATCACAGCATCAGAATCTTAGGCCTTA AAATCATTTAGTCTGGCC

AGGTGCAGTGGCTCACGCCTGTAGTCCTAGCACTTTGGGAAGCCAAGGCAGGTGGAT TGCTTGAGCTCAGGAGTT

TGAGGCCACCCTGGGCAACGTGGCAAAATCCAGTCTCTACAAAAAACACAAAAATTA GCTGGGCACAGTGGTGCG

CACCTGCTGTCCCAGCTACTCGGGAGGCTGAGGTGGGAGGATCACCTGAGCCTGGGA GGTCAAGGTTGCAGCAAA

CCAAGATCCTGCCACTGCACTCCAGCCTGGGCAACAGAGCAAAACCCTGTCAAAAAA AAAAAAAAAATCTGGTCT

AAGCACCACCTTTGATGGAGGCAGGAATCCTGCAGCAGCCTGGAGCCAACCCAATGG CTCCCTGCCTCTGGTGCT

CTATTTCTTATCACCATACAGGTCTCTAAGGTCACCTTTAGGTCTAACATTGTATGA ATGATTGTCAGCAACTTT

CCAATTTTTTTCATATACATATATTTTTTTTATTTTATTTTTTACTTTTTCTTTTTT TGAGACAGGATCTCACTC

TGTCACCCAGGCTGGAGTGCAGTGGCGTGATCATAGCTCACTGTAGCTTCAACCTCC TGGGCACAAGTGATCCTC

CCACCTCAGCTTCCAAGGTACCTGTGACTACAGGAATGCGCCACCATGCCCGGCTAA TTTTGTTATATATATATA

TATATATATTTGTACCTTGGTTTTCTCATCTGTAACACCAGGGGTTGAAGTGCTGTG ATATTTTTTGGTTCTAGG

ATAATTTGGAAATAGGGATCTCTTAACATCTTGGATACTGCCAATAGATCATTCACG AAATCCCTTTAGATGAGT

GTTATGTTTACTTAGGTCTCTAAAACAATTTGTAAGGTCAACAGATGGGGATAAGAA TGACCTGAAGCTGGTCGT

CTTTGTTCGCAACCAGAAACAGTCCTGCCTCAAAAAAAAAAAAAAAAAAAAGGGAGT AGGCAGGTGGCCATATTT

GTTTCTGGCAAGTGAGTGCTGAAGGAAAGGAGCTGAAGCCTCCCACAGTCATAACTG GTGCTGGCAGGCTACTGT

CTCGGTCTTGGGCGCCACTGATCTAAGGTCACGGCTCTGCTTGCTGCTCCCACCCGC TCCAGTTTAAAACCTGCG

GTTCCAGGGTTCTCCAGCCCCTCCCTTTTTCACGCTCCGAAGCCGAGAAGGCCCAAA GCGAAGACAGAGAGGACC

CGGAAGTAGGGAAAACCTCTGAGCACGTGATGGGGGAACACGCGGGTGCTGTCACGT GATCCGACAAACGGCCTC

TGCATAGTGCAGAACATTCTGCTGCTCTTAAAGGTACAGGCCTCAGGGTCCCTGCTG TAGACGGGGCGGGGGAGA

GTACGATGGGTGGGGCGTGGTGGGTCGTAGGGCGCTCGAGATGGAGCCCCCAGCTTC CTTGATGGATCGCGGGGC

GCGAGTGCCCTAGACAAGCCGGAGCTGGGACCGGCAATCGGGCGTTGATCCTTGTCA CCTGTCGCAGACCCTCAT

CCCTCCCGTGGGAGCCCCCTTTGGACACTCTATGACCCTGGACCCTCGGGGGACCTG AACTTGATGCGATGGGAG

GCTGTGCAGGCTCGCGGCGGCGCTTTTCGGATTCCGAGGGTGAGTATTCCCGCCCAC CCTCATGGAACGACCACC

TCTGGCTCGCAGCCCACCTGAGGGGAATGAGAGCTGACTCGGCCCCGGGAGGACACG CAAGGAGCGGTCGTGCAC

CTGAGAGTAGGTGGGGGTCATGCCCTCTTCTCGCTCTCCCAGGGGAGGAGACCGTCC CGGAGCCCCGGCTCCCTC

TGTTGGACCATCAGGGCGCGCATTGGAAGAACGCGGTGGGCTTCTGGTGAGTGGCCT GAGACTTCAGCGAGTGAC

AATGGCATGAGAAGAGGGAAGTGACCGGAGGAAAGGGGGAAGAAAGAGTTAAGCTGC GCAAAGGAGCTGAACCCA

CAAATATTTACTGAATCCACTGGTTTGCCCCAGGGGCAGCATGTAAAATTTCCAAAC TTCACTCCTTATCACCTT

CCGGATTTAGCAAGATCCTCATACCTACCACCTGTAGGATTATTTACTTTTTTCTTC CAGCCAATTCAGCCTTAA

CGTTTTTTAGTGCACATATTTTTAAAAAGAAAACTTTAAATCACGACTCCACTTGGA AAACCAGCGTTTTCCTAG

TGAACGTTTAAATAAATGCATCACTACTAAAACTTTTTTATTTTTTTAATTTATTTT ATTTTATCTTATTTTTTT

TTCTTTTTGAGACAGGGTCTTGCTCTGTTTCCCAGGTTGGAATGCAGTAGTGCAATC ACAGCTCCCTGCAGCCTG

GAACTCCTGGGCTCAAGTGATTCTCCCACCTCAGCCTCCAAGTAGGTGTGGCTACAG GTGTGCACCACCGCATCT

GCCTAATTTTTATATTTTTTTATAGAGATGGGGGTCTCACTATGGGACCTGGGCTGG TCTCAAACTCGTGGCCTC

AAGTGATCCTCCTACCTCAACCTTCCAAAGTGCTGAGATTACAAGCATGAGCCACCA TGCCGGGCCTGAACCTTT

_{TTTTTTTTTTTTTTTTTTGAGACGGAGTCTCTCTCTGTCCTCCAGGCTGGAGTGC AGTGGCGCAATCTCGGCTCA}

CTGCAAACTCCGCCTCCCAGGTTCACACCATTCTTTTGCCTCAGCCTCCGGAGTAGC TGAGACTACAGGCGCCTG CCACCACGCCCGGCTATTTTTTTGTATTTTTAGTAGAGATGGGGTTTCACCGCGTTATCC AGGATGGTCTCGATC

TCCTGACCTCGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTG AGCCACCGCGCCCAGCTT

_{TTTTTTTTTTTTTTTTTAATTTTATTTTATTTTTGAGACAGAGTCTCACTCTATC ACCCAGGCTGGAGAGCAGTG}

GCACAATCTCGGCTCACTGAAACCTCCACCTCCAAGGTTCAAGCTATTCTCCTGTCT CAGCTCCCCGAGTAGCTG

GGATTACAGGTGCGTGCCACCAGACACAGCTAATTTTTTGTATTTTTAGTAAAGACA GGATTTCACCATGTTGGT

CAGGCTGGTCTAGAACTCCTGACCTTAGGTGATCCGCTTGCCTCCACTTCCCAAAGT GCTGGGATTACAGGCGTG

AGCCACCGTGCCCCGCCCTGACATAGGGGCTTTGGGATCACAGACTTGGATTCACTT CCAGCCTCCAAGGCCTCT

CCCAGAAACTCTCTGTGACCACTCTCCTTTTTTTTTTTTTTTTTTGAGATAGAGTCT CGCTCTTGTAGCCCAGGC

TGGAGTGCAATGGCACAATCTCGGCTTACTGCAACCTCCGCCTTTCGGGTTCAAGTG GTTCTCCTGCCTCAGCCT

CCTGAGTAGCTAGGATTACAGGCATGTGCAACTTCGCCCAGCTAATTTTGTATTTTT TAGTAGAGACGGGGTTTC

TCCATATTGCTCAGGCTGGTCTCGAACTCCTGACCTCAGGCGATCCGCCCGCCTCGG CCCCTCAAAGTGCTGGAA

TTACAGGTGTGAGCCACTGCACTCGGCCTCTTCCTTTTTATTTTTTATTTTTGTGAC AGGGTCTTGCTCTGTCAC

CCAGGCTAGAGTGCAGTGGCATGATCACAGTTCACTGCAGCCTCTGACTCCTGGACT CAAGCAATCCTCCCACCT

CAGCCTCCCAAGTAGCTGGGACTACAGTGCAAGCCACCACACCTGGCTAATTTTTAA ATTTTTTGTAGAGATGGG

GGTCTCACTATCTTGCCCAAGCACCAAAGCCTGTATTTTTGACCCCAACATTGAATG AGGATGGGATCTGGTGAT

AGAGGGAAAAAAAAGAGGGGGCTGATTGAAGGGCACAGGTAAGGGAAGGTTTGGCAC AAAGGCTACAGATGGCTG

CAGGATGAGGAGGGAGTTGAGACCTGTCCTGCTCCTTCCAGGCTGCTGGGCCTTTGC AACAACTTCTCTTATGTG

GTGATGCTGAGTGCCGCCCACGACATCCTTAGCCACAAGAGGACATCGGGAAACCAG AGCCATGTAAGTGACTCT

CTACCACCACCACCATGGTTAGTCCCTGTGGGAAGATGAGGGGGTGGGACAAGGTGG GGTAAAGTTTCCAGTCTC

TGGCTGGGCACAGTGGCTCACACCTGTAATCCCAGCAGTTTGGGAGGCTGAGGCGGG CGGATCACTTGAAGTCAG

GAGTTCGAGACCAGCCTGGCCAACATGGTGAAACTCCGACTCTACTAAAAATACAAA AACTACCTGGGTGTGGTG

GCACACACCTGTAGTCTGAGCTATTCAGGAGGCTGAGGCAGGAGAATTGCTTGAAGC CAGGAGGTGGAAGTTGCA

ATGAGCCAAGATCACACCACTGCACTCTAGCTTGGGCAACAGAGTGAGACACCATCT CAAAAAAAAGAAAGCTCT

TGAATGTGTTATCTAAACAAAGTCCATTTACAGGGCCATCTGTGGCCTGTGGTCTAG TAGTGTGAGACTCATATG

GAAACCCCCTCTTCATTGTGCATGCAGAGACGCTGAGGTCCTGAGAAGTCAGTCACT GCTGACCCAAAGCCATCC

TTCAAGTGAAGGCAGAGCTGGGACGGGAGCCAGGCTCTGTGTGTCTATCCCTCTGCC TCCAGGGTGAAAGACATG

CTTTTCTCCCATTAGGTGGACCCAGGCCCAACGCCGATCCCCCACAACAGCTCATCA CGATTTGACTGCAACTCT

GTCTCTACGGCTGTGCGTACTCATCTCACCTGGTCCTTGCCTGACCCAGGGCCCCTG GGTCTATAGACCCCACTC

CCAGCCCTTCACTACCCAGCTGGGACTGTCATGATTAAAACAGTTGAGACCTGGGCT GGGCGCACTGGCTCACAC

TTGTAATCCCAGCACTTTGGGAGACCAAGGTGGGAGGATCACTTGAGCCTAGGAGTT TGAGACCAGCCTGGGCAG

TATAGTGAGACCCCCATCTTAAAGAAAAAAATTAAAAAATATATATATATAAAATAA TTGAGACCTTAATATAAT

TTCTGAGGCTGAGGCGGATGGATCACTTGAGACCAGGAGTTCAAGACCAGCCTGGCC AACATGGTGAAACCCCAT

CTGTACTAAAAATACAAAAATTAGCCAGGCATGGTGGTGCACGCCTGTAATCCCAGC TACTCAGGAGGCTGAGGC

AGGAGAATCACTTGAACCCAGGAGGTGGAGGTTGTAGTGAGCCAAGATCGAGCCACT ACACTCCAGCCTGGGTGG

CAGAATGAGACTCACTCTCAAAATATATATATATATAGTTTCTGAGTCCTTTCTGTC TGCACCATATATTAGCTC

ATTTAAGCCACACAGCAGTCCTGTGAGCTAGGTGCTATGATATTCCCATTTTCCAGA TGAGGAAACTGAAGCTCA

GAGAGTATAAACTCTTGACACACAACTAAGGAGTGGGAGAGCTGAGACTTGAACCCA GGCGTGCCTGACTCCAGA

GCCTGTGTTTGTAGCAGGCCTGTTTGGCCAGCTCCTGCCTCTCCTTGGCCACGTGGT TGGGAGGGTTGTCCCCTG

GAAGCTCTGCGGTCTCACTCTATTCTCCTGTCCCAGGCTGTGCTCCTGGCGGACATC CTCCCCACACTCGTCATC

AAATTGTTGGCTCCTCTTGGCCTTCACCTGCTGCCCTACAGGTCTGGGTGAGGGTAG TGGGAGGCAGGGTGGGCA

GGAGCTGAGAAAGGGGAGGCTGGGATGGCTGAGATGCTGAGAGTAGAGACCGACCTT CCCCCTCCCTTCCCTTCT

CACCCCCTCAGCCCCCGGGTTCTCGTCAGTGGGATTTGTGCTGCTGGAAGCTTCGTC CTGGTTGCCTTTTCTCAT

TCTGTGGGGACCAGCCTGTGTGGTGAGTGTGTGGTTCTGTGTCAGATGGGGAGCCCC GAGGAACCACATCAGAGC

ATTTGTGGGAAGAGTCTCCCCAGCCTCCCAGAGGAAAGGGATTCATTCTGTCACCCT TAGAAGCCTGCTAGGGCT

ATCAGCAGTAGGCGATGGGAGACTGGGACAATTTGGAGGGGTAGGCAGTGGAGGAGA TGGGAGAAAATGGATGAA

TTAGATGGAGATTGAGGTGAACAAAGTCAAGACTCTGTGATGGACCAGGCACAGTGA CTCATGCCTATAATCCCA

GCACTTTGGAAAGCCAAGGCAGGCAGATCACCTGAGGTCAGGAGTTCGAAACCAGCC TGGCCAACATGGAGAAAC

CCCGTCTCTACCAAAAATACAAAAATTAGCTGGGTGTGGTGGCAGGAGCCTGTAATC CCAGCTACTCGGGAGGCT

GAGGCAGGAGAATCTCTTGAACCTGGGAGGCAGAGGTTGCAGTGAGCCGAGATCACG CCACTGTACTCCAGCCTG

GGTGACAGGGCGAGACTCCGTCTCCAAAAAGAAAAGAAAAGAAAAAGACTGATGAAG GGGCAGAGACATCAAGGG

TGCATGTCTGCCCCTGGTCTGATAACTGGGTGGATGGAGGTGCCACTCTCCATGAAG GGACACGCAGGGGAGTGG

GGCTCTGCTTCAGACCTGGAACCTGGCCTATGCATGGGATCTATTGGAGCCTCTATG AGCTGATACTGAGGAGGC

CATGGCCAGACACATTAGAGGCCTGGGCAGTGTGGCAAGGTGTGGTGTGACCATCCC AGTGCTTGTCCTCCCCCC

AGGTGTGGTCTTCGCTAGCATCTCATCAGGCCTTGGGGAGGTCACCTTCCTCTCCCT CACTGCCTTCTACCCCAG

GTAAGCAGGTGGAGCAGGGAGTGTGGGGAGAGGCTGTCCCATGGTCAGCCTAGGTCC TCCTGAATGTTCCTGTGT

TCTCCTTCCCAGGGCCGTGATCTCCTGGTGGTCCTCAGGGACTGGGGGAGCTGGGCT GCTGGGGGCCCTGTCCTA

CCTGGGCCTCACCCAGGCCGGCCTCTCCCCTCAGCAGACCCTGCTGTCCATGCTGGG TATCCCTGCCCTGCTGCT

GGCCAGGTGAGCTGCCCTGAGCCGGGAGGGAGAGGGGTCCAAGGAGAGAAAACTTGG CCATGGCTGGGTGTGGTG

GCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGAGGGCAGATCGCCTAAGGT CAGGAAACCAGCCTGGCC

AACATGGTGAAACCCCGTCTCTACTAAAAATACAACAATTAGCCAGGTGTGGTGGCG GGTGCCTGTAGTCCCAAC

TACTCAGGAGGCTAAGGCAGGAGAATCGCTTGAACCCCGGAGGCAGTGGTGGCAGTG AGCCGAGATCGTGCCATT

GCACTCCAGCCCGGGCGACAGAGTTAGACTCTGTCTCAGGAAGAAAAAAAAAAAAAG AAAGAAAAGAAAACTTGA

TTATGATTGCAATCTTCAAGTCCCTACCTTGCTGTGAAGGGAGGCGGAATCTGGACT CTGATAGCCCCAGGTGTG

AGTCCTGGAGCTGCCACTTTTTAGCTTTGTAGCGTTGAACAAGTTACTCCACCTCTC TGAACCCTCAGTTTCCCC ATATCTCAAATGGCAGTTGTTCTTGCTTTCCTTGGAGGTGATGAGGGTAATGCATTCAGC ACAGTGTGGTTCCCA AGGTGATTAGAAGTAGGATGAGGGTGGACTTTATTTGATTAGTTCCTTTTTTTTTTTTTT TTTTTTTTAATCAGT GTTGACCAGGTTGGCCTCGAATGTGTAGCCTTGCCTGCCTGAGTGCCAGGGCAACAGGCC TGAACCATGGCGACT CCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTCATGGTGTCACTCAGGC TGGAGTGCAGTGACT GGTGTGATCTCAGCTCACTGCAGCCTCGGCCTCCCGGGCTCTAGTGATTCTCCTGCCTCA GCCTCCCGAGTAGCT GGGACTACAGGCCCATGCCACCATGCCTGGCTAATTTTTTATATTTTTAGTAGAGACGGG GTTTCACTGTATTGG CCAGGCTGGTCTCAAACTCCTGACCTCAAGTGATCCACCTGCCTTGGCCTCCCATAATGC TAGGAATACAGGCGT GAGCCACCGCGCCCGGCCTGATTAGTTCTGTGTATTTTCATGCATATTACAAAACACTTT GGCCGGGCATGGTGG CTCACATCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGCACCACGAGGTCAGGAG TTTGAGACCAGCCTG GCCAATATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGCTTGGTGGCG CTTGCCTGTAGTCCC AGCTACTTGGGAGGCTGAGGAGAAGAATCGCTTGAACCCAGGAGGCAGAGGTTGCAGTGA GCCGTGATTGTGCCA CTGCACTCCAGGCTGGGCAACAGAGCGAGACTCCGTCTAAAAAAAAAAAAAACACACACT TTGAACTAGCCAGAC ACACCTGGCCCTTCACAAGATTAGTGCTTAGGACAAGTCTAAGTAGAAAAAGTGAGTCCA TTTAAAGAAAAATAT TAAGTAAAAATATATATATACAGGAGGAATATGGATATGGCAAATAACATGAAGCCAGAA CCTAAGTGACTAAAA TCGAGGAAGTTCTGGCCTAGCACAGAGTGAACGTATAATTAATGGGAGCTAGAGCGACCA TTAGAATAATAATGG CCAGAAAACAGAACTGACTCGCTAGGTATGAGCCAGCAGTCCGAACAAGGGCCTGCTGAG CACAGTGGCTCACAG CTGTAATCCCAGTGCTTTAGGAGGCTGAGGCTGGAGGACCACTAGAGGCTAGAAATTTGA GACCAGCCTGGGCAA CATAGCGAGACTCCATTTATACAAAAAATGGAAAATATGAGGCGGGCTTGGTGGCACGTG CCTGTAGTCCCCGCT ACTTGGGAGGCTGAGGCGGGAGGATTGCTTGAGCCTGGGAGGTCGCGGCTGCAGTGAGCT ATGATTGCACCACTG CATTCCAGCCTGGATGCTGGAGAAAGACCCTGTCTCTGAAAAAAATCAAAACAAAATGAA GGCCCATGAATAGGA ATAAGGAGATAAATTCAGGTCCAAAGAAAGAAGCCTTTGTCCACAATTGGAGCTCTCCCG CACAGCGTAGGAGCC TTAGAGGCAGTGAGCTACCCATCTTTGAAAGTGTTCAAAGGTAGAGGTGTTCCCTGGGAA AAGTGGCCTAGATGG TCCCTGGGGACCTTCCCAGCCAGTAGGGTTTTCTGACCCTGCCTTCATCCTACTCCTAGC TATTTCTTGTTGCTC ACATCTCCTGAGGCCCAGGACCCTGGAGGGGAAGAAGAAGCAGAGAGCGCAGCCCGGCAG CCCCTCATAAGAACC GAGGCCCCGGAGTCGAAGCCAGGTAGGAGACACAGACCCTCAGAGAGGTCACTTTCTTTC TCTCTGGGTTTGGCC TTTTCCTCTCTGCAATAGGCAAAGTTAAGAGGGGAAAGAGAGTGAGTGTACTGGTTATGG GAAAAGCCTTTGTCT ATTTGAGAAATACTTGTTGAGGACGAGCACAGTGGCTCACGCCTGTAATATCCCAGCACT TTGGGAGGCTGAGGC AGGAGGACCACTTGAGCTCAGAAGTCTGAGTCCAGCCTGGGCAACAGAGCAAGACCTTGT CGCAAAAAAAAAAAA AAAAAAAAAAAAAA G AAAA G AAA G G AA G G AA GGGAGGGAGA G AA G AA GGGATATT AA TTGAGTACTTACCATGTG CCAGGCATTCCAATCACAAGTGCTGAGGCCCTGCGGTGGGGATAAGCTGGGATGTTCTAG AACCAGAGAATGGCC AGTGAGACTGGGCAGGGTGGGCCAATGGCCATCTGTGGAGCTGTACAAGAGACATTGGCC GGGCGTGGTGGTTCA TACCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGATGGATCATTTGAGATCAAGAGTT TGAGACCAGTCTGGC CAACATGGTAAAACCCCGTCTCTACTAAAAATAAAAAAATTAGCCAGGTGTGGTGGTGCA TGCCTGTAGTCCCAG CTACTTGGGAAGCTGAGGCACGAGAATCACTTGAACCCAGGAGGCAGAGGCTGCAGTGAG CTGAGATTGCACCAC TGCACTGCAGCCTGGGCAACTGAGTGAGACTCTGTCTCAAAAAATAAAAAAAATTAAAAA TCAAAGAGACGTGAA GAGGTCTCACCTGGTTAGCTTTTATTTTCATCATGATAAAGTATGTATATTACTTGAAGT TTACCATTTTATTAT TTTTTATCTTACTTTATTTTTTTGAGGTGGAGTTTCGCTCTTGTTGCCCAGGCTGGAGTG CAATGGCGCAATCTC AGCTCACTACAACCTCTGCCTCCTGGGTTCAAGTGATTCTCCTGCCTCAGCCACCCGAGT AGCTGGGACTACAGA CACCTGCCACCACACATGGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCTCCATGT TGGCCAGGGTGGTCT TGAACTCCTGACCTCAGGTGATCCGCCCACCTCAGTCTCCCAAGGTGCTGGGATTACAAG CATGAGCCACTGCGC CCAGCCATTTTAACCATTTTTAAGTGTCCAATCCAGTGGCATGGAAGTGAGTTCCCACTG TTGCGTGGTCTGGTT AATTCTGTCATACCGGTGCCTCTTTGCCGAGTCTTCAGTGTGAAAACTTCATCTGCCCTT CTCTCTCTGTTCCCC TGCAGGCTCCAGCTCCAGCCTCTCCCTTCGGGAAAGGTGGACAGTGTTCAAGGTTCGGAT GATGGCTGGGGTATG TCCCTGTGGGGCTTGCTCACCTCCAGGCCCCCCGCTTATATCTTCTGCCTTTTCCAGGGT CTGCTGTGGTACATT GTTCCCTTGGTCGTAGTTTACTTTGCCGAGTATTTCATTAACCAGGGACTTGTAAGTGAG GGGTGCTAGGAGGGG TGTGGAGGTGGCGATTGGGGCTGGGACCCACACAGCCCCGTCCATCTCCCCTGTCTGGTA TTTGTTGCAGTTTGA ACTCCTCTTTTTCTGGAACACTTCCCTGAGTCACGCTCAGCAATACCGCTGGTAAGAGGA GCGAGGGCAGTGGGC TGGGAGGGCGCCGTGGTGATGCAGCTGCCCTGCCCAGTAGGCACCGGGGGGAGCGGGATG GTCCCTGGAGGAGCC TCCTCTCCTCCCCCCAACCCTAACCTCAGGTACCAGATGCTGTACCAGGCTGGCGTCTTT GCCTCCCGCTCTTCT CTCCGCTGCTGTCGCATCCGTTTCACCTGGGCCCTGGCCCTGCTGCAGGTACCAAACCCC TGCCCCTCACTTCAC TCCCACCTTGGCTCCCAGCTTGGCTCCCAGCTTCCCCAAACCCCCTGCTTCCCACTATCA GTGGGAAGTGTAAAT _{TTTTTTTTTTTTTTTTGAGACAGAGTCTCGCTCTGTCGTCCAGGCTGGAGTGCAG TAGCGCAATCTTGGCTTACT} GCAACCTCTGCCTCCCGGGTTCAAGTGACTCTCCTGCCTCAGTCTCCTGAGTAGCTGGGA TTACAGGCGCCCGCC ACCATACCTGGCTAATTTTTGTATTTTTAGTAGAGATGGGATTTTGCCATATTGGCTGGT CTTGAACTCTTGACC TCAGGTGATCTGCCGGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGGAAGTGTAAATTG TACACTATCATTTCT TAGCACAGGGAACCAAAGTCCAGCAGAGCTCCATGTAAAATGTATAGCCTCAGGAGCCAA GCAAAACTGAGCTTC AGTACTCAGCCACTGTGTGACTTAGGGCAGGTCTCAGAACCTCTCTGAGCCTCAATTTCC TCATGTATAAATTGG GTGTGGCCAATACCTATTTTTCAGGGTAGTTGTAAGAAATAGAGATGAGAGAAAGAATCG AAGAACAAGATTTTG TAGTGTGTGTGTGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT TTTAAGCATTGGGAT TGGGCCGGGCCAGGTGGCTCATGCCTGTAATCCCAGCACTATGGGAGGCTGAGGCAGGTG GATCATTTGAGCTTG GTAGTTCAAGTCCAGCCTGGGCAACATGGCACAATCACATCTCTACAAAAATTAGCCAGG TGCAGTGGCGCACAC CTGTAGTCTTAGCTACTGGGAAGCTGAGGTGGGAGGATCACTTGAACCCATGAGGTCAAG GCTGCAGTAACCAAG GTCATGCCACTGCACTCCAGCCTAGGCAATAGAGCGAGACCCTGTCTCCCAGAAAAAGAA AAAAGAAAGGAAAAG TCGGAACCTTCCTCTGTCGCCTAGGCTGGAGTACAGTGGCATGATCATAGGTCATTTACC TCAGGCAAAGAGACA GGCTTTCAAATAATGTTTCTAACTATAAGAAAATGTACAGTTCTTTTATTCTAGATAGTT AATTATAATAGAATT

GGTGACTTTGAAGTCAGTGGGTTATTTATCTTGAGGGTGGTGAGCAAATTCAACTGC CCTTGGCAAACAGTAGTT

TACAGCTTTAAGGGCGCAAACACCCTTCCTTACAACAGTCTAATAAAAGGAGTTTCA TATAACCAGCATTCATTG

TTTCAAGGTTACAAATCCACAGCCCCTGCAGGTAACTGTTACAATAAACTGTCAACT GTGACAGGAAAGTCTTCT

GCCTTTGAAATTTAGGAACTTGGCCAGTCCTGGTGGCTCACACCTGTAATCCCAGCA CTTTGGGAGGCCAAGGCA

GGTGAATCACCTGAGGTCAGGATCAGGAGTTTGAGACCAGTCTGGCCAACACAGTGA AACCCCGTCTCTATTAAA

AATGCAAAAATTAGTTGGGCGTGGTGGCACCTGCCTGTCATCTCAGCTACTCGCTAG GCTGAGGCAGGAGAATTG

CTTGAACTCCGGATATGGAGGTTGCAGTGAGCCGAGATTGCAGTACTGTACTCCAGC CTGGGCCACAGAGCGAGA

CTCCATCTCCAAAAAAAAAGACTTAGGAACTCAATTCTGTGATAGCAAAATACAGAA ATAGAGATTATACTTTAG

GCTAGGTGTTGTGGCTCACACCTGTAATCCCAATGCTTTTGGAGGCTAGGGTGGGAG GATCACTCGAAGCCAGGA

GCTTGAGACCAGACTGGACAACATAGTGAGACCCCCCACCTCTACAAAAAATTAAAA AAAAAAATAAGCCAGGTG

GGAGAATTGCTTGAGCCCAGAAGTTTGAGAGAGCAGCCTGGGTAACATCACGAGACC TCATCTCTACAAAAAACA

ACAACAGGGTGTGGTGACATTCACCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAG GAGGATCGCTTTAGCCCA

GGAGTTCCAGGCTGCAGTGAGCCATAATCATGCCACTGTACCCCAGCCTGGGTGAGA CCCTATCTCAAAAAAAAA

GATTACACTTTCATATTATGGCAAAAGCATGTAAATATCCTCCAGCCTCCTTCCTTA TTTATCTGAGCAGTGCTG

CAAGCCCAGGGTTTGTACCCACAAAACCCCAAATTTATAACTCAGGGTGCTTGTAGA TACCACTGAAACATGTAG

CTTATGGTTTTAAGCTACATGTTTTTAGCTATAATGTTTTTTGTTTTGTTTTGTTTT TTGAGATGGAGTCTCGCT

CTGTTGCCCAGGCTGGAATGTAGCGGTGCAATCTTGGCTCACTGCAACCCCTGCCTC CCAGATTCAAGCGATTCT

CAAGCCTCAGCCTTCCGAGTAGCTGGGATTACAGGTGCGCCACCACGCCCAGCAAAT TTTTTGTTTTGTTTTGTT

TTGTTTTGTTTTTTAGAGACAGAGTTTTGCTCTTGTAGCTCAGGCTAGAGTACAATG GCGTGATCTTGGCTCACT

GCAACCTCCGCCTCCCAGGTTCAGGCGATTCTTCTGCCTCAGCCTCCTGCGTAGCTG GGATTACAGGCACGTGCC

ACCACACCTGGCTAATTTTTGTATTTTTTAGTAGAGATGGGGTTTCGCCATGTTGGC CAGGCTGGTCTTGAACTC

CTGACCTCAGGTGATCCACCCGCCTTGGCCTTCCAAAGTGCTGGGATTACAGGTGTG AGCCACCACACCTGGCTA

GAGGTGTGTCCTTTTTAATGCTCATCTTTAAGTCTACCTCAGATTTAATGAATTAGG ACCTCTAGGGTAAGCCCC

GCTTGGGGCATTTGTACCTAGATCCCCAGGCAATTCTTACATATACGAAAAGTATGA ACCGCCACGAGGAGGCCT

ACACACTGCACAAGAGGCAATGTGGCGTGGTCAGTGCTGGGAGGATGGCACCTCCTT GAGAGACTGGGGCACAGA

GGTGGGACCTACACCCTGGGGTGGGGATCGAAGAGGCTTCCTGGCCAGGTGCGGTGG CTCCTGCCTATAATCCCA

GCACTTTGGGAGGCTGAGGCAGGGGGGCAGCGACTGCTTGAGACCAGGAGTTCGAGC CCAGCTGAATAGGATGCC

CGGCTACTCCCACCTTGTTTTACCATAGTGAACCAACGGTTTTTCCAATAGGGGCAA TTTTGCTGCCCAGCGGAT

GTTTGGCAACGTCTGCAGGCATGCTTTGGTTGTCACAACTGAGGGATGCTATGGGCA TCTAGCGGGCAGAGGCCA

GGGATCCTGCTAAATGCGCTGCCATATACAGGACAGCCCTCACCATGCAGAACAACC AGCACCAAACGCCAAGAG

TGCTGCAATTGAGAAATCCTGGTTTCGATGGTCCCACTTCCTTTAAGAAGCCTCCAT TTTTTTTTTTTTTTTTTT

_{TTTTTTTTTTATGAGACAGGATCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGC ACAATCATAGCTTACTGCAG}

CCTCCACCTCCTAGGTTCAAGCAATCCTCCTGTCTCAGACTCCCGAGTAGCTGGGAC TGCAGGTGCGTGCCACCA

TGCTCAACTAATTTTTAAATTTTTTGTAGAGACGGAGTTTTGTTATGTTGCCCAGGC TGGTCTCGAACTCCTGGC

CTCAAGTGATCATCCCACCTTGGTCTCCCAATGTGCTGGTATTACCTGCATGAGCCA CTGCACCAGGCTGAGGTT

GAGAATTTTTTTTTTTTTTTTTTGGGATGGAGTCTCTCTCTGTTGCCCAGGCTGGAG TGCAGTGCTGCGATCTTG

GCTCACTGCAGCCTCTGCCTCCCAGGTTCAAGTGATTCTCCTGCCTCAGCCTTCCAA GTAGCTGGGACTACAGAA

CCACTACACCCAGATAATTTTTGTATTTTTAGTTGAGATGGGGTTTTACCATGTTTA GTAGAGACCAGGCCAGGC

TGGTCTCGAAATCCTGACCTCAGGTGATCTGCCCACCTCAGCCTCCCAAAGTGCTGG GATTACAGGTGTGAGCCA

CCACACCGGGCTGGTGTTGAGATTTTATCTAGACCTGGCAGCCCTCTCCTTCACCAG TAACTCCTAAAACCAGGG

ACCCCTGGAGGGGAGGCCACCTCTCCTTCCCTGCCCCGCCCTGGTCCCAGGCTTAGC CTCTCCCCCTGTTCACAG

TGCCTCAACCTGGTGTTCCTGCTGGCAGACGTGTGGTTCGGCTTTCTGCCAAGCATC TACCTCGTCTTCCTGATC

ATTCTGTATGAGGGGCTCCTGGGAGGCGCAGCCTACGTGAACACCTTCCACAACATC GCCCTGGAGGTCAGCATT

GGCCGGGCAAGGGCTGGGGGTGGCCTGTCCAGGGACACCCAGGGCAGGGATGTCTGG GACTGAAGCCTCACCCCT

GCTCTCTGCCCTCCCAGACCAGTGATGAGCACCGGGAGTTTGCAATGGCGGCCACCT GCATCTCTGACACACTGG

GGATCTCCCTGTCGGGGCTCCTGGCTTTGCCTCTGCATGACTTCCTCTGCCAGCTCT CCTGATACTCGGGATCCT

CAGGACGCAGGTCACATTCACCTGTGGGCAGAGGGACAGGTCAGACACCCAGGCCCA CCCCAGAGACCCTCCATG

AACTGTGCTCCCAGCCTTCCCGGCAGGTCTGGGAGTAGGGAAGGGCTGAAGCCTTGT TTCCCTTGCAGGGGGGCC

AGCCATTGTCTCCCACTTGGGGAGTTTCTTCCTGGCATCATGCCTTCTGAATAAATG CCGATTTTATCCATGGAC

TTCTTATATCGTTTTTGTCTCTAAAAAGAAACTTTTATTATGAAAGTAATACATGCC CACTTTCCTATATATAGA

CAGCATAAAGAAGGTATGTCAGCAGATTTCTCCCTTTTTTGTTTGTTTGTTTGTTTG TTTGTTTGTTTTGACAGA

GTCTCACTCTGTCACCTAGGCTGGAGTGCAATGGCGTGATCTTGGCTCACTGCAACC TCCACTTCCCGGTTCAAG

CAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGTGGTCACTACCATGT CTGGCTAATTTTTATATT

TTTAGTACAGACGACATTTTGCCATGTTGGCCAGGCTGGTCTCAAACTCATGACCTC AGGTGATCCGCCCACCTT

GGCCTCCCAAAGTGCTGAGATTACAGGTGTGAGCCACTGTACCTGGCCTCTGCCATC TTTGAGGCCTTACGTATT

CATTCATTCATGCATTCATTGTTTGGGACAATCTGTAAACAATCATGTTGGACATTC TTTCCAGCATGAAAGTTT

TCAGAGAAGTGGAAAGAATTGTGCAGTGAATCATCCTGAGTATGCGCCACCCTGTTC TATGTTAACATTTTGTTA

CAGTTGTTTTATCATGTAGTCCAGCCCCCATCAAATCTATGTATTCACTGTTCCATC AGTGAGTAGATAGAGCTA

ATGAATGGCAGGGTTGGCACCTGAGGCATTTTATTTTATTATTTTTTTTTATTTTTA GAGACAGGGTTGCACTCT

GTCACCCAGGCTGAAGTACAGTGGCACAGTCATGGCTCACTGTAGCCTTGACCTCCT AGACTCAAACAATCCTCA

CACCTTGGCCTCCCAAGCAGCTGGGACTGCAAGTGCATGCCACCAAGCCCAGCTAAT TTTTTTTTTTTTTTTTGT

AGAGACGCGATCTTCCTATGTTACCCAGGCTGGTCTTGAACTCTTGAGCTCAAGCAG TCCTGCCTTGGCCTCCCA

AAGTACTGAGATTACAAGCATGAGCCACTGCTCCTGGCATCCTGTTATTTAGGTACA GTTTTTATACACAGTAAA ATGTATAAATTGTTACATATTTTTACAGTAAACTGAGTCTGGGCCAGGCACTGTGGCTCA CACCTGTAATCCCAG

CACTTTGGGAGGCTGAGGCGGGCAGATCACCTGAGGTCAGGAGTTTGAGACCAGCCT GCACAACGTGACAAAACC

CCGTCTCTACGAAAAATATAAAAAATTAGCCAGGTGTGGTGGCTTGCACCTGTAGTC CCAGCTACTTGGAAGGCT

GAGGCGCAAGAATTGCTTGAACCCAGGAGGCGGAGGTTGTGGTGAGCTGAAATTGCA CCACTGCACTCCAGCCTG

GGCAACAGAGCAAGACTCTGTCTCAAAAAAAAAAACAAAAAAACTGAGTCTGACTTA GTGTTTCTCATACTTCAT

TGTTACCCTTTCTACTATATTTGCCATATCTGAACCTAACAGTACTTTTATTTACTT AATATTTTTCATTAAAGC

AACTAACCTTTAATATACATTCAGTTATTTTAAAGGGAAACATTATTAAAATCATGA GTTTGAGGTGATATATAT

GTATTTTCTACTTTACTTTAAATAGTTATGTAACTATTGGAGTTTAAAATGAACATG CAAGTTCCCCCTGAGACT

ACTCCATTACGACCAGTGATGTTTGTGGACCCAGATCTAGGAAATGCTGACCCACTG GTTAATAGCATGGGCTTT

GGGGGCAGAAGGAACTGACTTTCAGGTGCCAACCCTGCCATTCACTAGCTCTATTAC CTTGGGCAAGTCACTTCA

CCTCTCTGCACCTCAGTTTCCTGATCTGTCCAATGGGATAATAGTAGCACCAGCATT TCATAAGTTGCTTTTGGA

AACTACATGAGATCATGTCAACACTCAATAAACATCAGCTCTGATCATGCTATGATA TGACTCCTGCCTCTCACC

TTTGCATTGCATTATACCCGTGTTTCTCAAACCTTAGTGTGTACTGGAGGCCTTGTT TTTTGTTTTTGTTTTCTT

GGAGACCAAGAGTCTTGCCCTGTCACCCAGGCTGGAGTACAGTGGCATGATCATAGC TCACTGCAGTCTCAACCT

CCTGGGCTCAAGGGGTCCTCCTGCCTCAGCCTCCTGAGTATCTAAGACTGCAGGCAC GCACCACCATACCTGGCT

AATTTTTATATTTTTGGTAGAGATGGGGTTTCACCATGTTGTCCAAGATAGTCTTGT ACTCCAGAGCTCAAGTGA

TCCACCCACCTTGGCCTCCCAAAGTGCCAGGATCACAAGTGTGAGCCACTGCACCTG GCCCTGCCTGGCTAATTT

TTATTTTTTTTCTGTAGAGACGAGGTCTTGCTTTGTTGCTCAGGCTGGACTGGAACT CCTGGCCTCAAGTGATCC

ACCTGCTTCAGCCTCCCAAAGGTCTGGGATTATTACCCATGAGCCACCACACCCAGC CACAATATTTTATTTATC

CATTTGTTAGTTGATGGATATTGGGGTTTCAATTTTTTGACTTAACTATGAATCATG CTGCTATAAATGTTTGGG

TAAAGTTTTTGTGTGGACACATGTTTTCCATTTCTCTTGGATAATATCAAGGAGTGG AATTGCTGGGTTGATGGT

AATTTTGTTTGATCCTTTGAGGAACTACCCGACTGTTTCCCAAAGTGGCTGCAATTT TACATTCCACTGGCAGTG

TACGAGGACTCCACGTCCCCACCAATACTTGCTCTTTTCCATTCTTTTTCCACCGCA ACCTCCGTCTCCCCGGTT

CAAGTGATTCTCCTGCCTCAGCCTCCCTCTTTTTTTTTTATTTTTGAGACAGTCTCG CTCTGTTGCGCAGGATGG

AGTGCAGTGGTGCTATCTCGGCTCACTGCAATCTCAGTCTCCTGAGTTCAAGTGATT CTCCCACCTCAGCCTCCC

AAGTAGCTGGGATTACAGGCATGCGCCACTACACCTGGCTAATTCTTTTTTGTTGTT GTTTTTTGTTTTTGTTTT

GTTTTGTTTTTTCAGATGGAGTCCTGCTCTGTCACCAGGCTGGAGTGCAGTGACGCA GTCTCTGCTCACTGCAGC

CTCCGCTTCTGGGTTCAAGCTATTCTTCTGCCTCAGCCTCCCAAGTAGCTGGGATTA CAGGCACCCACCACCACA

CCTGGCTAATTTTTGTATTTGTTATGGAGACGAGGTTTCGCCATGTTGGCCAGGCTG GTCTCCAACTTCTAAACT

CAGGTGATCCACCGGCCTTGGCCTCCCAGAGTGCTGGGATTACAGGCATGAGCCACC GTGCCCAGCCTCTTTTCC

_{GTTTTTGTTTTTTTTTTTTCCTTTGGTTGTCTGGTTTGGTTTTTATTATAGCCTT TCTAGTGAGTGTGAAGTGGT}

ATCTCACTGTGGTTTTGATTTGCATGTCCCTGGTGACTAATGATGCTGAGCATCTTT TCATGTCCTTATTGACCG

TACATATAGTTCTTTGAGAAATGTCTTCTCAGATCCTTTGCCCATTTTTAAATGGGA TTGTCTTTTTTTATTAAA

GAGCCAGAGATCTGAAGTCTTTTTGAGTTGTAAGATTTCTTCATATATTCTAGACAG AAGTCCCTTATAAGATAC

ATGATTTGCAAATATTTTCTCCCATTCTGTGTTGTCTTTTTACTTTTTTGATATTGT CTTTTGGTACGGAAAGAC

ACTTATTTTGATGAAGTCCAATTTATCTATTTTTTGTTTGGTTGCTTGTGCTTCTGG TAACATATCTAAGTGGGA

GCCAGGTGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGAG GTTTGCTTGAGACCAGGA

GTTCAAGACCAGCATGAGCAACATTGCAAGGTTCTATCTCTATAAAAAATTAAAATA ATGATAAAAGGAGGCCGG

GCGTGGTGGCTCATGCCTATAATCCCAGCATTCTGGGAGGCAGGAGGACAACTTGGG CCAGGAGTTCAAGACCAG

TCTGGGCATCATGGCAAAAAAAGAAAAAAGAAAAAATTATCCTGGCAAGGTATGCCT GTAGTCCCAGCTACTTGG

GAGGCTGAGGCGGGAGAATTGCTTGAGCCCGAGAGGTGGAGGTTGTAGTGAGCTGAG ATTGAACCATTGCACTCC

AGCCTGGGTGACAGGAATGAAACCCTGTCTCAAAAAATAAAAATAAAATAAGCCAGG TGTAGTGCTGCGTGCCTG

TACTCCCAGCTACTCTGGAGGCTGAAGTGGGAGGATCACTTGAGCCTAGGAATTCGA GGCTGCAGTGAGCTATGA

TCATACCACTGCACCCCAATGTGGGCACAGAGCAAGACCCTATCTCTTAAAAAACAA ACAAAGAGGGGCGGGGTG

CAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCAGATCACGA GGTCAGGAGATCGAGACC

ATCCTGCCTAACACGGTGAAACCCCATCTCCTTTTTTTTTTTTTTTTTTTTTGAAAC AGAGTTTCATTCTTTTTG

CCCAGACTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCGGGGATT ACAAGGGTGTGCCACCAC

ACCCAGCTAATTTTTTATATTTTTAATAGAGGCGGGGTTTCACCGTGTTGGCCAGGC TGGCCTCAAACTCCTGAC

CTCAAGTGATCCGCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAACCA GCGCGCCCAGCCTCCTAC

ATTTTCTTCTAAGAGTTTTATAGTTTTAGCTCTTACATTTAGATCTTGTTTTCATTT TGAATTAACTTTTTTGCA

GGTGGTGTGATACAGCAGTCTGTGCAGAAAAAAGTTAACATAGTAGACCCTAACTGC TAACTTTATTTTTACTTT

TATTTTTTATTTTGTTTAGAGACAGGGTTTTTGTCACCCAGGATGGAGTGAAGTGGC GTGATCATAGCTCACCGT

AACCTTGAACTCCTGGGCTCCTCTGCCTCAGCCTCCCAATTAGCCACCACACCCAGC TACCTTTTGTACATTTTT

TTGGTAGAGACGGTGTCTCACTATGTTGCCCAGTCTGGTCTCAAACTCCTCAGCTCA AATGATCTGCCCACCTTG

ACCTCCAGAGATACTGGGATTACAGGCGTGGGCCACTGCACCAGACCCAGGTTTATT CTTACACCAATACAACAC

TGTCTTAATTACTATAGCTTTGTAGTGAGCTTTTGAAAATAGGAAATGTGAATCCTC CAACTTTTTTTTTTTGGA

GACGGAGTCTCGCTCTGTTGCTCAGACTGGAGTGCAGTGGCACTATCTCAGCTCACT GCAAACTCCATCTTCCAG

GTTCAAGTGATTCTCCTGCCTCAGCCTACCAAGTATCTGGGACTACAGGCAGGTGCC ACCATGCACAGATTCCTT

GGACATCCCTGAGAGGTCAATCATGAAGGTCAACTTGGTTTTCTCCCCCTCATTTGG GTTCAGAATTTAAAGTCC

ACACACACAGGCAGTAAGATGATTATAGATAAGGACATCATCACTCGGTTTCGGATG TTAAATTGTCTAGGTGGG

TTAGGGGTGATTTGAGATCACACAACCTTGTGCCACAAAGAGGAATTCCCAGGCCAG AGGGAGACATTTTATTGC

CATGTTATGATCTTATCATTGAGTTGAAAGGCAATCTTGTTTCATTTTGGATTCTTT CTTATGTTTATGTCTTAT

AAGGGCACTTTGAATTTCCAAGCAAATAATAATTTTGAATTAGCTTTTAATCATTGA CTTCTAGCACAGTTATAT

GATCAGAAACGTGCTGTGTGATTTGATTGCTCTCAAATATATTGAGATTTGCTGGAA CAAAATAAGTCAGGTTAA TTTTTGTAAATGTACCATGCTTGCTTAAAATGAATGTATCTACATTTGTTCCTGAGATAC GGGTTGATGGACGGA

TGGCTACATGGATGTGATGGAGATGGTTTACTATCGGGACCTTCCGCATCCTGCTGA TGTTTTGTTGCTTAGGAT

ATGAATGGCTGAGCGGAGGCTGTAAAACCTGGCACTCTGCTTGGGTATGAGGTTCTT CCTGCCATCCTGCCATCA

TTTGTTTTTTATGTTTTGTCGCCAAAAGTGACCTTGAGGAACCCTGGGAGCTCAGGA AGGAAGGAGCGCCCAGAA

GCAGGGACAGGGAGCTGGTTGGGGAGGACCAGAAATCAGGTTTGTGAAGGTTCCAGA GAGGACCTGTCCTTGCGA

GGAGTGTGGGAGACTGAGATGGGGGAGGGGTCATTGGAATGATGCGGGCGCTACTTG GCATTGTCCATTGTGAGG

CACCACCGGGGTCATCAGGGATTGGTGGAGAGGGAGTATAAAGCCCCAGGTTTGCTA AGGGAGGGCCCAGACCGA

AGAAGGTTTGGCGGATAGCAGAACCTTTTTGTCTCCCTCTAATTGCTCCTAAGCCTC ACGCTCCCTTGCCCCGCG

TGTCCTGTTGCTTCCCTGATCTTCTCCGTGACCTGTAGCTAAACCTTCCACCAGCGC TTGAGAACTTAATTTGAA

CCGGATCCTTTCCCAGACCCCTTTCTTCTTCTCCTCCTCCACCTCCTCCAGGTGCCC AACAGCCCCCTTCTCCTC

CTTTCCCTTCCCTTACTTCCCCCCTTCCCCTCCCCTTCCCCTCCCCCTCCCCTCCCC TTCCCCTCCCCTTCCCCT

TCCCCTCCCCCTCCCCTTCCCCTCCCCCTCCCCTTCCCCTCCCCCTCCCCTCCCCCG CCCCAACTCAGATCCGGC

CCCGGTCCCCGTCCCCTTCCCTCCCCCCTGCCCTAAGCCACCTCCACCTCTGTCCTG GCCGCCTCAGGGCACCCT

GAAAGGATCAGGACATGCGGCTGCGCTTTTGGCTCCTCATTTGGCTCCTGCTGGGAT TTATCAGCCATCAGCCCA

CCCCTGTGAGTAGACGCTGGACCCGCGGGGTTTCTTCCTTTTTACTGGGCTGTGTCA CGCGGCATGAAATTACAC

AGCTCAGGCCTGTAATCCCAGCACTTTAGGGGGCCGAGGTGGGCAGATCACTTGAGT CCAGGAGTTGAAGACTAG

CCAGGGCATCATGGCGAAACCCCATCTCTACAAAAAATTCCAAAAAAGATTAGTCGG GCCTGGTGGTGCGTACCT

GTTATCCCAGTTACTGGAGAGGCTGAGGTGGGAGGATCGCTTGGGCCGAGGAGCTGG ACGTTGCAGTGAGCTGAG

ATGGCCCCGCTGCACTCTTGTCTCTAACAAACAAAATGGACCAAAACAAAGTGAAAT GTCATTTGATTTGTGTCA

TCTGGTTTGATGACTTTTTTTTTTTTTTTTTTAGACAGAGTCTCACTCTGTCGCCCA GGCTGGAGTGCAGTGGCA

AGATCTCGGCTCACTGCAACCTCCGCTTCCGGGGTTCAAGCAATTGTCCTGCCTCAG CCTCCTGAGTAGCTCAGA

TTACAACGCCTGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTCG CCAGGATAGTCTCCATCT

CTTGACCTCGTGATCCGCCTGCCTCGGCCTCCCAGTGCCGGGATTACAGGCGTGAGC CACCGCGCCTGGCCAAAA

TATATAACCTTAAGTGTAAGTTTACTAACTTTGGAAAGTACATACACCAGCATAAAC CGACCCCCTTTCAAGATC

TACATTATTTTATTTATTTATTTATTTTTTTGAGACAGTTTCTCCCTTGTTGCCCAG GCTGGAGTGCAATGGGGC

AATATCAGCTCACCGCAACCTCTGCTTCCCAGGTTTGAGCGATTCTCCTGCCTCAGC CTCCCGGGTGGCTGGGAT

TACAGACATGTGCCACCACTCCCAGCTAATTTTGTATTTTTAGTAGAGATAGGGTTT CTCCATGTTGGTCAGGCT

GGTTTTGAACTCCCGACCTCAGGTGATCCGCCCGCCTCGGCCTCCCAAAGTGTTGGG ATTACAGGCGTGAACCAC

CGTGCCCAGCCAAGATCTACACTATTATGTCACCCCAGAAAGTGAACTCTCAGTCTT CCCAGCCAGTCTCTTTCT

TATCATAGGTTAGCTTGCTTATTCTGGAATTTCGCGTATACAGATGCGTGCCATGCC ATAGGTACTCTTTTGTGT

CTGCTTTGTTCTGCTCAACACCATGTTTCTGAAATCATTACCATTGTTGTATGGTTC TCTAACTCCATCATTTCC

ATTTCAGACTCAGCATATGCTGAGTTCAACCTGTTGAAGGGCTATCTCTGTTTAATT CACCATCTTGAAAGAAAC

ATTTAAAATTGAGATGTTTTCAAGAATATATAGTTAAATCCTGAGGAATCGATGTAG AAATGTTATCACAAGCTG

TCTGAACTTACTCAGGGGAAGTCTTCGTCTTCACTCACATAAGAGTCTACTGGAATT AATATCAACAATCTTAGA

GAAATCCCACACTATTCATGCCATTTTCATGATCTCCACCTTGGTAATTTTTTTTTT TTTTTTTTTTTTTTGAGA

CAGAGTCTCACTCTGTCACCCAGGCTGAAGTGCAGTGGTGCGATCTCGGCTCACTGC AACCTCTGCCTCCTGGGT

TCAAGTGATTCTTCTGCCTCAGCCTCCCAAGTAGCTGGAACTGTAGGCACGTGCCAC CATGCCCTGCTAATTTTT

TGTAATTTTAGTAGAGATGGGTTTCACCGTGTTAGCTAGGATGGTCTCAATCTCCTG ATCTCGTGGTCCACCCAC

CTCGGCTTCCCAAAGTGCTGAGATTGCAGGCGTGAGCCACCACACCCGGCCCACCTT GTTAATTTTTAAGCACTA

AAATTTGATACTTATTTGTGAATGAAGTAATCTCTTCATTGTATTTTTTTTTTTTAC TTATGCTGAGCTTCAAAT

GACAAAGATTCATATAATCCAAGAGAGAAGTATTATTTAGAGGGATTCTTTTACCAT GTGATATATAATAAATGC

ATCCAATGTTATACATCAATTTAAAAAACAAGTAAATAACTTTAAAGAAAAGATAAC TACTGGCCAGGTGCAGTG

GCTCACACCTGTATTCCCAGCACTTTGGGAGGCCGAGGCAGGTGGATCAAGAGGTCA CGAGTTGGAGACCAGCCT

GGCCAAGATGGTGAAACCCTGTTTCTACTCAAAATACAAAAATTAGCCGAGTGCGGT GGCAGGCGCCTGTAATCC

CAGTTACTCAGTAGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCA GTGAGCTGAGATCATGCC

ACTGCAATCTAGCCTGGGTGACAGAGCAAGACTTTGTCTCCAAACAAAAAGAAAAGA TAATTACTTTATACTTAG

CTTGTCTTAGCCATGAGTGACGGGCTGCATGTGGCCCAGGACAGTTTTGAATGCAGT TCAACACAAATTTGTAAA

CTTTCTTAAAACATTAGGAGATTTTGGCCAGGTACAGTGGCTCATGCCTGTAATCCC AGCACTTTGGGAGGCTGA

GGCGGGCAGATTACCTGAGGTCAGGAGTTCGAGACCACCCTGGCCAACATGGCAAAA TCCCATCTCCACAAAAAA

TACAAAAATTTGCTGAGTGCATTGTCAGGCACCTGTACTCCCAGCTACTCAGGAGGC TGAGGCAGGAGAATCACT

TGAACCTGAGAGGCAGAGGTTGCAGTGAGCCGAGAGCACGCCACTGCACTCCAGCCT GGGTGACAGAGTGAGACC

CCATCTCAAAAACAAAACACCAAACAAAAACAAAAACAAAAAAAAATGGCTGGGCAC GGTGGCTCACACCTGTAA

TCCCAGCACTTTGGGAGGCCGAGGCAGGCAGATCGCCTGCCAGGAGTTCAAGGCCAG ACTGGCCAACATGGTGAA

ACCTCATCTCTACTAAAAATACAAAAATGAGTCGGGCATGGTGGCAGAGACCTGTAA TCTCAGCTACTCGGGAGG

CTGAGGGAGGAGAATGGCTTGAGCCCAGGAGCTGGAGGTTGCAGTGAGCCGAGATTG CACCACTGCACTCCAGCC

TGGGCGACTGAGTGGAGCGGAACTCTGTCTCCAAAAAAAAAAAAAAAAGAGTTTTTT TTTAGATCATCAGCTATT

GTTAGTGTTAGTGTATGTTATGTGTGGCTCAAGACAACTTTGTTTCTTTTAATATAG GCAGGGAAGTGAAAAGAT

TGGATATCCCTGCTTTATACCAAGAAAGACAACACCCCACATTTGCAATGCCTAAAA ACACTACCAGCCATCTGA

AAAACATGAGACTTCTAACTTCTGTTCTTTTTTGTAGCAGTGGAATCCCACGGTGAT ATCTGAGGGATGTGGTTA

CCTTTTGGAGGAGGTTGACGGTTTCTAAGGATGATTCTTTCTGAGTGAAATATTGTC GGTGTCATTGACCTTTTC

ATTATTTCAACTATTATTATTCCAGGTTATCAATAGTCTGGCTGTCTATCGTCATCG TGAGACTGACTTTGGTGT

AGGAGTTCGAGACCACCCTGGCCAACATGGCAAAACCCCATCTCCACAAAAATTGGA TAATTTGATAATTATCAT

TATTGGGTTTCTGAGACGTTACACATTTAACGTTCTCTTCTGCACAAGTTGCCTTTG TGTGAGTATACTAACTTT

CTGTAGAGGTATACTTGTAATCACAAATAAGAATAAATTATATAAAACAATTCACGT TTCTGGACTTCATTATGA ATATGTGGTTTTACCCAAAAAATCAGGGAAAT GATT TAT TAGCATAAGAAT TAT GAAAATGTCTGC CAT TTACAT TATGAAAATTAAATAGGTCGGTGTTTGTTTAATAGAATGTCAACAGAGCTTTTGGTCAAA AATAAGTTTTTTTAG CCTTTGTGCTATTTATCACAAATGGAGTATGAGGTTTCGTCACTTAAATAGGAAATTCTT TCTAAACTCTTCTGC TTTATAGTTCTATCGTATGGGTGGAAGGAAAGCTTCCAATCTCCTCTCTGAAGATTCACT GCAGAAATGAGCTGA CAACAGACAGCTTAACAGGAAAAGAAAAACATAGAACAGGCATAAACATGGGAACCAGCT GAAAAATGAGACTGC TAGAAGGGCCGGATGGTTGATGCTTAAAGAGCACCCTCTTCTGAGGGGAGAGGGAGATAG ATGGAGATGTAGGCC ATTTAGAGGGGCAGCAAATGATTTTTAGGGGAAATGAAAGAGGCCAAGGAACAAACAATT GGCCTGAGACAAAGT TCCTCTGAGGTCATAGGGACGAGGTGACAAACTGCCGGAAGGTGAAGGGCAGAACTGCAC TGCGTCTCATGATGC AGAGAAAGCCCCAGAGAATCTCTTAGAACTGCCCTCCAAGAGAATCAATGAAAAGTGTGT CTGGGCAGGGTAATT TTGAATGACATCATTCAAAGTGCATGTTCCCACTTGCAACTGGAGAGAGATCAGTATGTC AAAAGTCTGTACTTG GTAAGAATTTGGCTGCTAAGTTGTGCCATAATTTGTCTTTTGAGCCTTTTTTCCTTTGGG TAAGTTGAGCTCTAC ATTTTGTCTTGCCATTCATGACAGTAAAAATGTGGTTTTCTGGGGGCTGAACCTCCTGAA CAATGATCCAAGATA AAAGTACTAATACCACAATGCTTTTTTATATTCAAGGGAAGAGGAAGTATGTTTCAGTTT TACCACCTAGATAAT TACACGTCATTTGGCACTGCCTTTCAAGATATGTAGAAAACAGAAAATATATGAGTTATG AAGATATCTAGGCAC ATTTAACATTCTCTATGCCACTTAGTCCTGAACAGAGAATTTTCGGTATAAATTGGAGGA AGCTTTTTTTTTTTT TTTTTCTTTTCTCACCCCCGAGACGAGTCTCCCTCTGTTGCCCAGGCTGGAGTATAATGG TGTGATCTCGGCTCA CTGCAACCTCCACCTCCTGGCTTCAAGCGATTCCCCTGCCTCAGCCTCTCAAGTAGCTGG GATTACAGGTGCCCA CCACCATGCCCAGCAAATTTTTGTATTTTTAGTAGAGTCGGGGTTTTACCATGTTGGCCA GGCTAGTCTCAAAAC CCGACCTCAAATGATCCACCCGCCTCAGCCTCCCAAAGTGCTGGGATTACAAGCGTGAGC CACCACGTGAGCCAG GGGAAGTTTTTAAATTTACCACTTTTTAACAGTTCCATTTAGGAAAGTTCAGTTGAGCTG CTGGACTTGGACAAC TTCGCACCTCTCATCTTTGTCCTTGTCATCTAGTCATCTATACCATTACCTCCTAAGCAG GGACATCATGGGTGC CATGAAGCATTCATGCGTGATGGCATTTCTTTGCTTGTCATTTCTTCATGTGTTTGACAT TTCTCCTAGCTCCAA ACTGGGCCAGCTACCTTTCCTATGAAATCTAGTAGTAGCTGTGGGATTGACGTGGTTGCT CTTTTCATCTTTTTA GATTACCCATTGCTTCTCTCGAAATCCTAGTACATGATTTTTTTTTTTTATCCTATGTGC AGAAATCAGGAAAAA ACAAATTCTACAAAGAATTTGAAAGATATTATTTCAGGCCAGGTGTGGTGGCTCATGCCT GTAATCCCAGCACTT TGGGAGGCTGAGGCAGGTGGATGACTTGAGGTCAGGAGTTCAAGACCAGATGGGCCAACA TGGTGAAACCCCATC TCTACTAAAAAGACAA

Gene sequence: Homo sapiens CLN3

(RefSeqGene (LRG_689) on chromosome 16; nucleotides 5,001-30,650 of NCBI Reference Sequence: NGJ308654.2; SEQ ID NO: 2)

(25650 nucleotides in length)

ATAACTGGTGCTGGCAGGCTACTGTCTCGGTCTTGGGCGCCACTGATCTAAGGTCAC GGCTCTGCTTGCTGCTCC

CACCCGCTCCAGTTTAAAACCTGCGGTTCCAGGGTTCTCCAGCCCCTCCCTTTTTCA CGCTCCGAAGCCGAGAAG

GCCCAAAGCGAAGACAGAGAGGACCCGGAAGTAGGGAAAACCTCTGAGCACGTGATG GGGGAACACGCGGGTGCT

GTCACGTGATCCGACAAACGGCCTCTGCATAGTGCAGAACATTCTGCTGCTCTTAAA GGTACAGGCCTCAGGGTC

CCTGCTGTAGACGGGGCGGGGGAGAGTACGATGGGTGGGGCGTGGTGGGTCGTAGGG CGCTCGAGATGGAGCCCC

CAGCTTCCTTGATGGATCGCGGGGCGCGAGTGCCCTAGACAAGCCGGAGCTGGGACC GGCAATCGGGCGTTGATC

CTTGTCACCTGTCGCAGACCCTCATCCCTCCCGTGGGAGCCCCCTTTGGACACTCTA TGACCCTGGACCCTCGGG

GGACCTGAACTTGATGCGATGGGAGGCTGTGCAGGCTCGCGGCGGCGCTTTTCGGAT TCCGAGGGTGAGTATTCC

CGCCCACCCTCATGGAACGACCACCTCTGGCTCGCAGCCCACCTGAGGGGAATGAGA GCTGACTCGGCCCCGGGA

GGACACGCAAGGAGCGGTCGTGCACCTGAGAGTAGGTGGGGGTCATGCCCTCTTCTC GCTCTCCCAGGGGAGGAG

ACCGTCCCGGAGCCCCGGCTCCCTCTGTTGGACCATCAGGGCGCGCATTGGAAGAAC GCGGTGGGCTTCTGGTGA

GTGGCCTGAGACTTCAGCGAGTGACAATGGCATGAGAAGAGGGAAGTGACCGGAGGA AAGGGGGAAGAAAGAGTT

AAGCTGCGCAAAGGAGCTGAACCCACAAATATTTACTGAATCCACTGGTTTGCCCCA GGGGCAGCATGTAAAATT

TCCAAACTTCACTCCTTATCACCTTCCGGATTTAGCAAGATCCTCATACCTACCACC TGTAGGATTATTTACTTT

TTTCTTCCAGCCAATTCAGCCTTAACGTTTTTTAGTGCACATATTTTTAAAAAGAAA ACTTTAAATCACGACTCC

ACTTGGAAAACCAGCGTTTTCCTAGTGAACGTTTAAATAAATGCATCACTACTAAAA CTTTTTTATTTTTTTAAT

TTATTTTATTTTATCTTATTTTTTTTTCTTTTTGAGACAGGGTCTTGCTCTGTTTCC CAGGTTGGAATGCAGTAG

TGCAATCACAGCTCCCTGCAGCCTGGAACTCCTGGGCTCAAGTGATTCTCCCACCTC AGCCTCCAAGTAGGTGTG

GCTACAGGTGTGCACCACCGCATCTGCCTAATTTTTATATTTTTTTATAGAGATGGG GGTCTCACTATGGGACCT

GGGCTGGTCTCAAACTCGTGGCCTCAAGTGATCCTCCTACCTCAACCTTCCAAAGTG CTGAGATTACAAGCATGA

GCCACCATGCCGGGCCTGAACCTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTCT CTCTGTCCTCCAGGCTGG

AGTGCAGTGGCGCAATCTCGGCTCACTGCAAACTCCGCCTCCCAGGTTCACACCATT CTTTTGCCTCAGCCTCCG

GAGTAGCTGAGACTACAGGCGCCTGCCACCACGCCCGGCTATTTTTTTGTATTTTTA GTAGAGATGGGGTTTCAC

CGCGTTATCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCGCCCGCCTCGGCCTC CCAAAGTGCTGGGATTAC

AGGTGTGAGCCACCGCGCCCAGCTTTTTTTTTTTTTTTTTTTAATTTTATTTTATTT TTGAGACAGAGTCTCACT

CTATCACCCAGGCTGGAGAGCAGTGGCACAATCTCGGCTCACTGAAACCTCCACCTC CAAGGTTCAAGCTATTCT CCTGTCTCAGCTCCCCGAGTAGCTGGGATTACAGGTGCGTGCCACCAGACACAGCTAATT TTTTGTATTTTTAGT

AAAGACAGGATTTCACCATGTTGGTCAGGCTGGTCTAGAACTCCTGACCTTAGGTGA TCCGCTTGCCTCCACTTC

CCAAAGTGCTGGGATTACAGGCGTGAGCCACCGTGCCCCGCCCTGACATAGGGGCTT TGGGATCACAGACTTGGA

TTCACTTCCAGCCTCCAAGGCCTCTCCCAGAAACTCTCTGTGACCACTCTCCTTTTT TTTTTTTTTTTTTGAGAT

AGAGTCTCGCTCTTGTAGCCCAGGCTGGAGTGCAATGGCACAATCTCGGCTTACTGC AACCTCCGCCTTTCGGGT

TCAAGTGGTTCTCCTGCCTCAGCCTCCTGAGTAGCTAGGATTACAGGCATGTGCAAC TTCGCCCAGCTAATTTTG

TATTTTTTAGTAGAGACGGGGTTTCTCCATATTGCTCAGGCTGGTCTCGAACTCCTG ACCTCAGGCGATCCGCCC

GCCTCGGCCCCTCAAAGTGCTGGAATTACAGGTGTGAGCCACTGCACTCGGCCTCTT CCTTTTTATTTTTTATTT

TTGTGACAGGGTCTTGCTCTGTCACCCAGGCTAGAGTGCAGTGGCATGATCACAGTT CACTGCAGCCTCTGACTC

CTGGACTCAAGCAATCCTCCCACCTCAGCCTCCCAAGTAGCTGGGACTACAGTGCAA GCCACCACACCTGGCTAA

TTTTTAAATTTTTTGTAGAGATGGGGGTCTCACTATCTTGCCCAAGCACCAAAGCCT GTATTTTTGACCCCAACA

TTGAATGAGGATGGGATCTGGTGATAGAGGGAAAAAAAAGAGGGGGCTGATTGAAGG GCACAGGTAAGGGAAGGT

TTGGCACAAAGGCTACAGATGGCTGCAGGATGAGGAGGGAGTTGAGACCTGTCCTGC TCCTTCCAGGCTGCTGGG

CCTTTGCAACAACTTCTCTTATGTGGTGATGCTGAGTGCCGCCCACGACATCCTTAG CCACAAGAGGACATCGGG

AAACCAGAGCCATGTAAGTGACTCTCTACCACCACCACCATGGTTAGTCCCTGTGGG AAGATGAGGGGGTGGGAC

AAGGTGGGGTAAAGTTTCCAGTCTCTGGCTGGGCACAGTGGCTCACACCTGTAATCC CAGCAGTTTGGGAGGCTG

AGGCGGGCGGATCACTTGAAGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAA ACTCCGACTCTACTAAAA

ATACAAAAACTACCTGGGTGTGGTGGCACACACCTGTAGTCTGAGCTATTCAGGAGG CTGAGGCAGGAGAATTGC

TTGAAGCCAGGAGGTGGAAGTTGCAATGAGCCAAGATCACACCACTGCACTCTAGCT TGGGCAACAGAGTGAGAC

ACCATCTCAAAAAAAAGAAAGCTCTTGAATGTGTTATCTAAACAAAGTCCATTTACA GGGCCATCTGTGGCCTGT

GGTCTAGTAGTGTGAGACTCATATGGAAACCCCCTCTTCATTGTGCATGCAGAGACG CTGAGGTCCTGAGAAGTC

AGTCACTGCTGACCCAAAGCCATCCTTCAAGTGAAGGCAGAGCTGGGACGGGAGCCA GGCTCTGTGTGTCTATCC

CTCTGCCTCCAGGGTGAAAGACATGCTTTTCTCCCATTAGGTGGACCCAGGCCCAAC GCCGATCCCCCACAACAG

CTCATCACGATTTGACTGCAACTCTGTCTCTACGGCTGTGCGTACTCATCTCACCTG GTCCTTGCCTGACCCAGG

GCCCCTGGGTCTATAGACCCCACTCCCAGCCCTTCACTACCCAGCTGGGACTGTCAT GATTAAAACAGTTGAGAC

CTGGGCTGGGCGCACTGGCTCACACTTGTAATCCCAGCACTTTGGGAGACCAAGGTG GGAGGATCACTTGAGCCT

AGGAGTTTGAGACCAGCCTGGGCAGTATAGTGAGACCCCCATCTTAAAGAAAAAAAT TAAAAAATATATATATAT

AAAATAATTGAGACCTTAATATAATTTCTGAGGCTGAGGCGGATGGATCACTTGAGA CCAGGAGTTCAAGACCAG

CCTGGCCAACATGGTGAAACCCCATCTGTACTAAAAATACAAAAATTAGCCAGGCAT GGTGGTGCACGCCTGTAA

TCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGGTGGAGGT TGTAGTGAGCCAAGATCG

AGCCACTACACTCCAGCCTGGGTGGCAGAATGAGACTCACTCTCAAAATATATATAT ATATAGTTTCTGAGTCCT

TTCTGTCTGCACCATATATTAGCTCATTTAAGCCACACAGCAGTCCTGTGAGCTAGG TGCTATGATATTCCCATT

TTCCAGATGAGGAAACTGAAGCTCAGAGAGTATAAACTCTTGACACACAACTAAGGA GTGGGAGAGCTGAGACTT

GAACCCAGGCGTGCCTGACTCCAGAGCCTGTGTTTGTAGCAGGCCTGTTTGGCCAGC TCCTGCCTCTCCTTGGCC

ACGTGGTTGGGAGGGTTGTCCCCTGGAAGCTCTGCGGTCTCACTCTATTCTCCTGTC CCAGGCTGTGCTCCTGGC

GGACATCCTCCCCACACTCGTCATCAAATTGTTGGCTCCTCTTGGCCTTCACCTGCT GCCCTACAGGTCTGGGTG

AGGGTAGTGGGAGGCAGGGTGGGCAGGAGCTGAGAAAGGGGAGGCTGGGATGGCTGA GATGCTGAGAGTAGAGAC

CGACCTTCCCCCTCCCTTCCCTTCTCACCCCCTCAGCCCCCGGGTTCTCGTCAGTGG GATTTGTGCTGCTGGAAG

CTTCGTCCTGGTTGCCTTTTCTCATTCTGTGGGGACCAGCCTGTGTGGTGAGTGTGT GGTTCTGTGTCAGATGGG

GAGCCCCGAGGAACCACATCAGAGCATTTGTGGGAAGAGTCTCCCCAGCCTCCCAGA GGAAAGGGATTCATTCTG

TCACCCTTAGAAGCCTGCTAGGGCTATCAGCAGTAGGCGATGGGAGACTGGGACAAT TTGGAGGGGTAGGCAGTG

GAGGAGATGGGAGAAAATGGATGAATTAGATGGAGATTGAGGTGAACAAAGTCAAGA CTCTGTGATGGACCAGGC

ACAGTGACTCATGCCTATAATCCCAGCACTTTGGAAAGCCAAGGCAGGCAGATCACC TGAGGTCAGGAGTTCGAA

ACCAGCCTGGCCAACATGGAGAAACCCCGTCTCTACCAAAAATACAAAAATTAGCTG GGTGTGGTGGCAGGAGCC

TGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCTCTTGAACCTGGGAGGCA GAGGTTGCAGTGAGCCGA

GATCACGCCACTGTACTCCAGCCTGGGTGACAGGGCGAGACTCCGTCTCCAAAAAGA AAAGAAAAGAAAAAGACT

GATGAAGGGGCAGAGACATCAAGGGTGCATGTCTGCCCCTGGTCTGATAACTGGGTG GATGGAGGTGCCACTCTC

CATGAAGGGACACGCAGGGGAGTGGGGCTCTGCTTCAGACCTGGAACCTGGCCTATG CATGGGATCTATTGGAGC

CTCTATGAGCTGATACTGAGGAGGCCATGGCCAGACACATTAGAGGCCTGGGCAGTG TGGCAAGGTGTGGTGTGA

CCATCCCAGTGCTTGTCCTCCCCCCAGGTGTGGTCTTCGCTAGCATCTCATCAGGCC TTGGGGAGGTCACCTTCC

TCTCCCTCACTGCCTTCTACCCCAGGTAAGCAGGTGGAGCAGGGAGTGTGGGGAGAG GCTGTCCCATGGTCAGCC

TAGGTCCTCCTGAATGTTCCTGTGTTCTCCTTCCCAGGGCCGTGATCTCCTGGTGGT CCTCAGGGACTGGGGGAG

CTGGGCTGCTGGGGGCCCTGTCCTACCTGGGCCTCACCCAGGCCGGCCTCTCCCCTC AGCAGACCCTGCTGTCCA

TGCTGGGTATCCCTGCCCTGCTGCTGGCCAGGTGAGCTGCCCTGAGCCGGGAGGGAG AGGGGTCCAAGGAGAGAA

AACTTGGCCATGGCTGGGTGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGG CCAAGGAGGGCAGATCGC

CTAAGGTCAGGAAACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATA CAACAATTAGCCAGGTGT

GGTGGCGGGTGCCTGTAGTCCCAACTACTCAGGAGGCTAAGGCAGGAGAATCGCTTG AACCCCGGAGGCAGTGGT

GGCAGTGAGCCGAGATCGTGCCATTGCACTCCAGCCCGGGCGACAGAGTTAGACTCT GTCTCAGGAAGAAAAAAA

AAAAAAGAAAGAAAAGAAAACTTGATTATGATTGCAATCTTCAAGTCCCTACCTTGC TGTGAAGGGAGGCGGAAT

CTGGACTCTGATAGCCCCAGGTGTGAGTCCTGGAGCTGCCACTTTTTAGCTTTGTAG CGTTGAACAAGTTACTCC

ACCTCTCTGAACCCTCAGTTTCCCCATATCTCAAATGGCAGTTGTTCTTGCTTTCCT TGGAGGTGATGAGGGTAA

TGCATTCAGCACAGTGTGGTTCCCAAGGTGATTAGAAGTAGGATGAGGGTGGACTTT ATTTGATTAGTTCCTTTT

_{TTTTTTTTTTTTTTTTTTAATCAGTGTTGACCAGGTTGGCCTCGAATGTGTAGCC TTGCCTGCCTGAGTGCCAGG}

GCAACAGGCCTGAACCATGGCGACTCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTG AGACGGAGTCTCATGGTG TCACTCAGGCTGGAGTGCAGTGACTGGTGTGATCTCAGCTCACTGCAGCCTCGGCCTCCC GGGCTCTAGTGATTC TCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCCCATGCCACCATGCCTGGCTAAT TTTTTATATTTTTAG TAGAGACGGGGTTTCACTGTATTGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGTGATC CACCTGCCTTGGCCT CCCATAATGCTAGGAATACAGGCGTGAGCCACCGCGCCCGGCCTGATTAGTTCTGTGTAT TTTCATGCATATTAC AAAACACTTTGGCCGGGCATGGTGGCTCACATCTGTAATCCCAGCACTTTGGGAGGCCGA GGCGGGCGCACCACG AGGTCAGGAGTTTGAGACCAGCCTGGCCAATATGGTGAAACCCCGTCTCTACTAAAAATA CAAAAATTAGCCAGG CTTGGTGGCGCTTGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGAGAAGAATCGCTTGA ACCCAGGAGGCAGAG GTTGCAGTGAGCCGTGATTGTGCCACTGCACTCCAGGCTGGGCAACAGAGCGAGACTCCG TCTAAAAAAAAAAAA AACACACACTTTGAACTAGCCAGACACACCTGGCCCTTCACAAGATTAGTGCTTAGGACA AGTCTAAGTAGAAAA AGTGAGTCCATTTAAAGAAAAATATTAAGTAAAAATATATATATACAGGAGGAATATGGA TATGGCAAATAACAT GAAGCCAGAACCTAAGTGACTAAAATCGAGGAAGTTCTGGCCTAGCACAGAGTGAACGTA TAATTAATGGGAGCT AGAGCGACCATTAGAATAATAATGGCCAGAAAACAGAACTGACTCGCTAGGTATGAGCCA GCAGTCCGAACAAGG GCCTGCTGAGCACAGTGGCTCACAGCTGTAATCCCAGTGCTTTAGGAGGCTGAGGCTGGA GGACCACTAGAGGCT AGAAATTTGAGACCAGCCTGGGCAACATAGCGAGACTCCATTTATACAAAAAATGGAAAA TATGAGGCGGGCTTG GTGGCACGTGCCTGTAGTCCCCGCTACTTGGGAGGCTGAGGCGGGAGGATTGCTTGAGCC TGGGAGGTCGCGGCT GCAGTGAGCTATGATTGCACCACTGCATTCCAGCCTGGATGCTGGAGAAAGACCCTGTCT CTGAAAAAAATCAAA ACAAAATGAAGGCCCATGAATAGGAATAAGGAGATAAATTCAGGTCCAAAGAAAGAAGCC TTTGTCCACAATTGG AGCTCTCCCGCACAGCGTAGGAGCCTTAGAGGCAGTGAGCTACCCATCTTTGAAAGTGTT CAAAGGTAGAGGTGT TCCCTGGGAAAAGTGGCCTAGATGGTCCCTGGGGACCTTCCCAGCCAGTAGGGTTTTCTG ACCCTGCCTTCATCC TACTCCTAGCTATTTCTTGTTGCTCACATCTCCTGAGGCCCAGGACCCTGGAGGGGAAGA AGAAGCAGAGAGCGC AGCCCGGCAGCCCCTCATAAGAACCGAGGCCCCGGAGTCGAAGCCAGGTAGGAGACACAG ACCCTCAGAGAGGTC ACTTTCTTTCTCTCTGGGTTTGGCCTTTTCCTCTCTGCAATAGGCAAAGTTAAGAGGGGA AAGAGAGTGAGTGTA CTGGTTATGGGAAAAGCCTTTGTCTATTTGAGAAATACTTGTTGAGGACGAGCACAGTGG CTCACGCCTGTAATA TCCCAGCACTTTGGGAGGCTGAGGCAGGAGGACCACTTGAGCTCAGAAGTCTGAGTCCAG CCTGGGCAACAGAGC AA GACCTTGTCG C AAAAAAAAAAAAAAAAAAAAAAAAAA G AAAA G AAA G G AA G G AA GGGAGGGAGA G AA G AA G G G ATATTAATTGAGTACTTACCATGTGCCAGGCATTCCAATCACAAGTGCTGAGGCCCTGCG GTGGGGATAAGCTGG GATGTTCTAGAACCAGAGAATGGCCAGTGAGACTGGGCAGGGTGGGCCAATGGCCATCTG TGGAGCTGTACAAGA GACATTGGCCGGGCGTGGTGGTTCATACCTGTAATCCCAGCACTTTGGGAGGCTGAGGCA GATGGATCATTTGAG ATCAAGAGTTTGAGACCAGTCTGGCCAACATGGTAAAACCCCGTCTCTACTAAAAATAAA AAAATTAGCCAGGTG TGGTGGTGCATGCCTGTAGTCCCAGCTACTTGGGAAGCTGAGGCACGAGAATCACTTGAA CCCAGGAGGCAGAGG CTGCAGTGAGCTGAGATTGCACCACTGCACTGCAGCCTGGGCAACTGAGTGAGACTCTGT CTCAAAAAATAAAAA AAATTAAAAATCAAAGAGACGTGAAGAGGTCTCACCTGGTTAGCTTTTATTTTCATCATG ATAAAGTATGTATAT TACTTGAAGTTTACCATTTTATTATTTTTTATCTTACTTTATTTTTTTGAGGTGGAGTTT CGCTCTTGTTGCCCA GGCTGGAGTGCAATGGCGCAATCTCAGCTCACTACAACCTCTGCCTCCTGGGTTCAAGTG ATTCTCCTGCCTCAG CCACCCGAGTAGCTGGGACTACAGACACCTGCCACCACACATGGCTAATTTTTGTATTTT TAGTAGAGATGGGGT TTCTCCATGTTGGCCAGGGTGGTCTTGAACTCCTGACCTCAGGTGATCCGCCCACCTCAG TCTCCCAAGGTGCTG GGATTACAAGCATGAGCCACTGCGCCCAGCCATTTTAACCATTTTTAAGTGTCCAATCCA GTGGCATGGAAGTGA GTTCCCACTGTTGCGTGGTCTGGTTAATTCTGTCATACCGGTGCCTCTTTGCCGAGTCTT CAGTGTGAAAACTTC ATCTGCCCTTCTCTCTCTGTTCCCCTGCAGGCTCCAGCTCCAGCCTCTCCCTTCGGGAAA GGTGGACAGTGTTCA AGGTTCGGATGATGGCTGGGGTATGTCCCTGTGGGGCTTGCTCACCTCCAGGCCCCCCGC TTATATCTTCTGCCT TTTCCAGGGTCTGCTGTGGTACATTGTTCCCTTGGTCGTAGTTTACTTTGCCGAGTATTT CATTAACCAGGGACT TGTAAGTGAGGGGTGCTAGGAGGGGTGTGGAGGTGGCGATTGGGGCTGGGACCCACACAG CCCCGTCCATCTCCC CTGTCTGGTATTTGTTGCAGTTTGAACTCCTCTTTTTCTGGAACACTTCCCTGAGTCACG CTCAGCAATACCGCT GGTAAGAGGAGCGAGGGCAGTGGGCTGGGAGGGCGCCGTGGTGATGCAGCTGCCCTGCCC AGTAGGCACCGGGGG GAGCGGGATGGTCCCTGGAGGAGCCTCCTCTCCTCCCCCCAACCCTAACCTCAGGTACCA GATGCTGTACCAGGC TGGCGTCTTTGCCTCCCGCTCTTCTCTCCGCTGCTGTCGCATCCGTTTCACCTGGGCCCT GGCCCTGCTGCAGGT ACCAAACCCCTGCCCCTCACTTCACTCCCACCTTGGCTCCCAGCTTGGCTCCCAGCTTCC CCAAACCCCCTGCTT CCCACTATCAGTGGGAAGTGTAAATTTTTTTTTTTTTTTTTGAGACAGAGTCTCGCTCTG TCGTCCAGGCTGGAG TGCAGTAGCGCAATCTTGGCTTACTGCAACCTCTGCCTCCCGGGTTCAAGTGACTCTCCT GCCTCAGTCTCCTGA GTAGCTGGGATTACAGGCGCCCGCCACCATACCTGGCTAATTTTTGTATTTTTAGTAGAG ATGGGATTTTGCCAT ATTGGCTGGTCTTGAACTCTTGACCTCAGGTGATCTGCCGGCCTCAGCCTCCCAAAGTGC TGGGATTACAGGGAA GTGTAAATTGTACACTATCATTTCTTAGCACAGGGAACCAAAGTCCAGCAGAGCTCCATG TAAAATGTATAGCCT CAGGAGCCAAGCAAAACTGAGCTTCAGTACTCAGCCACTGTGTGACTTAGGGCAGGTCTC AGAACCTCTCTGAGC CTCAATTTCCTCATGTATAAATTGGGTGTGGCCAATACCTATTTTTCAGGGTAGTTGTAA GAAATAGAGATGAGA GAAAGAATCGAAGAACAAGATTTTGTAGTGTGTGTGTGTGTGTGTGTGCGTGTGTGTGTG TGTGTGTGTGTGTGT GTGTGTGTGTTTTAAGCATTGGGATTGGGCCGGGCCAGGTGGCTCATGCCTGTAATCCCA GCACTATGGGAGGCT GAGGCAGGTGGATCATTTGAGCTTGGTAGTTCAAGTCCAGCCTGGGCAACATGGCACAAT CACATCTCTACAAAA ATTAGCCAGGTGCAGTGGCGCACACCTGTAGTCTTAGCTACTGGGAAGCTGAGGTGGGAG GATCACTTGAACCCA TGAGGTCAAGGCTGCAGTAACCAAGGTCATGCCACTGCACTCCAGCCTAGGCAATAGAGC GAGACCCTGTCTCCC AGAAAAAGAAAAAAGAAAGGAAAAGTCGGAACCTTCCTCTGTCGCCTAGGCTGGAGTACA GTGGCATGATCATAG GTCATTTACCTCAGGCAAAGAGACAGGCTTTCAAATAATGTTTCTAACTATAAGAAAATG TACAGTTCTTTTATT CTAGATAGTTAATTATAATAGAATTGGTGACTTTGAAGTCAGTGGGTTATTTATCTTGAG GGTGGTGAGCAAATT CAACTGCCCTTGGCAAACAGTAGTTTACAGCTTTAAGGGCGCAAACACCCTTCCTTACAA CAGTCTAATAAAAGG AGTTTCATATAACCAGCATTCATTGTTTCAAGGTTACAAATCCACAGCCCCTGCAGGTAA CTGTTACAATAAACT GTCAACTGTGACAGGAAAGTCTTCTGCCTTTGAAATTTAGGAACTTGGCCAGTCCTGGTG GCTCACACCTGTAAT

CCCAGCACTTTGGGAGGCCAAGGCAGGTGAATCACCTGAGGTCAGGATCAGGAGTTT GAGACCAGTCTGGCCAAC

ACAGTGAAACCCCGTCTCTATTAAAAATGCAAAAATTAGTTGGGCGTGGTGGCACCT GCCTGTCATCTCAGCTAC

TCGCTAGGCTGAGGCAGGAGAATTGCTTGAACTCCGGATATGGAGGTTGCAGTGAGC CGAGATTGCAGTACTGTA

CTCCAGCCTGGGCCACAGAGCGAGACTCCATCTCCAAAAAAAAAGACTTAGGAACTC AATTCTGTGATAGCAAAA

TACAGAAATAGAGATTATACTTTAGGCTAGGTGTTGTGGCTCACACCTGTAATCCCA ATGCTTTTGGAGGCTAGG

GTGGGAGGATCACTCGAAGCCAGGAGCTTGAGACCAGACTGGACAACATAGTGAGAC CCCCCACCTCTACAAAAA

ATTAAAAAAAAAAATAAGCCAGGTGGGAGAATTGCTTGAGCCCAGAAGTTTGAGAGA GCAGCCTGGGTAACATCA

CGAGACCTCATCTCTACAAAAAACAACAACAGGGTGTGGTGACATTCACCTGTAATC CCAGCTACTCGGGAGGCT

GAGGCAGGAGGATCGCTTTAGCCCAGGAGTTCCAGGCTGCAGTGAGCCATAATCATG CCACTGTACCCCAGCCTG

GGTGAGACCCTATCTCAAAAAAAAAGATTACACTTTCATATTATGGCAAAAGCATGT AAATATCCTCCAGCCTCC

TTCCTTATTTATCTGAGCAGTGCTGCAAGCCCAGGGTTTGTACCCACAAAACCCCAA ATTTATAACTCAGGGTGC

TTGTAGATACCACTGAAACATGTAGCTTATGGTTTTAAGCTACATGTTTTTAGCTAT AATGTTTTTTGTTTTGTT

TTGTTTTTTGAGATGGAGTCTCGCTCTGTTGCCCAGGCTGGAATGTAGCGGTGCAAT CTTGGCTCACTGCAACCC

CTGCCTCCCAGATTCAAGCGATTCTCAAGCCTCAGCCTTCCGAGTAGCTGGGATTAC AGGTGCGCCACCACGCCC

AGCAAATTTTTTGTTTTGTTTTGTTTTGTTTTGTTTTTTAGAGACAGAGTTTTGCTC TTGTAGCTCAGGCTAGAG

TACAATGGCGTGATCTTGGCTCACTGCAACCTCCGCCTCCCAGGTTCAGGCGATTCT TCTGCCTCAGCCTCCTGC

GTAGCTGGGATTACAGGCACGTGCCACCACACCTGGCTAATTTTTGTATTTTTTAGT AGAGATGGGGTTTCGCCA

TGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCACCCGCCTTGGCCTT CCAAAGTGCTGGGATTAC

AGGTGTGAGCCACCACACCTGGCTAGAGGTGTGTCCTTTTTAATGCTCATCTTTAAG TCTACCTCAGATTTAATG

AATTAGGACCTCTAGGGTAAGCCCCGCTTGGGGCATTTGTACCTAGATCCCCAGGCA ATTCTTACATATACGAAA

AGTATGAACCGCCACGAGGAGGCCTACACACTGCACAAGAGGCAATGTGGCGTGGTC AGTGCTGGGAGGATGGCA

CCTCCTTGAGAGACTGGGGCACAGAGGTGGGACCTACACCCTGGGGTGGGGATCGAA GAGGCTTCCTGGCCAGGT

GCGGTGGCTCCTGCCTATAATCCCAGCACTTTGGGAGGCTGAGGCAGGGGGGCAGCG ACTGCTTGAGACCAGGAG

TTCGAGCCCAGCTGAATAGGATGCCCGGCTACTCCCACCTTGTTTTACCATAGTGAA CCAACGGTTTTTCCAATA

GGGGCAATTTTGCTGCCCAGCGGATGTTTGGCAACGTCTGCAGGCATGCTTTGGTTG TCACAACTGAGGGATGCT

ATGGGCATCTAGCGGGCAGAGGCCAGGGATCCTGCTAAATGCGCTGCCATATACAGG ACAGCCCTCACCATGCAG

AACAACCAGCACCAAACGCCAAGAGTGCTGCAATTGAGAAATCCTGGTTTCGATGGT CCCACTTCCTTTAAGAAG

CCTCCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTATGAGACAGGATCTTGCTCTGT CACCCAGGCTGGAGTGCA

GTGGCACAATCATAGCTTACTGCAGCCTCCACCTCCTAGGTTCAAGCAATCCTCCTG TCTCAGACTCCCGAGTAG

CTGGGACTGCAGGTGCGTGCCACCATGCTCAACTAATTTTTAAATTTTTTGTAGAGA CGGAGTTTTGTTATGTTG

CCCAGGCTGGTCTCGAACTCCTGGCCTCAAGTGATCATCCCACCTTGGTCTCCCAAT GTGCTGGTATTACCTGCA

TGAGCCACTGCACCAGGCTGAGGTTGAGAATTTTTTTTTTTTTTTTTTGGGATGGAG TCTCTCTCTGTTGCCCAG

GCTGGAGTGCAGTGCTGCGATCTTGGCTCACTGCAGCCTCTGCCTCCCAGGTTCAAG TGATTCTCCTGCCTCAGC

CTTCCAAGTAGCTGGGACTACAGAACCACTACACCCAGATAATTTTTGTATTTTTAG TTGAGATGGGGTTTTACC

ATGTTTAGTAGAGACCAGGCCAGGCTGGTCTCGAAATCCTGACCTCAGGTGATCTGC CCACCTCAGCCTCCCAAA

GTGCTGGGATTACAGGTGTGAGCCACCACACCGGGCTGGTGTTGAGATTTTATCTAG ACCTGGCAGCCCTCTCCT

TCACCAGTAACTCCTAAAACCAGGGACCCCTGGAGGGGAGGCCACCTCTCCTTCCCT GCCCCGCCCTGGTCCCAG

GCTTAGCCTCTCCCCCTGTTCACAGTGCCTCAACCTGGTGTTCCTGCTGGCAGACGT GTGGTTCGGCTTTCTGCC

AAGCATCTACCTCGTCTTCCTGATCATTCTGTATGAGGGGCTCCTGGGAGGCGCAGC CTACGTGAACACCTTCCA

CAACATCGCCCTGGAGGTCAGCATTGGCCGGGCAAGGGCTGGGGGTGGCCTGTCCAG GGACACCCAGGGCAGGGA

TGTCTGGGACTGAAGCCTCACCCCTGCTCTCTGCCCTCCCAGACCAGTGATGAGCAC CGGGAGTTTGCAATGGCG

GCCACCTGCATCTCTGACACACTGGGGATCTCCCTGTCGGGGCTCCTGGCTTTGCCT CTGCATGACTTCCTCTGC

CAGCTCTCCTGATACTCGGGATCCTCAGGACGCAGGTCACATTCACCTGTGGGCAGA GGGACAGGTCAGACACCC

AGGCCCACCCCAGAGACCCTCCATGAACTGTGCTCCCAGCCTTCCCGGCAGGTCTGG GAGTAGGGAAGGGCTGAA

GCCTTGTTTCCCTTGCAGGGGGGCCAGCCATTGTCTCCCACTTGGGGAGTTTCTTCC TGGCATCATGCCTTCTGA

ATAAATGCCGATTTTATCCATGGACTTCTTATATCGTTTTTGTCTCTAAAAAGAAAC TTTTATTATGAAAGTAAT

ACATGCCCACTTTCCTATATATAGACAGCATAAAGAAGGTATGTCAGCAGATTTCTC CCTTTTTTGTTTGTTTGT

TTGTTTGTTTGTTTGTTTTGACAGAGTCTCACTCTGTCACCTAGGCTGGAGTGCAAT GGCGTGATCTTGGCTCAC

TGCAACCTCCACTTCCCGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCGAGTAGCTG GGATTACAGGTGGTCACT

ACCATGTCTGGCTAATTTTTATATTTTTAGTACAGACGACATTTTGCCATGTTGGCC AGGCTGGTCTCAAACTCA

TGACCTCAGGTGATCCGCCCACCTTGGCCTCCCAAAGTGCTGAGATTACAGGTGTGA GCCACTGTACCTGGCCTC

TGCCATCTTTGAGGCCTTACGTATTCATTCATTCATGCATTCATTGTTTGGGACAAT CTGTAAACAATCATGTTG

GACATTCTTTCCAGCATGAAAGTTTTCAGAGAAGTGGAAAGAATTGTGCAGTGAATC ATCCTGAGTATGCGCCAC

CCTGTTCTATGTTAACATTTTGTTACAGTTGTTTTATCATGTAGTCCAGCCCCCATC AAATCTATGTATTCACTG

TTCCATCAGTGAGTAGATAGAGCTAATGAATGGCAGGGTTGGCACCTGAGGCATTTT ATTTTATTATTTTTTTTT

_{ATTTTTAGAGACAGGGTTGCACTCTGTCACCCAGGCTGAAGTACAGTGGCACAGT CATGGCTCACTGTAGCCTTG}

ACCTCCTAGACTCAAACAATCCTCACACCTTGGCCTCCCAAGCAGCTGGGACTGCAA GTGCATGCCACCAAGCCC

AGCTAATTTTTTTTTTTTTTTTTGTAGAGACGCGATCTTCCTATGTTACCCAGGCTG GTCTTGAACTCTTGAGCT

CAAGCAGTCCTGCCTTGGCCTCCCAAAGTACTGAGATTACAAGCATGAGCCACTGCT CCTGGCATCCTGTTATTT

AGGTACAGTTTTTATACACAGTAAAATGTATAAATTGTTACATATTTTTACAGTAAA CTGAGTCTGGGCCAGGCA

CTGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCAGATCACCT GAGGTCAGGAGTTTGAGA

CCAGCCTGCACAACGTGACAAAACCCCGTCTCTACGAAAAATATAAAAAATTAGCCA GGTGTGGTGGCTTGCACC

TGTAGTCCCAGCTACTTGGAAGGCTGAGGCGCAAGAATTGCTTGAACCCAGGAGGCG GAGGTTGTGGTGAGCTGA AATTGCACCACTGCACTCCAGCCTGGGCAACAGAGCAAGACTCTGTCTCAAAAAAAAAAA CAAAAAAACTGAGTC

TGACTTAGTGTTTCTCATACTTCATTGTTACCCTTTCTACTATATTTGCCATATCTG AACCTAACAGTACTTTTA

TTTACTTAATATTTTTCATTAAAGCAACTAACCTTTAATATACATTCAGTTATTTTA AAGGGAAACATTATTAAA

ATCATGAGTTTGAGGTGATATATATGTATTTTCTACTTTACTTTAAATAGTTATGTA ACTATTGGAGTTTAAAAT

GAACATGCAAGTTCCCCCTGAGACTACTCCATTACGACCAGTGATGTTTGTGGACCC AGATCTAGGAAATGCTGA

CCCACTGGTTAATAGCATGGGCTTTGGGGGCAGAAGGAACTGACTTTCAGGTGCCAA CCCTGCCATTCACTAGCT

CTATTACCTTGGGCAAGTCACTTCACCTCTCTGCACCTCAGTTTCCTGATCTGTCCA ATGGGATAATAGTAGCAC

CAGCATTTCATAAGTTGCTTTTGGAAACTACATGAGATCATGTCAACACTCAATAAA CATCAGCTCTGATCATGC

TATGATATGACTCCTGCCTCTCACCTTTGCATTGCATTATACCCGTGTTTCTCAAAC CTTAGTGTGTACTGGAGG

CCTTGTTTTTTGTTTTTGTTTTCTTGGAGACCAAGAGTCTTGCCCTGTCACCCAGGC TGGAGTACAGTGGCATGA

TCATAGCTCACTGCAGTCTCAACCTCCTGGGCTCAAGGGGTCCTCCTGCCTCAGCCT CCTGAGTATCTAAGACTG

CAGGCACGCACCACCATACCTGGCTAATTTTTATATTTTTGGTAGAGATGGGGTTTC ACCATGTTGTCCAAGATA

GTCTTGTACTCCAGAGCTCAAGTGATCCACCCACCTTGGCCTCCCAAAGTGCCAGGA TCACAAGTGTGAGCCACT

GCACCTGGCCCTGCCTGGCTAATTTTTATTTTTTTTCTGTAGAGACGAGGTCTTGCT TTGTTGCTCAGGCTGGAC

TGGAACTCCTGGCCTCAAGTGATCCACCTGCTTCAGCCTCCCAAAGGTCTGGGATTA TTACCCATGAGCCACCAC

ACCCAGCCACAATATTTTATTTATCCATTTGTTAGTTGATGGATATTGGGGTTTCAA TTTTTTGACTTAACTATG

AATCATGCTGCTATAAATGTTTGGGTAAAGTTTTTGTGTGGACACATGTTTTCCATT TCTCTTGGATAATATCAA

GGAGTGGAATTGCTGGGTTGATGGTAATTTTGTTTGATCCTTTGAGGAACTACCCGA CTGTTTCCCAAAGTGGCT

GCAATTTTACATTCCACTGGCAGTGTACGAGGACTCCACGTCCCCACCAATACTTGC TCTTTTCCATTCTTTTTC

CACCGCAACCTCCGTCTCCCCGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCTCTTT TTTTTTTATTTTTGAGAC

AGTCTCGCTCTGTTGCGCAGGATGGAGTGCAGTGGTGCTATCTCGGCTCACTGCAAT CTCAGTCTCCTGAGTTCA

AGTGATTCTCCCACCTCAGCCTCCCAAGTAGCTGGGATTACAGGCATGCGCCACTAC ACCTGGCTAATTCTTTTT

TGTTGTTGTTTTTTGTTTTTGTTTTGTTTTGTTTTTTCAGATGGAGTCCTGCTCTGT CACCAGGCTGGAGTGCAG

TGACGCAGTCTCTGCTCACTGCAGCCTCCGCTTCTGGGTTCAAGCTATTCTTCTGCC TCAGCCTCCCAAGTAGCT

GGGATTACAGGCACCCACCACCACACCTGGCTAATTTTTGTATTTGTTATGGAGACG AGGTTTCGCCATGTTGGC

CAGGCTGGTCTCCAACTTCTAAACTCAGGTGATCCACCGGCCTTGGCCTCCCAGAGT GCTGGGATTACAGGCATG

AGCCACCGTGCCCAGCCTCTTTTCCGTTTTTGTTTTTTTTTTTTCCTTTGGTTGTCT GGTTTGGTTTTTATTATA

GCCTTTCTAGTGAGTGTGAAGTGGTATCTCACTGTGGTTTTGATTTGCATGTCCCTG GTGACTAATGATGCTGAG

CATCTTTTCATGTCCTTATTGACCGTACATATAGTTCTTTGAGAAATGTCTTCTCAG ATCCTTTGCCCATTTTTA

AATGGGATTGTCTTTTTTTATTAAAGAGCCAGAGATCTGAAGTCTTTTTGAGTTGTA AGATTTCTTCATATATTC

TAGACAGAAGTCCCTTATAAGATACATGATTTGCAAATATTTTCTCCCATTCTGTGT TGTCTTTTTACTTTTTTG

ATATTGTCTTTTGGTACGGAAAGACACTTATTTTGATGAAGTCCAATTTATCTATTT TTTGTTTGGTTGCTTGTG

CTTCTGGTAACATATCTAAGTGGGAGCCAGGTGCAGTGGCTCATGCCTGTAATCCCA GCACTTTGGGAGGCCAAG

GCGGGAGGTTTGCTTGAGACCAGGAGTTCAAGACCAGCATGAGCAACATTGCAAGGT TCTATCTCTATAAAAAAT

TAAAATAATGATAAAAGGAGGCCGGGCGTGGTGGCTCATGCCTATAATCCCAGCATT CTGGGAGGCAGGAGGACA

ACTTGGGCCAGGAGTTCAAGACCAGTCTGGGCATCATGGCAAAAAAAGAAAAAAGAA AAAATTATCCTGGCAAGG

TATGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCGGGAGAATTGCTTGAGCCCGA GAGGTGGAGGTTGTAGTG

AGCTGAGATTGAACCATTGCACTCCAGCCTGGGTGACAGGAATGAAACCCTGTCTCA AAAAATAAAAATAAAATA

AGCCAGGTGTAGTGCTGCGTGCCTGTACTCCCAGCTACTCTGGAGGCTGAAGTGGGA GGATCACTTGAGCCTAGG

AATTCGAGGCTGCAGTGAGCTATGATCATACCACTGCACCCCAATGTGGGCACAGAG CAAGACCCTATCTCTTAA

AAAACAAACAAAGAGGGGCGGGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTG GGAGGCTGAGGCGGGCAG

ATCACGAGGTCAGGAGATCGAGACCATCCTGCCTAACACGGTGAAACCCCATCTCCT TTTTTTTTTTTTTTTTTT

TTGAAACAGAGTTTCATTCTTTTTGCCCAGACTCCCGGGTTCAAGCGATTCTCCTGC CTCAGCCTCCTGAGTAGC

GGGGATTACAAGGGTGTGCCACCACACCCAGCTAATTTTTTATATTTTTAATAGAGG CGGGGTTTCACCGTGTTG

GCCAGGCTGGCCTCAAACTCCTGACCTCAAGTGATCCGCCCACCTCGGCCTCCCAAA GTGCTGGGATTACAGGCG

TGAACCAGCGCGCCCAGCCTCCTACATTTTCTTCTAAGAGTTTTATAGTTTTAGCTC TTACATTTAGATCTTGTT

TTCATTTTGAATTAACTTTTTTGCAGGTGGTGTGATACAGCAGTCTGTGCAGAAAAA AGTTAACATAGTAGACCC

TAACTGCTAACTTTATTTTTACTTTTATTTTTTATTTTGTTTAGAGACAGGGTTTTT GTCACCCAGGATGGAGTG

AAGTGGCGTGATCATAGCTCACCGTAACCTTGAACTCCTGGGCTCCTCTGCCTCAGC CTCCCAATTAGCCACCAC

ACCCAGCTACCTTTTGTACATTTTTTTGGTAGAGACGGTGTCTCACTATGTTGCCCA GTCTGGTCTCAAACTCCT

CAGCTCAAATGATCTGCCCACCTTGACCTCCAGAGATACTGGGATTACAGGCGTGGG CCACTGCACCAGACCCAG

GTTTATTCTTACACCAATACAACACTGTCTTAATTACTATAGCTTTGTAGTGAGCTT TTGAAAATAGGAAATGTG

AATCCTCCAACTTTTTTTTTTTGGAGACGGAGTCTCGCTCTGTTGCTCAGACTGGAG TGCAGTGGCACTATCTCA

GCTCACTGCAAACTCCATCTTCCAGGTTCAAGTGATTCTCCTGCCTCAGCCTACCAA GTATCTGGGACTACAGGC

AGGTGCCACCATGCACAGATTCCTTGGACATCCCTGAGAGGTCAATCATGAAGGTCA ACTTGGTTTTCTCCCCCT

CATTTGGGTTCAGAATTTAAAGTCCACACACACAGGCAGTAAGATGATTATAGATAA GGACATCATCACTCGGTT

TCGGATGTTAAATTGTCTAGGTGGGTTAGGGGTGATTTGAGATCACACAACCTTGTG CCACAAAGAGGAATTCCC

AGGCCAGAGGGAGACATTTTATTGCCATGTTATGATCTTATCATTGAGTTGAAAGGC AATCTTGTTTCATTTTGG

ATTCTTTCTTATGTTTATGTCTTATAAGGGCACTTTGAATTTCCAAGCAAATAATAA TTTTGAATTAGCTTTTAA

TCATTGACTTCTAGCACAGTTATATGATCAGAAACGTGCTGTGTGATTTGATTGCTC TCAAATATATTGAGATTT

GCTGGAACAAAATAAGTCAGGTTAATTTTTGTAAATGTACCATGCTTGCTTAAAATG AATGTATCTACATTTGTT

CCTGAGATACGGGTTGATGGACGGATGGCTACATGGATGTGATGGAGATGGTTTACT ATCGGGACCTTCCGCATC

CTGCTGATGTTTTGTTGCTTAGGATATGAATGGCTGAGCGGAGGCTGTAAAACCTGG CACTCTGCTTGGGTATGA

GGTTCTTCCTGCCATCCTGCCATCATTTGTTTTTTATGTTTTGTCGCCAAAAGTGAC CTTGAGGAACCCTGGGAG CTCAGGAAGGAAGGAGCGCCCAGAAGCAGGGACAGGGAGCTGGTTGGGGAGGACCAGAAA TCAGGTTTGTGAAGG

TTCCAGAGAGGACCTGTCCTTGCGAGGAGTGTGGGAGACTGAGATGGGGGAGGGGTC ATTGGAATGATGCGGGCG

CTACTTGGCATTGTCCATTGTGAGGCACCACCGGGGTCATCAGGGATTGGTGGAGAG GGAGTATAAAGCCCCAGG

TTTGCTAAGGGAGGGCCCAGACCGAAGAAGGTTTGGCGGATAGCAGAACCTTTTTGT CTCCCTCTAATTGCTCCT

AAGCCTCACGCTCCCTTGCCCCGCGTGTCCTGTTGCTTCCCTGATCTTCTCCGTGAC CTGTAGCTAAACCTTCCA

CCAGCGCTTGAGAACTTAATTTGAACCGGATCCTTTCCCAGACCCCTTTCTTCTTCT CCTCCTCCACCTCCTCCA

GGTGCCCAACAGCCCCCTTCTCCTCCTTTCCCTTCCCTTACTTCCCCCCTTCCCCTC CCCTTCCCCTCCCCCTCC

CCTCCCCTTCCCCTCCCCTTCCCCTTCCCCTCCCCCTCCCCTTCCCCTCCCCCTCCC CTTCCCCTCCCCCTCCCC

TCCCCCGCCCCAACTCAGATCCGGCCCCGGTCCCCGTCCCCTTCCCTCCCCCCTGCC CTAAGCCACCTCCACCTC

TGTCCTGGCCGCCTCAGGGCACCCTGAAAGGATCAGGACATGCGGCTGCGCTTTTGG CTCCTCATTTGGCTCCTG

CTGGGATTTATCAGCCATCAGCCCACCCCTGTGAGTAGACGCTGGACCCGCGGGGTT TCTTCCTTTTTACTGGGC

TGTGTCACGCGGCATGAAATTACACAGCTCAGGCCTGTAATCCCAGCACTTTAGGGG GCCGAGGTGGGCAGATCA

CTTGAGTCCAGGAGTTGAAGACTAGCCAGGGCATCATGGCGAAACCCCATCTCTACA AAAAATTCCAAAAAAGAT

TAGTCGGGCCTGGTGGTGCGTACCTGTTATCCCAGTTACTGGAGAGGCTGAGGTGGG AGGATCGCTTGGGCCGAG

GAGCTGGACGTTGCAGTGAGCTGAGATGGCCCCGCTGCACTCTTGTCTCTAACAAAC AAAATGGACCAAAACAAA

GTGAAATGTCATTTGATTTGTGTCATCTGGTTTGATGACTTTTTTTTTTTTTTTTTT AGACAGAGTCTCACTCTG

TCGCCCAGGCTGGAGTGCAGTGGCAAGATCTCGGCTCACTGCAACCTCCGCTTCCGG GGTTCAAGCAATTGTCCT

GCCTCAGCCTCCTGAGTAGCTCAGATTACAACGCCTGGCTAATTTTTGTATTTTTAG TAGAGACGGGGTTTCACC

ATGTTCGCCAGGATAGTCTCCATCTCTTGACCTCGTGATCCGCCTGCCTCGGCCTCC CAGTGCCGGGATTACAGG

CGTGAGCCACCGCGCCTGGCCAAAATATATAACCTTAAGTGTAAGTTTACTAACTTT GGAAAGTACATACACCAG

CATAAACCGACCCCCTTTCAAGATCTACATTATTTTATTTATTTATTTATTTTTTTG AGACAGTTTCTCCCTTGT

TGCCCAGGCTGGAGTGCAATGGGGCAATATCAGCTCACCGCAACCTCTGCTTCCCAG GTTTGAGCGATTCTCCTG

CCTCAGCCTCCCGGGTGGCTGGGATTACAGACATGTGCCACCACTCCCAGCTAATTT TGTATTTTTAGTAGAGAT

AGGGTTTCTCCATGTTGGTCAGGCTGGTTTTGAACTCCCGACCTCAGGTGATCCGCC CGCCTCGGCCTCCCAAAG

TGTTGGGATTACAGGCGTGAACCACCGTGCCCAGCCAAGATCTACACTATTATGTCA CCCCAGAAAGTGAACTCT

CAGTCTTCCCAGCCAGTCTCTTTCTTATCATAGGTTAGCTTGCTTATTCTGGAATTT CGCGTATACAGATGCGTG

CCATGCCATAGGTACTCTTTTGTGTCTGCTTTGTTCTGCTCAACACCATGTTTCTGA AATCATTACCATTGTTGT

ATGGTTCTCTAACTCCATCATTTCCATTTCAGACTCAGCATATGCTGAGTTCAACCT GTTGAAGGGCTATCTCTG

TTTAATTCACCATCTTGAAAGAAACATTTAAAATTGAGATGTTTTCAAGAATATATA GTTAAATCCTGAGGAATC

GATGTAGAAATGTTATCACAAGCTGTCTGAACTTACTCAGGGGAAGTCTTCGTCTTC ACTCACATAAGAGTCTAC

TGGAATTAATATCAACAATCTTAGAGAAATCCCACACTATTCATGCCATTTTCATGA TCTCCACCTTGGTAATTT

_{TTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTCACTCTGTCACCCAGGCTGAAG TGCAGTGGTGCGATCTCGGC}

TCACTGCAACCTCTGCCTCCTGGGTTCAAGTGATTCTTCTGCCTCAGCCTCCCAAGT AGCTGGAACTGTAGGCAC

GTGCCACCATGCCCTGCTAATTTTTTGTAATTTTAGTAGAGATGGGTTTCACCGTGT TAGCTAGGATGGTCTCAA

TCTCCTGATCTCGTGGTCCACCCACCTCGGCTTCCCAAAGTGCTGAGATTGCAGGCG TGAGCCACCACACCCGGC

CCACCTTGTTAATTTTTAAGCACTAAAATTTGATACTTATTTGTGAATGAAGTAATC TCTTCATTGTATTTTTTT

TTTTTACTTATGCTGAGCTTCAAATGACAAAGATTCATATAATCCAAGAGAGAAGTA TTATTTAGAGGGATTCTT

TTACCATGTGATATATAATAAATGCATCCAATGTTATACATCAATTTAAAAAACAAG TAAATAACTTTAAAGAAA

AGATAACTACTGGCCAGGTGCAGTGGCTCACACCTGTATTCCCAGCACTTTGGGAGG CCGAGGCAGGTGGATCAA

GAGGTCACGAGTTGGAGACCAGCCTGGCCAAGATGGTGAAACCCTGTTTCTACTCAA AATACAAAAATTAGCCGA

GTGCGGTGGCAGGCGCCTGTAATCCCAGTTACTCAGTAGCTGAGGCAGGAGAATCGC TTGAACCCGGGAGGCGGA

GGTTGCAGTGAGCTGAGATCATGCCACTGCAATCTAGCCTGGGTGACAGAGCAAGAC TTTGTCTCCAAACAAAAA

GAAAAGATAATTACTTTATACTTAGCTTGTCTTAGCCATGAGTGACGGGCTGCATGT GGCCCAGGACAGTTTTGA

ATGCAGTTCAACACAAATTTGTAAACTTTCTTAAAACATTAGGAGATTTTGGCCAGG TACAGTGGCTCATGCCTG

TAATCCCAGCACTTTGGGAGGCTGAGGCGGGCAGATTACCTGAGGTCAGGAGTTCGA GACCACCCTGGCCAACAT

GGCAAAATCCCATCTCCACAAAAAATACAAAAATTTGCTGAGTGCATTGTCAGGCAC CTGTACTCCCAGCTACTC

AGGAGGCTGAGGCAGGAGAATCACTTGAACCTGAGAGGCAGAGGTTGCAGTGAGCCG AGAGCACGCCACTGCACT

CCAGCCTGGGTGACAGAGTGAGACCCCATCTCAAAAACAAAACACCAAACAAAAACA AAAACAAAAAAAAATGGC

TGGGCACGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCAGA TCGCCTGCCAGGAGTTCA

AGGCCAGACTGGCCAACATGGTGAAACCTCATCTCTACTAAAAATACAAAAATGAGT CGGGCATGGTGGCAGAGA

CCTGTAATCTCAGCTACTCGGGAGGCTGAGGGAGGAGAATGGCTTGAGCCCAGGAGC TGGAGGTTGCAGTGAGCC

GAGATTGCACCACTGCACTCCAGCCTGGGCGACTGAGTGGAGCGGAACTCTGTCTCC AAAAAAAAAAAAAAAAGA

_{GTTTTTTTTTAGATCATCAGCTATTGTTAGTGTTAGTGTATGTTATGTGTGGCTC AAGACAACTTTGTTTCTTTT}

AATATAGGCAGGGAAGTGAAAAGATTGGATATCCCTGCTTTATACCAAGAAAGACAA CACCCCACATTTGCAATG

CCTAAAAACACTACCAGCCATCTGAAAAACATGAGACTTCTAACTTCTGTTCTTTTT TGTAGCAGTGGAATCCCA

CGGTGATATCTGAGGGATGTGGTTACCTTTTGGAGGAGGTTGACGGTTTCTAAGGAT GATTCTTTCTGAGTGAAA

TATTGTCGGTGTCATTGACCTTTTCATTATTTCAACTATTATTATTCCAGGTTATCA ATAGTCTGGCTGTCTATC

GTCATCGTGAGACTGACTTTGGTGTAGGAGTTCGAGACCACCCTGGCCAACATGGCA AAACCCCATCTCCACAAA

AATTGGATAATTTGATAATTATCATTATTGGGTTTCTGAGACGTTACACATTTAACG TTCTCTTCTGCACAAGTT

GCCTTTGTGTGAGTATACTAACTTTCTGTAGAGGTATACTTGTAATCACAAATAAGA ATAAATTATATAAAACAA

Intron 4: Homo sapiens CLN3 (849 nucleotides in length) GTGCGTACTCATCTCACCTGGTCCTTGCCTGACCCAGGGCCCCTGGGTCTATAGACCCCA CTCCCAGCCCTTCAC TACCCAGCTGGGACTGTCATGATTAAAACAGTTGAGACCTGGGCTGGGCGCACTGGCTCA CACTTGTAATCCCAG CACTTTGGGAGACCAAGGTGGGAGGATCACTTGAGCCTAGGAGTTTGAGACCAGCCTGGG CAGTATAGTGAGACC CCCATCTTAAAGAAAAAAATTAAAAAATATATATATATAAAATAATTGAGACCTTAATAT AATTTCTGAGGCTGA GGCGGATGGATCACTTGAGACCAGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAACCC CATCTGTACTAAAAA TACAAAAATTAGCCAGGCATGGTGGTGCACGCCTGTAATCCCAGCTACTCAGGAGGCTGA GGCAGGAGAATCACT TGAACCCAGGAGGTGGAGGTTGTAGTGAGCCAAGATCGAGCCACTACACTCCAGCCTGGG TGGCAGAATGAGACT CACTCTCAAAATATATATATATATAGTTTCTGAGTCCTTTCTGTCTGCACCATATATTAG CTCATTTAAGCCACA CAGCAGTCCTGTGAGCTAGGTGCTATGATATTCCCATTTTCCAGATGAGGAAACTGAAGC TCAGAGAGTATAAAC TCTTGACACACAACTAAGGAGTGGGAGAGCTGAGACTTGAACCCAGGCGTGCCTGACTCC AGAGCCTGTGTTTGT AGCAGGCCTGTTTGGCCAGCTCCTGCCTCTCCTTGGCCACGTGGTTGGGAGGGTTGTCCC CTGGAAGCTCTGCGG TCTCACTCTATTCTCCTGTCCCAG (SEQ ID NO: 3)

Exon 5: Homo sapiens CLN3

(80 nucleotides in length)

GCTGTGCTCCTGGCGGACATCCTCC TACAG (SEQ ID NO: 4)

Intron 5: Homo sapiens CLN3 (120 nucleotides in length)

GTCTGGGTGAGGGTAGTGGGAGGCAGGGTGGGCAGGAGCTGAGAAAGGGGAGGCTGG GATGGCTGAGATGCTGAG AGTAGAGACCGACCTTCCCCCTCCCTTCCCTTCTCACCCCCTCAG (SEQ ID NO: 5)

Exon 6: Homo sapiens CLN3

(86 nucleotides in length)

CCCCCGGGTTCTCGTCAGTGGGATTTG CAGCCTGTGTG (SEQ ID NO: 6)

Intron 6: Homo sapiens CLN3 (805 nucleotides in length)

GTGAGTGTGTGGTTCTGTGTCAGATGGGGAGCCCCGAGGAACCACATCAGAGCATTT GTGGGAAGAGTCTCCCCA GCCTCCCAGAGGAAAGGGATTCATTCTGTCACCCTTAGAAGCCTGCTAGGGCTATCAGCA GTAGGCGATGGGAGA CTGGGACAATTTGGAGGGGTAGGCAGTGGAGGAGATGGGAGAAAATGGATGAATTAGATG GAGATTGAGGTGAAC AAAGTCAAGACTCTGTGATGGACCAGGCACAGTGACTCATGCCTATAATCCCAGCACTTT GGAAAGCCAAGGCAG GCAGATCACCTGAGGTCAGGAGTTCGAAACCAGCCTGGCCAACATGGAGAAACCCCGTCT CTACCAAAAATACAA AAATTAGCTGGGTGTGGTGGCAGGAGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAG GAGAATCTCTTGAAC CTGGGAGGCAGAGGTTGCAGTGAGCCGAGATCACGCCACTGTACTCCAGCCTGGGTGACA GGGCGAGACTCCGTC TCCAAAAAGAAAAGAAAAGAAAAAGACTGATGAAGGGGCAGAGACATCAAGGGTGCATGT CTGCCCCTGGTCTGA TAACTGGGTGGATGGAGGTGCCACTCTCCATGAAGGGACACGCAGGGGAGTGGGGCTCTG CTTCAGACCTGGAAC CTGGCCTATGCATGGGATCTATTGGAGCCTCTATGAGCTGATACTGAGGAGGCCATGGCC AGACACATTAGAGGC CTGGGCAGTGTGGCAAGGTGTGGTGTGACCATCCCAGTGCTTGTCCTCCCCCCAG (SEQ ID NO: 7)

Intron 8: Homo sapiens CLN3 (2228 nucleotides in length)

GTGAGCTGCCCTGAGCCGGGAGGGAGAGGGGTCCAAGGAGAGAAAACTTGGCCATGG CTGGGTGTGGTGGCTCAC

GCCTGTAATCCCAGCACTTTGGGAGGCCAAGGAGGGCAGATCGCCTAAGGTCAGGAA ACCAGCCTGGCCAACATG

GTGAAACCCCGTCTCTACTAAAAATACAACAATTAGCCAGGTGTGGTGGCGGGTGCC TGTAGTCCCAACTACTCA

GGAGGCTAAGGCAGGAGAATCGCTTGAACCCCGGAGGCAGTGGTGGCAGTGAGCCGA GATCGTGCCATTGCACTC

CAGCCCGGGCGACAGAGTTAGACTCTGTCTCAGGAAGAAAAAAAAAAAAAGAAAGAA AAGAAAACTTGATTATGA

TTGCAATCTTCAAGTCCCTACCTTGCTGTGAAGGGAGGCGGAATCTGGACTCTGATA GCCCCAGGTGTGAGTCCT

GGAGCTGCCACTTTTTAGCTTTGTAGCGTTGAACAAGTTACTCCACCTCTCTGAACC CTCAGTTTCCCCATATCT

CAAATGGCAGTTGTTCTTGCTTTCCTTGGAGGTGATGAGGGTAATGCATTCAGCACA GTGTGGTTCCCAAGGTGA

TTAGAAGTAGGATGAGGGTGGACTTTATTTGATTAGTTCCTTTTTTTTTTTTTTTTT TTTTTAATCAGTGTTGAC

C

_T A TG TG TT TT TG TG TC TC TT TC TG TA TA TT TG TT TG TT TA TG TC TC TT TT G _A GC G _A CT CG GC G _A CT GG TA CG TT C _A GC TC GA GG TG GG TC CA AA CC TA C _A GG GC GC CT TG GA GA AC GC TA GT C _A GG GC TG GA AC CT TC GC GC TT GT TT G ACAGGCCCATGCCACCATGCCTGGCTAATTTTTTATATTTTTAGTAGAGACGGGGTTTCA CTGTATTGGCCAGGC TGGTCTCAAACTCCTGACCTCAAGTGATCCACCTGCCTTGGCCTCCCATAATGCTAGGAA TACAGGCGTGAGCCA CCGCGCCCGGCCTGATTAGTTCTGTGTATTTTCATGCATATTACAAAACACTTTGGCCGG GCATGGTGGCTCACA TCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGCACCACGAGGTCAGGAGTTTGAG ACCAGCCTGGCCAAT ATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGCTTGGTGGCGCTTGCC TGTAGTCCCAGCTAC TTGGGAGGCTGAGGAGAAGAATCGCTTGAACCCAGGAGGCAGAGGTTGCAGTGAGCCGTG ATTGTGCCACTGCAC TCCAGGCTGGGCAACAGAGCGAGACTCCGTCTAAAAAAAAAAAAAACACACACTTTGAAC TAGCCAGACACACCT GGCCCTTCACAAGATTAGTGCTTAGGACAAGTCTAAGTAGAAAAAGTGAGTCCATTTAAA GAAAAATATTAAGTA AAAATATATATATACAGGAGGAATATGGATATGGCAAATAACATGAAGCCAGAACCTAAG TGACTAAAATCGAGG AAGTTCTGGCCTAGCACAGAGTGAACGTATAATTAATGGGAGCTAGAGCGACCATTAGAA TAATAATGGCCAGAA AACAGAACTGACTCGCTAGGTATGAGCCAGCAGTCCGAACAAGGGCCTGCTGAGCACAGT GGCTCACAGCTGTAA TCCCAGTGCTTTAGGAGGCTGAGGCTGGAGGACCACTAGAGGCTAGAAATTTGAGACCAG CCTGGGCAACATAGC GAGACTCCATTTATACAAAAAATGGAAAATATGAGGCGGGCTTGGTGGCACGTGCCTGTA GTCCCCGCTACTTGG GAGGCTGAGGCGGGAGGATTGCTTGAGCCTGGGAGGTCGCGGCTGCAGTGAGCTATGATT GCACCACTGCATTCC AGCCTGGATGCTGGAGAAAGACCCTGTCTCTGAAAAAAATCAAAACAAAATGAAGGCCCA TGAATAGGAATAAGG AGATAAATTCAGGTCCAAAGAAAGAAGCCTTTGTCCACAATTGGAGCTCTCCCGCACAGC GTAGGAGCCTTAGAG GCAGTGAGCTACCCATCTTTGAAAGTGTTCAAAGGTAGAGGTGTTCCCTGGGAAAAGTGG CCTAGATGGTCCCTG GGGACCTTCCCAGCCAGTAGGGTTTTCTGACCCTGCCTTCATCCTACTCCTAG (SEQ ID NO: 8)

Exon 9: Homo sapiens CLN3

(86 nucleotides in length)

CTATTTCTTGTTGCTCACATCTCCTGAGGCCCAGGACCCTGGAGGGGAAGAAGAAGC AGAGAGCGCAGCCCGGCA GCCCCTCATAAGAACCGAGGCCCCGGAGTCGAAGCCAG (SEQ ID NO: 9)

Intron 9: Homo sapiens CLN3

(1333 nucleotides in length)

GTAGGAGACACAGACCCTCAGAGAGGTCACTTTCTTTCTCTCTGGGTTTGGCCTTTT CCTCTCTGCAATAGGCAA AGTTAAGAGGGGAAAGAGAGTGAGTGTACTGGTTATGGGAAAAGCCTTTGTCTATTTGAG AAATACTTGTTGAGG ACGAGCACAGTGGCTCACGCCTGTAATATCCCAGCACTTTGGGAGGCTGAGGCAGGAGGA CCACTTGAGCTCAGA AGTCTGAGTCCAGCCTGGGCAACAGAGCAAGACCTTGTCGCAAAAAAAAAAAAAAAAAAA AAAAAAAGAAAAGAA AGGAAGGAAGGGAGGGAGAGAAGAAGGGATATTAATTGAGTACTTACCATGTGCCAGGCA TTCCAATCACAAGTG CTGAGGCCCTGCGGTGGGGATAAGCTGGGATGTTCTAGAACCAGAGAATGGCCAGTGAGA CTGGGCAGGGTGGGC CAATGGCCATCTGTGGAGCTGTACAAGAGACATTGGCCGGGCGTGGTGGTTCATACCTGT AATCCCAGCACTTTG GGAGGCTGAGGCAGATGGATCATTTGAGATCAAGAGTTTGAGACCAGTCTGGCCAACATG GTAAAACCCCGTCTC TACTAAAAATAAAAAAATTAGCCAGGTGTGGTGGTGCATGCCTGTAGTCCCAGCTACTTG GGAAGCTGAGGCACG AGAATCACTTGAACCCAGGAGGCAGAGGCTGCAGTGAGCTGAGATTGCACCACTGCACTG CAGCCTGGGCAACTG AGTGAGACTCTGTCTCAAAAAATAAAAAAAATTAAAAATCAAAGAGACGTGAAGAGGTCT CACCTGGTTAGCTTT TATTTTCATCATGATAAAGTATGTATATTACTTGAAGTTTACCATTTTATTATTTTTTAT CTTACTTTATTTTTT TGAGGTGGAGTTTCGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCGCAATCTCAGCTCAC TACAACCTCTGCCTC CTGGGTTCAAGTGATTCTCCTGCCTCAGCCACCCGAGTAGCTGGGACTACAGACACCTGC CACCACACATGGCTA _{ATTTTTGTATTTTTAGTAGAGATGGGGTTTCTCCATGTTGGCCAGGGTGGTCTTG AACTCCTGACCTCAGGTGAT} CCGCCCACCTCAGTCTCCCAAGGTGCTGGGATTACAAGCATGAGCCACTGCGCCCAGCCA TTTTAACCATTTTTA AGTGTCCAATCCAGTGGCATGGAAGTGAGTTCCCACTGTTGCGTGGTCTGGTTAATTCTG TCATACCGGTGCCTC TTTGCCGAGTCTTCAGTGTGAAAACTTCATCTGCCCTTCTCTCTCTGTTCCCCTGCAG (SEQ ID NO: 10)

Gene sequence: Mus musculus CLN3

(NCBI RefSeq NC 000073.7 (126170379..126184989, complement)) (14611 nucleotides in length)

AGGTTCTCAGAGGCTCCCCACTGCTTGCTTCCTCTTCACCGTGCTCCCAGATCTCCT GGGCTGTATTACTGCTCC

TGTCTCTGTGAGTCCCAGGCTCTGCCTCAGGCTTCAGTGGGACACTTGGCTCCTGGC CCCTGGCATCCTCAGCTG

CCTTCCAGGACCCAGTGTCCTGCCTTTCTGCTTGGGGCCTTGCAGAGCTATCTGCTC TCCACCCCCAGGTCTGTT

CACCAGCTAAACTTCCTTCTTGGCATCTTCTAGTCTCTTCGGGTGCTTCTACCCCCT CACTCTGGCCACTTCTAA

GTCCCTCCAGTACCTCCTGGGCTTCCTCTCCTACAACCTTCCCCTCAGTCACCTCTC CCAGTTCCTCAGCTGCCT

GTGTCTGCCTCTCTACTGTGGGGACTGTGTCTCCTACGAGGTAGGACACAAACGCAC TGAAGGAGTCCTAGAAGA

GAAGAAAGAAGAGTGAAGGCACTCAGGGGAGGTCTAACCACTTCCCCAGGAGTACAC TGTTAACAAAGCCCAGGT AAGAATCAGGGATCTGGACGTCTGCTAAGGGACCCCGACAAGGCCTAGCTTGAATAAGTG GGTACACTTGTAGAG

GCAGGAGATTGAAACCCAGGAAGGACTGGGCTAGGAGTCTGGCTGACTGTAGGAAAG GGAGCTCAGGCTTCAGGA

GAGGCTGTAAGGGAGAGGAAAGGTGAGACTACGAGGAATGGTGGAAGTTTCGGGTAT CTCTCTGCCCCTCTCACC

AGTGCTCCCCTCAAAGCCTGATGTAGCCCAGGGAGACGAAGCCGGAGAAAATCCATC CTGCAGGTCTCTGTGTGT

CCAGAAAGCTCACAATGACCATCCGTCCCCACCCACGAAGTCCTCCCCAGCACCCAA GACACTTCCTCTTCCCCT

GACTCTCCCTGACAAATCTCCACCCCAGGCCCTGTGCACCTCCCCCCTGCTCATGGA TGACTTCATGACAGGCAG

GGGGATGGCTTAACAGTGTCCAGGTGACTCAGACTCGGGTGCACATCTGGAACTAAA GGGCAGAGGATCGGGCCA

GAAAGGGGAAAGGGAAAAAAAAAGTGGAAACAGGAGACCTCAGAATTGGGTATGGGG GAGGGGGCATTCGGCAAG

TGCAGCCTGAGTGACTCAGGTCTTGTATTCGCACCTCAGCCATTTCCGCCTCCTCCA AGCCACAGCCTTCAGTGA

GAGGAAGCTAGGGTTTGTGGTTCCTGATTTATTTGGGGGAGAAGCTGTAGAAGTGAG GATAAGGCCACTGGAAGT

TCTGGGTTCCCATTTTCAACGCCATTAAGATTTTTCTGGGGATGAAAGGTCTGGATT GGTGCTCAATGCCCACAC

CCTCACCTATGGTTATTCTTGTACTGTGGTCATGTGTCCTCACAGCTTACTGTGAGT TCCTTAAGGACAGAGGCT

GCAGAATTTCTAGAGACTCGTTTGGGTAGGAGGATCCTTTTTCTCACTGTTTGAGTG ATCTAGGAAGAACACCAC

AGGAGGGCATATTTCAAACGGCAAATCTTAATGACAGGTGGTTGAAAAGACCCGGGA GAAGTGGGCAGAGGCTGC

TGGAATCCATAGCAGAGGAATAGGCTTGATGAAGAATGGTCAAGATGCTGCCCTGCC ATCGAGTGGAAGGTAAGG

GAAGGACTCCTTATTTGGAACTGTGAGGTGACACATTCCAACTATCCTCTCAGCCTC ATTCTCCTAACTTCCAAT

TAGGACAAACGGCTGACATCACAGACCAGGAAAAACACCCATAGCAGCTTTCTCAGT GAACTTAGCACCTTTGCT

CTATTTCTTACTGCTGAGTCTCTGAAGTCAGCTAAAAGTCTAGCCTCTGTTGCACAG TGGGGCGGAGGGACCTTG

GTTTTCTCTTGTAAAACCAGGGGTTGGACAACTTTCGTGGTATTTGTTGTTCTAGGA TAGTAAGGCAAAGGGGAT

TTAACCGTAACATCGTGAATATTGCCACAGATAATTCATAGAATGCTGTTTTGTGGG TGTTTATTTTTAATCGAT

CTACTCTCAGCTACTTCCCAGATGCTTATTATAGGTCTTTACAATAGGTCTGTAAGG ACAACAGATAACACTTGA

GGCTGGTCGCCTTTGCGCTCAAGCAGAACTTGTCCTTGGGACGTTGGGTGGGGTAGT GGGACATCCGCAGCAAAG

TAGCCATAATTGTTTCAGGCAAGTAAGAGCTGCAGGAAAGAAGCTGAAGCCTTCCGC GCCATGACTAGTACTGGC

AGATTTGGTCGGCGTCTAGAGCGCCGCTTTTCATGGCTCTACTTGTGGCTCCCTTCG ACTCCAATTCAAAATCAG

TGCTTCCAGCGTTCTCTCGAACTCTCCTTCTTTTATTCTCCGAAGTTTGAGAAGTCC AAAGGCGAAAAAGAGAAA

AAAGGACCCGGAAGTAAGAAGCAACCCTCAAGCACGTGATGGAAGGCTCACGGGTAC TGTCACGTGACCCCCAAC

CTGCTCGTCTACCTTGCGGAGAGTTCAGCTGCTCTTAAAGGTACAGGCCTGGGGGCG GGGGCTTTGCTTTAGAGG

GGCAGGGGGCGGGGTCAGGCGGGTGGGGCGTGGGTGAACACGAGCTGGAGATCTGGG GATCCTTCGACGGACATG

TCGTGTATAGCAGACAGCGGAACCTGGGACTGACCGCGGGGCATTGATCCTTCGCAC CCACCTGTCCCAGACTTT

AATCTGTTTTCTTGAAGCTAGCTCGGAACACACGCTGACTTTGGGCCCTTTGGGGGA CCCGAACTCAATGTTATG

GGAAGTTCTGCGGGCTCGTGGAGGCGCCTTGAGGATTCTGAGAGTGAGTGCTCTCAT CGGTTGCCTTGGGAATGA

CCACCTCTGCCCGCGGCTCTCCCGAGGAGTTGGGATGCTGGCCCTGCCCCCTCAACA CCATGCGGGATAGGCGGG

GCCTTCGTGTACCCGAGTGAGTGGGGGGTCATGCTTTCTCTACCTCCCAGGGGAGGA GACCGACTCAGAGCCCCA

GGCCCCTCGGTTGGATAGTCGGAGTGTCCTTTGGAAGAATGCAGTGGGTTTCTGGTA AGTGGTCTGAGAGTTGTG

CAAAAGTAAAATGCCAGCTGGGCGTGGTGGCACACGCCTTTAATCCCAGCATTTGGG AGGCAGAGGCAGGCGGAT

TTCTGAGTTCGAGGCCAGCCTGGTCTACAGAGTGAGTTCCAGGACAGCCAGGGCTAC ACAGAGAAACCCTGTCTC

GAAAAACAAACAAACAAACAAACAAACAAAGTAAAGTGCGATTGGAGCAAAGATGAG AGCCAAGTTGTGAAAAGC

AGTTGAATACACAGATACTTAATCAACTGTGAACTTTCCAAGTTCCATTAAATTCCC ACCTTCACCTATTGATCA

GATCTTAGTATCTGTAGAGTTATTTATTAAATGTTTTCCTCCAGCTAACTTTGTTTT TTTTCCTCTTCCTTCCTT

TCTTTCTTTCTTTCTTTCTTCCTCTTCCTCCTCCTCCTCTTCCTCTTCTTTTTCTTC TTTTTTTTGGAAAGGTCT

TTAAACCCTACAAGCAGTAAGGGCTTACTTTGACCTTTCTAATCCTGCTTTTGCCTT CCAGGTGCTGCAGTTACA

GGCCCTACTGTAGCACAATGCCCAGAACACCCATTGTTTCTGTTTGCAGTGCAGGGT ATTAAAAGCAGGTCCTTC

CCGGGCTCACGCACTGTTGTACAAGTGAGCTCTGTCTCCAGCCAATTTGCTTTTGCC AAGTTCTGAGCACAGCTC

TGGGAGATGAAAATAAGAACAATATGCCCTGCTATTATTTTGGTTCCCTTGAGATCA GCAGGTGAAAATTTGTCT

CCGTTTTGCACAGGAATACGCTAGATATTTGCATTGTACCACAGTGTTTCCGTTGAT GAGAAATGCCCAGGGATC

AGAATCTTAGTGTAAGCGTGGGAGAAATAGTGCTTAGGAGGACTTGGCACTTGGCCA TGAGCTGGTCTAATCTCA

AGGCCTGTTTTGATAGTGGAGAAGGAAAAAAAAAAGGTGTATCTTTAAGGACTGTAA GTGGATAGAAAGCCAGAC

AGTGAAGATGTGTCCTCATTTCTAGGATCTTGGGTCTTTGCAACAATTTCTCATATG TGGTGATGCTGAGCGCTG

CCCATGACATCCTCAAGCAGGAGCAGGCGTCTGGAAACCAGAGCCATGTAAGTAAGC CCCTCTTCACCACCACCA

TAGGTCAGTAAGTGAGCCCCTCTGTACCACTACCATAGGTCAGTCCGCATGGGGAAT ACTAAGAACCCACTCAGG

GCCAGAGGACCCCTGAAAAACTCCTAGAGTAGCTCTGGGTGGGATTGGGGAGCTAAA GGTCTTAGTCTCCCACAT

CCTCATGGCAGGAGGGATCTTAAAGAGAGCAACGGGTACCAGGTTGGTGAACTCACA TAACTGGCTGGGTAAGAC

CTTATTTTGTGGTGTGTTTTGTCATTGTACCCTGTTGACATTTGAGGTCAGGTGACT GTTGTGAGTCTGTCCTGC

ATGTTGTAGATGTTTAGTAGCATCCCATGCCCCTTGTCAACCAAAAGTGACAGCCAA AAATGTCTCCAGATGTTA

CAAATGCTCCTGCCAGGTAACATTACCCAGGTGGAGAGCCTTTGGCGGCAGTGAGCT AGAGCACGCAGGCCCTAA

GAGGCGCAGCCTCCACTTTGCAGGTTCACTAGGAAATTTGACCCAAGCTGATATTTT CCAGAAACCCAGATTTCT

ATATGAATGCTCTTAAACATGGTGCATCCATCTTGGGGGGCTGTCTTTGGCCTCTGG CCCAAAGTTTGAGATGCT

TATAAACACTCTCTTCATTGTGGACATTGAAAAACTAAGGCCCCCTAGAAGCCTTGC CTACCCACAGCCATTTGT

GAAGTTAGGACAGAACTGGACCTGGGCCTTGGTTAGCCTCCTGCCGCTTTGTCGGAA AGACCCTCTTCTCTCCCA

TAGGTGGAACCAGGCCCAACACCCACACCCCACAACAGCTCATCTCGATTTGACTGC AACTCCATCTCCACAGCT

GTGAGTGTCCATGTGGGGACCCCACGATCCTTGCCTTTGCAATCTCGTTTCCCCACC CCCACCCCCATCCAGTTG

GGAATGTTGGGATTGCAGTGATGAATATAAATATGTAAATAGTAATTATTAGTTCAG GTGCCCCCCTTCCCCCAG

CTTTGTGAGTGAAACACTGTAAGTTTCCCTCTTTGGATGAGGAAACTGAAGGTCAGA GTGTATAAATATGCCCTG

ACAGCCATTTGAGGAGCCCCAGAGCAGGGAATGAATGGAGATCTATCTAACTCCAGA GCCTAACTTACAGCAGGT CTCACTGGCCGGCTCCCCGGCTGGGTTGGGAAGGTTGTCTCCGGGAAGGTCTAGGCTCTC ACTGTGTTCTCCTGT

CTCAGGCGGTGCTCCTAGCAGACATCCTTCCCACCCTTGTCATCAAACTCCTGGCGC CTCTTGGCCTTCACTTGC

TGCCTTACAGGTCTGGGTCGGGGTGGCAGGAGGGAGGCAGGGTGGGAGGAAGCGGGA GTCTGGAGAGTCTCGCAT

GGAGGTGGCCTTCTGAGTTCCTCCTCATCTCTTCTCAGCCCCCGGGTGCTCGTCAGT GGAGTTTGTTCTGCTGGG

AGCTTTGTTCTGGTTGCCTTCTCTCAGTCAGTGGGGTTAAGCCTGTGTGGTGAGTAC GAAGCTGGGAGAGGTTGA

TGGACTCAACAGAGTGATGTGCTGTAGTCTCCCCAGCTTCCTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTG

TGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGACACACACTTGGGAG ATTGTGAATTTGAGATTA

GTTGGGGCCACATAGTGAATTCAAGCCTATCCTAAGCTGCACAGCAAAACCCTATCT CAAAAAACAAAGACAAAA

AAAAACCAAAAAAACAAGGAGAGGGCCAGTCCAGGCTGATTTACATAGCAAGTTCCA GGCCAGCCTGGACTACAT

CCCCGAGACTTTGTCTCAAAGACAAACCTCCTAGAGCTTCCCTAACCAGAAGTGAGC TCTAGGAACAATTGGAGG

GTGTCAATAGGAAGGAATGGGGAGACTGAGTATAGGTGGGGATGAGCAGGGTTGAAT AATTGGGAATTATTAACT

GAATGAAGATGGGGGTACAGTCTCGGAGAGGACATGTGGAGGAGGGTGTGGCTTTTC AGATGAGACCCTAGCCTG

TCTGTTCAGTGACAGATCTGTTCTTAGCCCTGCTGAGCTGATGGAGTTGAGGCTGTA AACAAACCACCGGGGACT

GCGGCTTTCAGTTAAAGGTCACTGTTTGCTGTTTGTCCTCCCAGGAGTGGTTTTGGC CAGCATCTCCTCAGGGCT

AGGGGAGGTCACCTTCCTCTCACTGACTGCCTTCTACCCCAGGTAAGCGGCCTGGGC CAGGAGGACTGGGAGACG

AGTGCCCCCTGCTGGCTTCCATCTTCCTCAGTGCTCTTGTCTTCTTTTCAGTGCTGT GATCTCATGGTGGTCTTC

GGGTACCGGGGGTGCAGGGCTTCTTGGATCGCTGTCTTACCTGGGACTCACCCAGGC TGGCCTCTCCCCGCAGCA

CACCCTACTTTCTATGTTGGGGATCCCTGTTCTGCTGCTAGCCAGGTGAGTGTCCTT AGGCCCAAAGGATGAAAG

ATGGAAGGAGGAGCTTGATGCTAATCCAATCTTCAGAGCTCTTCAGCCTTGTTGTGA AGGGGTGTGGACTCTGGA

CTCTCGACACCCCTCAGGTGGAATCTCAGAGTGGGGCTGTGTTAGCTGTGCGACTCT GGACAACTTCATTCCTGA

CCTTCAGTTGTCTCTGTCTCAGGCTGGTTCTGTCTCATCCGCACAGGTCGGCTCCTG AGACTGGGGCTGGGACTG

AGTGCACCCCGGTTTATTTCTTCTGTGTGTATTTGTATATTTTAGAAAACATTATTA AGCCAGGTGTAGTGGTAC

ATGCCTTTCATCCCAGCACTCTGGAGGCATCATGTTATAGCTTAGGGCTATTGTGGG GTATCCACCAGAAAGACG

GCTCAGACATGGGTTTAAACCCAGTGGAAAGCCGATTAGTCACCTGGTAACTACACA CTGCATGTTCAGTGGCAT

TTCCATTGGAGTCAGTTGTCCTAAGGTCCGGTGGCCTGTTACAAGGCTATCTTGAAG AAAAGGAGTCAGTATGTG

TGTAATGCTGAACACCCACAGTCCTGGCTCTTGGGAGGTGATGGAAAAGGCTCATGT GTTTGAAGCCTACCTGGG

ATACACGTGGAGACCCTGGCTCAAAAGCTAACTGAGATAATTGAACATATGGCAAAT GCCTCTAATCCAAGCCCT

CAGGAGGCAGGAGGATCTCTGTGAGTTTGGGGTCAGCCTGGTCTACATCTTGAGCTC CAGGCCAGCCAGAGCTGC

ATAGTTAAGACCCTGTCTCAAAAAAAAAAAAAAAGAAAGAAAGAAATAAAGCAATCC CATGATCATACAGACCCT

GGGTAAACTCAGTCACAACACGAAACAAAAAGACACTGAGTATGAGAAAGGGACCTG TGATGTGGGAAAGGTCAC

AGGGTGTGGGGACCCGAGGGGGTGGGGAGAGTCACCAGAGGGCAGTATATATGTGAA TAAAAATGTCAAGAAACA

CATTTAGTAAGAATCTCTGTGAAGTGGTGAATAGATGAGAAGTACTGAGAACAGGGA AACGAGCAGGTGCTGTGA

CTGAGTCAAATGAGGTCCCTGGGTAGAAACAAAGAAGCTTTTCCAGCACAGGGTACC TAGAGGCTGGGAGCACCC

CATGCAAGTGTGAAGGGCCTGGAGGGGCGGGCCTGCAGGACCTCTGGGGCCTTTCCC GACAAGAGCCCCCTGACT

CGCTTCTCCATTCCATCCCAGCTATTTCTTGTTGCTCACGTCTCCTGAACCCCTGGA CCCTGGAGGGGAAAACGA

GGCAGAGACTGCTGCCCGGCAGCCTCTCATAGGCACCGAGACCCCAGAGTCAAAGCC AGGTAGGATGCAAAGGCC

CTCCCATCCCAGGGCCACTTTCCGCCTTGGGGTTTTGGTCTCAACAGTGGACACAGT TGAGGGAAACAGGCTGCT

AGGTGTACAGGTCTGGGTCAAAGCAGTCATGTATACTTACTAACTACTGACTGGATG CCAGACATTCCGATTGCT

GGGAATGGTAGTGCTCCAGATGAAGGAATGAGAGCATTTTCACAGGACATAAAACTG CGATGGGGCCCAGAGGGG

TGGCGCACACCTTGAATCCCAACACTCCGGAGGCAGCCACAATCAGATCTCTATGAT TTCAAGGCCGGTTGGGCT

ACATAGTAAGACCCTGCACAGACCCAGAAAGTAACGGTTAAGGAAATGGGGTGGGGA TGGCTAAGGGAGCTGCTT

TACAGGAGGTTCCAAGTGTGGGGGTTTTGTTGTTGTTTTGTTTTTTCTTCAAGTTTT TGCTTATTGGTTTTTAAA

TAGTCTCTCTGTATAGCCCTAGCTGGCCTAGAAATCACTGTGTACTCTCGAATGGCC TCAACCTCACAAATCCAC

CTGCCTCTGCCTCTCAAATGCTAGGAGTAAACGCATATGCCACCATACACCTGGCTA GGATTTATTTTTCTATTT

ATTGATGTGTATGTGTATTTGTGAACTTATACCGCGGATGACCAGCAGAGGGCATCA GATGCCTTGGAGTTGCAG

TTACAGGCAGTTGGGAGCTGGCCGATGTTGCCAGGATTCAAACGTGGACCCCCTCAT GGTTGAGCAACCAGTGCT

CGCTCGCTCTTTCTTTCCTTTTCTCTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTT TCTTTCTTTCTTTCTTTC

TTTCTTTCTTTCTTTCTTTCTTTCTTCCTTCCTTCCTTCCTTCCTTTCTTTCTTTCT TTCTTTCGGATTTATTTA

TTTATTTAATGTATATGAGCACAATGTAGTTGTCTTCAGACACACCAGAAGAGGGCA AGAGGGCATCAGATCTCA

TAACAGATGGTTGTGAGCCACCATGTGGTTGCTGGGAATTGAACTCAGGACCTCTGG AAGAGCAGTCAGTGCTCT

TAACCACTGAGCCATCTCTCCAGCCCCTCTTTCTTTTTTTTTTATTGTTTGTTTGTT TTGTTTTTGTTTTGTTTT

TTGTTTTTTTGTTGTTGTTGTTGAGGCAGAGCTTCTCTGTGTAGCTCTGGCTGTCCT GGAACTCACTCTCTAGAC

CAGAAGTCAAAAATTCTCCTGCCTCTGCTTTCCATGCGCTGGGATTCAGGGCGTGTG CCGCCACTTAACCCCAAG

CCACCTCTCCAACTCTGAGAAAGGCTCTACTTATTTATTTAATTTTGTAATTATTTG TTTGTTTGTTTGCTTGCT

TTTCGAGGAAGGGTTTCTCTGTGTAGCCCTGGCTGTCCTGGAACTCACACTGTATGT AATCCAGGCTGCCCTCAA

ACTTTCAGAGATCCACCTGCCTCAGCCTCCCAAGTGGGGATTAAAGGCATGCGCCAC CACTGCCCAGCGATTTGT

CTATTTTTATTTTATGTGCATTGGTGTTTTGCCTGTGTGCATGTCTATGGGAGGGTG TTGGATCCCCTGGAACTG

GAGTTACAGACAGTTGTGAGCTGCCACGTGGGTGCTGGGAATTGAGGTCCGCTGGAA GAGCAGCCATTACTCTTA

ACCACTGAGCCATCTCTTGAGCCCCCGAGAGAACCCCCAACAACAAACCTTTTATTT TTGAGAATGGAGCTGTCT

GGGCAAGAGCGTGTGCACAAGTGTGAGGTCTGACGTTCTGAGGATTGAGCTCTGCTC ATTCAGACTCGGCAGTGA

GTGCCCTCATCCACCGAGCATCCATCAGCCCAAGACAGCCTTTTGAAGGATATCTCT GGGCTGAAACTTGGCTGT

CAAGGTGTCAGCTTTGCAAATGCCTAGGTGATCATGTTCCAGGCACAGGAAACAGCT CAGGGTGAGGCCTTATGT

GAGAAAAGGAGCCTGGTTGTTCTAGAAATGAAAGGCTTAGCCAGAGTCTGGGATGGA GTCGGTAGATAAAAGCTG

TACCCTGATCTATAGTCTTGCTCATTAAAAAAAAAAAAAAAAAGTTTTATAAGTATA TGTGTGTATATACATATG TATACAGCGCGAAGTTTACCATTTTAGCGACTACTAAGTGTGCACTAAGTGGCATTTAAA TCCACACACTGCTGT

GCTGGCTGGTTGCTTGCTGCATGCTGGCACCTTAGCTAAGCGTTCAGCGTGCAACCT CCTCCCCTTCCCCCTGCT

CTGTAGGTGCCAGCTGGGACCTCTCCCTCCAGGAAAGGTGGACAGTGTTCAAGGTTT GAATGGTGGCTGCGGGTG

CCCCGTGAGGATCCTCTGCCTCTTGCCTTCCACTTACATCTCCCGACTCTTCCAGGG TCTCTTGTGGTACATCAT

CCCTCTGGTGCTGGTCTACTTTGCAGAATACTTTATCAACCAGGGACTTGTGAGTGA GGGGCGCCGGGGAGGGAT

GTGGGAGGAGAGATAGGGCTGAGGTTCCGCCATCTCCATCTGTCTGGTCTTCGCTGC AGTTCGAGCTCCTGTTTT

TCCGGAACACTTCCCTAAGCCATGCTCAGCAGTACCGATGGTGAGAGAAGTTGGCAG GTGGGCAGTAGGCTGGGA

GGATCCCTGATGTTGGGGTCCTGGGGTTGACCCCAAAAAGTGCCCTGAGAGCCTCTT CCTCTTCTCTGCCCCAAC

CTTAGGTACCAGATGCTATACCAGGCTGGTGTGTTCGCCTCCCGCTCTTCTCTCCAA TGTTGCCGAATACGGTTC

ACCTGGGTCCTAGCCCTGCTCCAGGTACTGAGTGCTGCCCTGTTTGTTCACACCCAC CTCGGCCTCCTGAGTTTC

TTACATTAGTGTACTCCAGCGTTCCTAAGTACAGCACTAAAGGCCAGCTGGACTCCA TATAAAATGTGTGGTGGC

ACCAGGAGGAACCAGTCATACTTGTGGGTAAATTCCAGTTTCTGTTCTCAAGAGCTG TGTGACTCAGAGCAGTCT

CTGAACCTCTATTGAGCCTCAGTTTTCCTATCAGGAAGTCAGATGGGGTGGAGCAGG ATTGTGCACCTAGCCTGT

CTGAGGCTGTGGGTCCCATCCCTAGTACTGCAAAAATTAAAATATTTAATAGCTGTG GGCAACACCTATTTCTCA

GTGTTTGTATAGCAATGTTGAGAAAAAAAAGGAAGAACAAGATATTGTTTTGATTTA TTTTGGTTTTGTTCTTGT

CTGCTTTATTTTTTTTTCTTTGTTTGTTTTTAAGGTAGGGTCTCACTGTGTAGTCCT GGGTGGCCTGGAATCCGA

TATGTAGACTAGGCTAGCCTGAAGCTCACAGATCTGCTTCATGAATGCGGGCGTTGA GGGCCTGTGCCTCGTGCC

TGGCCGTCGTGCTTTGTTCTCACAGTCGTTCTGACTGCACCTGGGGCCTTGTTCCTG CTGGTCCATGCTGATTGT

TACGAAACTTCTTAAGCCCTCAAGCTTGCTTTCATTTACTTACTTATTTACTTTAAA GATTTGTTTGTTTATATA

TAAACAAACTGTAGCTGTGTTCAGACACACCAGAAGAGGGCATCAGCTAGCCGGGCG TGGTGGCGCACGCCTTTA

ATCCCAGCACTCGGGAGGCAGAGGCAGGCGGATTTCTGAGTTCGAGGCCAGCCTGGT CTGAAAAGTGAGCTCCAG

GACAGCCAGGGCTACACAGAGAAACCCTGTCTCGAACCCCCCCCCAAAAAAAAAAAA CAAAAAAAAACAAAAAAA

AAAAACAGAAGAGGGCATCAGATCCCATCATAGATGGTTGTGAGCCACCATGTGGTT GCTGGGAATCGAACTCAG

GACCTCTGGCAGAGCAGTCAGTGCTCTTAACCGCTGAGCCATCGCTCTAGCCCTATT TATTTATTTTATACAATT

TAAATAAACACACTTATATTTATATTTATTTATTGGTGGGAGGGAGACACTTGCCAA GCCGTGTGTATGGAGGTC

AGAGGACAGCGTGCAGGATTGGTTCCCTTCTTCAACGTGTGGGTTCTGGTGATCAAA CTCAGGTCATACAGGCAA

GTACCTGTAGCTGAGCCATCTCTTCAGCCCAAACAGGGTTTCTCTGTATAGCCCTGG CTGTCCTGGAACTCACTC

TGTGGACCAGGCTGGCCTCGAACTCAGAAATCTGCCTGCCTCTGCCTCCCAAGTGCT GGGTTTAAAGGCGTGCGC

CACCACTGCCCAGCTCAGCCCAAACTTTTTATTTGGAGGCAAAGTCTTCAAAGCTGG CCTTGAACCTACATCTGA

GGCCCACATAAGCCTTGACCATTGTTTAGTTTATATATACACAAACCTTTGTCTATG TGTATATATGTGAACCAC

CTGTGTGCCTGGTGCCCATGGAGGCCAGAGAGGGTATCAGATCCCTGGAACTGGAAC TACAGATAGGTGTGGACC

GCCCTGTGGGTGCTGTGGAAGAACAGCGGGTGCCTTTAACTACTGAGCCATCTTTCC AGTCCAAAATTTTGTACT

CTTGACATTTGCAGCTAGATCATTCTGATGTGTTACTTGCTGTAGGTGTTTAGCAGT ATCCCTAGCTGGTGAGAC

GGCAAGTAGGTCTCCAGACATTGTCAAATGACCCATTGATTTCAGTGCAGTAATTAG GGGAGAACAGGTGATGTT

CAGGGGGTCCTTGGGTGCTGAGGATAGGACTCTGGTAGAGCATTTGCCTCTGAATCA CAAGCTCTCGTGTTAGCT

CTGCAGTGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTT GTGTGTGTGATACACATG

GTAGGGGTGTCTAGGTGTTAGGATTCATGCCATTAAAAAAGAAACAAAAAGGCAAGA AGGAGAGGAGGTGACTGA

TGACCACAGTGGAATGACCCTGGTCCAACTGAGAGCTCACCAGTGATGGGAAGTTGA GGAACATTACAGGCTAAG

AGTGACAGGGACAAGTCAAGGAAGGGGAGAAACCCACAAGGCATGTGAGGATGAGGA TGGGGTCATCGAGGGCCT

ACCAAGATGTCGGGGTCCCACAGGCCCAGCTGGGCACTGGGACCCTACACAGCCCTC TGCTTCAGCGGCATCTCC

TATAACCTGGCAGCTCTGAGACCTGCTGCCTCCCTGCCGTATTGTTGCCCAGTCTCA CGCCTCCCTTCCCTCTGC

CCACAGTGCCTCAACCTGGCCCTCCTGCTGGCAGATGTCTGCTTGAACTTCTTGCCC AGCATCTACCTCATCTTC

ATCATCATTCTGTACGAAGGGCTCCTGGGTGGGGCCGCTTACGTGAATACCTTCCAC AACATTGCTCTGGAGGTC

TGTGCCAGTTGGACAAGGGCTGTGGGTGGCCTTAGGAGCCTTGTGGATGGAAGCCCT AACCCTTGTTCTTTGCTC

TCCCAGACCAGTGACAAGCACCGAGAGTTTGCCATGGAAGCTGCCTGTATCTCTGAC ACCTTGGGAATCTCCCTG

TCGGGGGTCCTGGCCCTGCCTCTGCATGACTTCCTCTGTCACCTCCCTTGACAGGAG TTGCTCGACACACACTGA

TCTGCAGGCACATGAGCAGATCACACATCTTCGAGCTCTGCCACAGCCTTTCCCTGC CCCACTGCAGCAAGGAGC

CCCTGATGTTTCCCACTCCTGAGCTGGCCTCAGAGTTTTCTCCTACCCTCTGCCCTT CTAATAAATGCTTATTTT

AACAGTTTCCTTTTTGTGCAGTCTCTGAAACGGAAAGTTTACATAAGCCCACTGTGA AAGATATATGTATTTATG

AATGTATAAAGTATTTCAGACAAGGTGTGGCACACACCTGTAATTCCAGCACTCAGC AGACTGGAGAATCCAAGT

TCACGTCTGACTGGGATATACGGTGAGACCCTGAGGACTAGGGCTATAGTTCAGTGG TAGAGTACTTGCCCAGGA

TGTCCTGGGCTCTCAGTTTTATCCCTGGAAAACAATTCAAAAGCAGCGTATGCTAAT ATAAGCCTGTAGTCCCAG

GATTGGGGAGGTAGAGACAGGAGGATCCAGAGTTCATGGTCCTCAGCTACATAGGAA ACCAAGGTCAGCCTGGCC

TACATAAGATCTTGTTTTACAATAATTACATTGTGTATTTCTGGCTTCTTTATAAAA AAAAATCAAATGTCCATA

GGTATGTAGATTTATATCTGGGTCTTCGATTCCATTGATCAACTTGTCTGTTTTTAT GCCAATACTGTGTGGGTT

TTATTACTATAACTCTGTAGTAGTGCTTGAAATAAGGATGGTGATACCTCCAGGAGT TCTATTACTGTACAGAGT

TGTTTTAGCAATCCTGTGTTTCTTGTTTTTTTCATATGAAATTGAGTATTGTTCTTT CAAGGTCTATAAAGAATT

GTGTTGGAATTTTGATGGACAACTGATTTCTTTCTTCAATGTCTTAAAGTTTTTATC ATATAACTCTTTGACTTG

CTTGGTTAGATTGACCCTACAATATTCATAGAAGACGAAGTGGGTAGTAGCCCTGAA CGAATGTCACAGGAGACA

ACTTCCTGAACAGAACACCAATAGTTCAGGCACTAAAATTGACAATAAACCAGATCT CATG (SEQ ID NO:

773)