COMPOSITIONS AND METHODS FOR CELLULAR DELIVERY OF RNA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent

Application No. 63/047,024 entitled "Compositions and Methods for Treating Cancer", filed on July 1 , 2020. The specification and claims thereof are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 1 , 2021 , is named 32064-1037-PCT_SL and is 436 Kbytes in size.

BACKGROUND

[0003] RNA interference (RNAi) is a natural process wherein protein expression is blocked by the degradation of mRNA molecules. This process is believed to provide a defense against the translation of foreign genetic material, for example from viruses, and a regulatory mechanism for native proteins. The foundational discoveries for RNAi were made during the 1990s. Micro-RNAs (miRNAs) were discovered in 1993 in nematodes and the phenomenon of gene silencing by long double stranded RNA (dsRNA) interference (RNAi) in 1998 ¹ · ² . Small interfering RNAs (siRNAs), double stranded RNA sequences of about 20 nucleotides, wherein on strand had significant Watson- Crick complementarity to a mRNA sequence in a cell, were subsequently found to be effective for in vitro gene silencing. ³ · ⁴ The mechanisms through which RNAi targets and degrades messenger RNAs were later partly elucidated. ^5-8 The mechanisms through which RNAi targets and degrades messenger RNAs were later partly elucidated. ^5-8

[0004] MiRNAs and siRNAs share common mechanisms of action but differ in their structure and, in the case of synthetic siRNAs, their origin. MiRNA precursors (pre-miRNAs) are naturally transcribed in the nucleus from DNA as single stranded RNA molecules with regions of selfcomplementarity connected by a hairpin turn motif. After leaving the nucleus, the human nuclease Dicer cleaves off the hairpin turn motif, leaving a dsRNA of about 22 nucleotides. This edited dsRNA is an siRNA, with a guide strand that targets a complementary mRNA sequence and a complementary passenger strand. The guide strand is incorporated into a multiprotein RNA-induced silencing complex (RISC), with nuclease activity, that degrades complementary, or partly complementary mRNA sequences in a catalytic fashion. Synthetic siRNAs, by contrast, are man-made molecules that often contain chemically altered nucleotides and must be introduced into the cell by specialized formulations. Otherwise, synthetic siRNAs function similarly to those generated within the cell from miRNAs in the RISC complex.

[0005] In the realm of RNAi therapeutics, it is important to differentiate miRNAs, siRNAs, antisense oligonucleotides (ASOs), and other molecules such as aptamers. ⁹ The relationship between miRNAs and siRNAs was discussed in [0002] ASOs are small (13-30 nucleic acid), single stranded nucleic acid molecules capable of hybridizing with complementary mRNAs. ¹⁰ However, unlike miRNAs and siRNAs, which are ultimately incorporated into the catalytic RISC complex and have prolonged effects on mRNAs, antisense oligonucleotides (ASOs) block normal mRNA function by simple steric hinderance or by RNase H enzymatic degradation. ¹¹

[0006] Many diseases involve the aberrant expression of native proteins or the expression of defective proteins, such as those containing mutations and blocking the production of such proteins could confer therapeutic benefits. For this reason, RNAi continues to be of interest in basic science and drug development. The application of RNAi to therapeutics has several attractive features: 1)

RNAi drugs can be highly specific for a targeted mRNA, minimizing off-target effects that are often observed in drugs that target protein receptors; 2) Pharmacological effect occurs at the post- transcriptional stage on mRNA, in contrast to drugs that affect DNA genome, that often have toxic side effects; 3) RNAi is a catalytic process, which gives rise to a prolonged pharmacological effect using a small dose (low nanomolar or picomolar) of drug.

[0007] A combination of factors greatly complicates the development of RNAi therapeutics such as siRNAs. ^12-14 Some of these factors are pharmacokinetic while others are related to the intracellular trafficking. Systemic iv administration of unmodified siRNA molecules is not practicable for several reasons. In plasma, unmodified siRNAs are degraded at 3’- and 5’-ends by nucleases. ¹⁵ This same nuclease instability occurs intracellularly. As highly polar small molecules with little plasma protein binding, siRNAs undergo ready renal excretion. siRNAs also trigger the innate immune system, which can lead to severe side effects. Being negatively charged molecules, siRNAs cannot efficiently permeate the cell membrane. Those siRNA molecules that enter the cell are transported to the endosome where the early endosomal membrane presents an additional barrier to egress into the cytosol. As endosomes mature to late endosomes and lysosomes, they become increasingly acidic and acquire nucleases which degrade siRNAs. The endosomal escape problem is generally considered to be the most formidable. ¹² · ¹⁶

[0008] The physicochemical characteristics of siRNAs also complicate oral drug delivery.

The presence of nucleases in the fluids of the digestive tract compromises the bioavailability of unmodified siRNAs. Moreover, the charged, hydrophilic, nature of siRNAs prevents passive transport across the intestinal lumen. Research in siRNA oral delivery has focused on polymer nanoparticle formulations and conjugates. ^17-19 [0009] Much effort has been devoted to formulations and drug delivery systems for siRNA therapeutics. ²⁰ · ²¹ These approaches can be broadly categorized as encapsulations (e.g., liposomes, lipid nanoparticles, polyamine, lipoplexes), sustained release polymer formulations, ²² · ²³ and covalent conjugates where the passenger strand is linked to diverse molecules (e.g., lipids, folate, porphyrin, antibodies, N-acetylgalactosamine (GalNac)). Liver targeted siRNAs have shown the most promise, using both lipid nanoparticle and covalent conjugate approaches. ²⁴ therapeutic (patisiran) for a rare genetic disorder. In 2018, patisiran, a lipid nanoparticle encapsulated siRNA targeting transthyretin mRNA was approved for treatment of hereditary Transthyretin Amyloidosis twenty years after the discovery of RNAi. ²⁵ · ²⁶ Subsequently, the FDA approved a liver targeted siRNA GalNac conjugate (givosiran) in 2019 for treatment of acute hepatic porphyria. ²⁷ Liver targeting of siRNA drugs is favored by the natural function of the liver for uptake of xenobiotics, the discontinuous endothelium that facilitates uptake of nanoparticle formulations, and the abundance (~100,000/cell) of asialoglycoprotein receptors (ASGRs), and the efficiency by which these receptors bind ligands (e.g., GalNac), internalize them by endocytosis, and recycle to the cell membrane (15 min). ²¹ · ²⁴

[0010] The use of vitamin conjugates for drug delivery has been known for many years. This approach takes advantage of the fact that many vitamins have receptors to transport them into cells. ²⁸ Abnormal metabolism in cancer cells can result in overexpression of receptors and make vitamin conjugates an attractive approach in oncology, particularly with folic acid, biotin and vitamin B12 (cobalamin, Cbl). Indeed, abnormally high levels of cobalamin and its associated plasma chaperone proteins are associated with cancer. ²⁹ - ³³

[0011] Cobalamin, an essential dietary nutrient, is bound to protein molecules from the point of ingestion through to its ultimate destination as an intracellular enzyme cofactor for methionine synthase (MS) and methymalonyl-CoA mutase (MUT). ^34-36 Cobalamin has a highly complex corrin structure wherein the central cobalt(lll) atom bears an axial substituent X which is commonly CN, Me, OH, 5’-adenosyl, and S-glutathionyl (Figure 1 A). ³⁷ Cyanocobalamin (X=CN) is a form that does not occur in nature; however, all the forms of vitamin B12 listed are processed intracellularly to ultimately deliver the enzymatic cofactors methylcobalamin to MS and adenosylcobalamin to MUT. ³⁶

[0012] Cobalamin is bound to chaperone proteins and cellular receptors from oral ingestion through delivery to its MS and MUT. In saliva, the chaperone protein transcobalamin I (haptocorrin, TCN1) binds cobalamin with high affinity and protects cobalamin from acidic hydrolysis in the acidic environment of the stomach through to the intestine, where TCN1 is degraded by proteases and cobalamin released. ³⁶ Cobalamin is then tightly and specifically bound by the chaperone protein intrinsic factor (IF) and enters the apical lumen through receptor mediated endocytosis into enterocytes and released on the basolateral side of intestinal lumen into circulation. ³⁶ After being transported through the intestinal lumen, cobalamin is bound with high affinity to either of two plasma chaperone proteins: transcobalamin I (TCN1 , haptocorrin) and transcobalamin II (TCN2). The majority of plasma cobalamin (~80%) is bound to TCN1 , and most of the remainder is bound to TCN2 (~20%), with very little unbound cobalamin present in plasma. TCN1 is a glycoprotein that occurs in plasma in two forms, with identical amino acid sequences, but differing in the type and number of post- translational sugar modifications, particularly sialic acid. ³⁸ The form predominantly present in plasma is more heavily sialylated and the TCN1/Cbl complex has a relatively long plasma half-life (10 days), whereas the less sialylated form of TCN1 , referred to as transcobalamin III in some literature, has a very short plasma half-life of about 5 min. ³⁹ · ⁴⁰

[0013] Cobalamin is required by all cells, but its mechanism of uptake differs according to the plasma chaperone protein to which Cbl is bound and according to cell type. CD320 receptor, a member of the low-density lipoprotein receptor (LDLR) family, is the principal receptor for cobalamin uptake in most cells. CD320 binds the TCN2-Cbl complex and internalizes that complex via receptor mediated endocytosis. ⁴¹ TCN2-Cbl is the only known natural ligand for CD320, which binds this complex with high affinity and specificity. ⁴¹ In vitro, endocytosis of TCN2-CD320 occurs within 10 minutes in HCC15 cells (Elzi, submitted for publication). The LRP2 (megalin), another receptor in the LDLR family, also binds and internalizes TCN2-Cbl and is heavily expressed in the kidney. ⁴² Unlike CD320, LRP2 binds and internalizes a host of proteins, cytokines, and hormones, and is believed to scavenge and recycle important biomolecules which might otherwise be lost in urine. By contrast, TCN1-Cbl is only known to be taken up by hepatocytes, which express very little CD320. ⁴³ Although its physiological function of TCN1-Cbl is not well understood, the desialylated form of TCN1/Cbl, also known as transcobalamin III, is a ligand for the asialoglycoprotein receptors (ASGRs), ³⁸⁴⁴ which are heavily expressed in the liver (~100,000 receptors per cell) and mediate the uptake of a variety of desialylated proteins ligands. ⁴⁵ · ⁴⁶ The plasma trafficking of and uptake of cobalamin is summarized in simplified form in Figure 2. In Figure 2 (A) there is a cell transmembrane receptor, which could represent CD320, LRP2, or ASGR, and an siRNA covalently linked to cobalamin (Cbl), complexed to a plasma chaperone protein TCNx, where x could represent 1 or 2 (e.g., TCN1 or TCN2). In Figure 2 (B) the ligand, that is, the complex of siRNA-Cbl bound to TCNx, binds to the cell membrane receptor; in the case of x = 2 (TCN2), the cell membrane receptor could represent CD320 or LRP2, whereas in the case of x = 1 (TCN1 , desialylated) the receptor could be ASGR. After binding of the ligand to the receptor, the entire complex is internalized through a process of receptor mediated endocytosis (RME) and the siRNA-Cbl complex is released into the cytosol (Figure 2C).

[0014] Cobalamin conjugates of radioisotopes, fluorescent molecules, and drugs have been described for a variety of diagnostic and therapeutic applications, primarily in oncology. ⁴⁷ · ⁴⁸ Covalent conjugation of cobalamin is generally performed at the side chain carboxy groups of the corrin core structure, which are designated by the letters a-e in Figure 1 B. Additionally, side chain modification can be performed at the ribose 5’-hydroxyl group on side chain f. For example, acidic hydrolysis of one or more of the side chain amide functional groups, followed by chromatographic purification, and peptide bond formation to a bifunctional linker (spacer molecule) which is in turn covalently bound to molecule with diagnostic or therapeutic application, such as radiotracer, a fluorescent dye, or a drug molecule. Thus L _a -L _e in Figure 1 B may represent covalent linkers in turn attached to a diagnostic agent or drug T _a -T _f . Conjugation is also possible at the 5’-OH group of the ribose moiety at position f, for example, by attachment of a carbamate or ester-functionalized linker L _f that is in turn attached to a diagnostic or therapeutic molecule T _f (Figure 1 B). Further, it is possible to perform conjugation at the cobalt atom of cobalamin by direct covalent bond formation at X (Figure 1 B), wherein X is comprised of a linker L _x joined to a diagnostic or therapeutic agent Tx.

[0015] A further aspect of cobalamin conjugates is their metabolic stability. While it is generally desirable that the linker (L) be stable in the extracellular space, for example in plasma, the linker structure may be designed to remain stable intracellularly or to cleave under intracellular conditions. As cobalamin is internalized to the endosome after receptor mediated endocytosis, linkers may be designed to cleave under endosomal conditions, wherein decreased pH or endosomal proteases cleave the linker. ⁴⁹ Where the chemical linker that joins cobalamin to the drug can be one that is relatively stable to intracellular metabolism (“non-cleavable”) or one that is easily cleaved under endosomal conditions according to established art (“cleavable”). ⁵⁰

[0016] The delivery of radiotracer conjugates for cancer diagnosis, both metal-based chelates and organic groups containing radioisotopes, has been reported. Cobalamin has been conjugated at the carboxy side chain functional groups, using a variety of covalent linkers, to a DPTA chelating moiety capable of binding a radioactive metal isotope (Collins, US 7,531 ,162 B2) and also to non-metallic radioisotopes on aryl functional groups (Collins, W02008136850A2). A conjugate was also prepared at the d sidechain of cobalamin to contain a histidine functionalized with radioactive ¹²⁵ l (Houts, US 4,465,775). In another example, the 5’-OH group of the ribose moiety was esterified (Figure 1 B, R ⁶ ) with a hydroxyphenyglutaryl group that was further functionalized with radioactive ¹²⁵ l (Bernstein, US 4,209,614, US 5,449,720).

[0017] Cobalamin fluorescent conjugates have been prepared. Grissom reported conjugation of fluorescein through a covalent linker to side chain carboxy groups and also to cobalt, resulting in fluorescent conjugates with selective uptake in certain cancer cells (Grissom, US 6,797,521 B2).

[0018] In oncology, cobalamin conjugates of diverse oncology drugs have been described, including organoplatinum drugs, ⁵¹ tubulin inhibitors, ⁵² doxorubicin, and paclitaxel. Doxorubicin and taxol have been conjugated to the 5’-hydroxy substituent of cobalamin (i.e., R ⁶ in Figure 1 B) using a metabolically stable linker in the case of doxorubicin and a linker that is cleavable under endosomal conditions in the case of taxol (Collins, US 7,232,805 B2). By contrast, reported doxorubicin was conjugated to the 5’-OH group of ribose (Figure 1 B, R6) through a metabolically reactive cathepsin cleavable linker (Weinshenker, WO 2005/0255 A2). Carborane conjugates for tumor targeted neutron capture therapy have been reported, wherein a carborane moiety is attached to side chains b, d, and e or at the cobalt atom (X) (Figure 1 B) (Collins, US6,806,363 B1). [0019] The ability of cobalamin to traverse the intestinal lumen is mediated by the chaperone protein intrinsic factor (IF), see above, which selectively binds cobalamin with high affinity to form a 1 :1 complex (Cbl-IF) and is a ligand for the cell membrane cubulin receptor, which in turn mediates the uptake of the Cbl-IF complex into intestinal epithelial cells through receptor-mediated endocytosis. ³⁵ This mode of intestinal transport has been utilized to transport cobalamin conjugates of various proteins through the intestine to the blood stream, a process which would not take place in the absence of conjugation. Thus, the conjugation of granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), and consensus interferon (IFN-Con) to the 5’-hydroxy group of cyanocobalamin, using a bifunctional glutaryl linker, afforded conjugates with demonstrated oral bioavailability in animal models (Habbefield, US 5,574,018). Carboxy side chain conjugates of cobalamin with the antibiotic neomycin and leutenizing hormone releasing hormone (LHRH) likewise greatly improved the oral bioavailability of these drugs (Russell-Jones, US 5,428,023).

[0020] The cellular delivery of cobalamin-nucleic acid conjugates has been reported for DNA plasmids and antisense oligonucleotide-peptide nucleic acid chimeras. Foreman reported the attachment of a polycationic polylysine linker to cobalamin through amide bonding at a side chain carboxylate, followed by non-covalent conjugation of plasmid DNA, based on the electrostatic attraction of the negative DNA plasmid to the polycationic linker (Foreman, WO1999065529A1).

BRIEF SUMMARY OF THE INVENTION

[0021] One embodiment of the present invention provides for a cobalamin complex or the pharmaceutically acceptable salt, stereoisomers, solvate or polymorph thereof comprising a siRNA sequence that is complementary to a nucleic acid sequence that codes for a target protein within a cell wherein the siRNA sequence is bound to a cobalamin and wherein the cobalamin is complexed with a cobalamin binding protein. The target can be for example a cobalamin binding receptor. The siRNA can be bound to cobalamin via a linker arm in another example. The linker can be selected from the linker of FIG. 3, FIG. 4 and/or FIG. 5 wherein n is 1-100; Q and Q’ are independently selected from O, NH, or methylene; and R is selected from H, alkyl, aryl, heteroaryl, alkoxy, and a stereogenic center is either in the R or S configuration. In one embodiment the cobalamin binding protein is a cobalamin plasma chaperone protein. For example, the cobalamin plasma chaperone protein is selected from a transcobalamin I, transcobalamin II, ortranscobalamin III. In one embodiment the complex can be of Formula B wherein the cobalamin is of formula B (Formula B); wherein one or more side chains of the cobalamin of Formula B at position a-d are modified with a covalent linker La-Ld selected from

position f is modified with a covalent linker Lf is selected from

and the siRNA molecule Ta-Tf is complementary to the nucleic acid sequence that codes for a target protein wherein the siRNA is bound to cobalamin through the selected linker to which the siRNA molecule is linked, and a group X in Formula B represents linker Lx selected from

an siRNA molecule Tx linked to Lx wherein Tx is selected from Ta-Tf. In one embodiment the cobalamin binding receptor is selected from a CD320 receptor or a LRP2 receptor. In one embodiment the siRNA is selected from siRNA sequences in TABLE 6, TABLE 2, TABLE 3, and/or TABLE 1 .

In a further example, the complex of Formula B wherein an X group that is bound to cobalt further comprises one selected from CN, Me, 5’-adenosyl, S-glutathionyl, alkoxy, lower alkyl, aryl, substituted aryl, and heteroaryl.

[0022] Another embodiment of the present invention provides for a method for introducing a complex as described in an embodiment into cells comprising providing to the cells an effective amount of the complex. For example, the complex is administered to a mammal, for example a human and for example the complex may be targeted to a liver or kidney of the mammal.

[0023] Yet another embodiment provides for a pharmaceutical composition comprising an embodiment of a complex or the pharmaceutically acceptable salt, stereoisomers, solvate or polymorph thereof. For example, the complex is adapted for administration selected from orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Further still the pharmaceutical composition is for treating a disorder wherein an effective amount of the complex together with a pharmaceutically acceptable carrier, additive or excipient wherein the disorder is selected from Cancer, ATTR amyloidosis (transthyretin, TTR), acute hepatic porphyria (AHP) (aminolevulinate synthase 1 , ALAS1), hepatitis B (HBV) or C (HBC), hypercholesteremia (ANGPTL3), hemophilia A and B (thrombin), primary hyperoxaluria (LDHA), paroxysmal nocturnal hemoglobinuria (PNH) (C5A receptor), antitrypsin deficiency liver disease (AAT), hypertension (angiotensin).

[0024] Yet another embodiment provides for a method for making a cobalamin complex comprising attaching an siRNA to a cobalamin wherein the siRNA is complementary to a portion of a nucleic acid sequence that codes for a target protein wherein the target protein is within a cell and the cell binds the complex.

[0025] One embodiment of present invention provides a method for drug delivery of one or more therapeutic siRNA molecules as covalent conjugates to cobalamin (Cbl) in humans or animals.

[0026] An aspect of an embodiment of the invention is a method for administration of siRNA molecules to humans or animals comprising the covalent conjugation of the passenger strand of such siRNA molecules to cobalamin through a covalent linker.

[0027] Another aspect of an embodiment is a method for delivery of siRNA molecules to cells in vitro as covalent conjugates wherein the siRNA molecule is covalently conjugated to cobalamin on the passenger strand.

[0028] Another embodiment of the invention is a method for the administration of siRNA molecules to humans or animals as covalent conjugates to cobalamin which are further complexed with a cobalamin plasma chaperone protein such as transcobalamin I, transcobalamin II, or transcobalamin III.

[0029] An aspect of an embodiment of the invention is conjugate compositions, illustrated in

Formula B, where a cobalamin molecule is covalently modified on the carboxy group of one or more side chain(s) (Figure 1 B, positions a-f) with a covalent linker (L _a -L _f ) and siRNA molecule (T _a -T _f ), and moreover, where the group X in Figure 1 B may represent a combination of linkers (L _x ) and siRNA molecules T _x .

[0030] Another aspect of an embodiment is a method for the treatment of a subject having a diseases characterized by abnormal cell proliferation, such as cancer, by administration of an siRNA molecules as described herein according to one embodiment of the present invention covalently conjugated to cobalamin at the passenger strand.

[0031] Another aspect of an embodiment is a method for targeted siRNA drug delivery to the liver by administration of one or more siRNA molecules covalently conjugated to cobalamin and further complexed to transcobalamin I.

[0032] Another aspect of an embodiment is a method for targeted drug delivery to the kidney by administration of one or more siRNA molecules covalently conjugated to cobalamin and further complexed to transcobalamin II. DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0033] The accompanying drawings, which are incorporated into and form part of the specification, illustrate one or more of the underlying principles and embodiments of the invention. These drawings are for purposes of illustrating the invention and are not to be construed as limiting the invention.

[0034] Figure 1A-B illustrates the chemical structure of cobalamin (Cbl). Figure 1A illustrates the structure of cobalamin (Cbl) as described herein, may encompasses structural variants where the apical X group bound to cobalt may include, but not be strictly limited to, CN, Me, 5’-adenosyl, S- glutathionyl, alkoxy, lower alkyl, aryl, substituted aryl, and heteroaryl. The side chains off the central corrin core structure of Cbl are designated arbitrarily with lower case letters. It is understood that under certain physiological conditions, such as binding to specific proteins, the apical adenosyl ligand on the alpha face of cobalamin may be coordinated to cobalt. Figure 1B illustrates the general structure of various Cbl conjugates, wherein covalent linkers are represented by L and drug molecules, or molecules otherwise possessing a biological activity, are represented by T. The subscripts are meant to designate the Cbl cleavage side chain or position at which the conjugation is present. The value of X is Figure 1B may represent the groups described in Figure 1A but may also include a linker bound to a drug molecule, or molecule with biological activity, designated as X = Lx- Tx, where Lx is a covalent linker and Tx is the molecule with biological activity, which could be a nucleic acid such as an siRNA.

[0035] Figure 2A-C illustrates the interaction between covalent conjugates of siRNA, assumed to be covalently linked at the sense (i.e., guide, or passenger) strand to cobalamin, as described in Figure 1 B, and further complexed to one of two plasma chaperone proteins TCNx, wherein x may be 1 (TCN1) or 2 (TCN2), and a cell expressing a transmembrane receptor, and the mechanism of cellular uptake for such conjugates. FIG.2A illustrates a covalent complex of an siRNA molecule and cobalamin (siRNA-Cbl), bound to a plasma chaperone protein (Cblx) is shown to be proximal to a cell expressing a transmembrane receptor for the complex, wherein the receptor could be CD320 or LRP2, which bind cobalamin complexes of TCN2, or ASGR, which binds cobalamin complexes of TCN1 , in its desialylated form. Figure 2B illustrates the ligand binds to the receptor and is internalized into the cell via a process of receptor-mediated endocytosis (RME) into the endosomal compartment (not shown). Figure 2C illustrates the conjugate siRNA-Cbl is released from the endosome into the cytosol.

[0036] Figure 3A-F illustrates linkers corresponding to L _a -L _e (see Figure 1 B) that covalently connect the 3’-phosphate group of an siRNA sense strand [siRNAjs to one (or more) of the peripheral carboxy groups of cobalamin (Figure 1 B). It is understood that the representation “[Cbl]-C(0)-“ in the figure denotes the cobalamin core structure and the carboxy on one of the side chains. For all structures in Figure 3A-F, n = 1-100, R = H, alkyl, aryl, heteroaryl, alkoxy, and all stereogenic centers could be either in the R or S configuration.

[0037] Figure 4A-F illustrates linkers corresponding to L _f in Figure 1 B. It is understood that

“[Cbl]-0-” denotes the cobalamin core structure and the oxygen of the ribose moiety as shown in Figure 1 B, to which the linkers L _f covalently connect the 3’-phosphate group of an siRNA sense strand [siRNAJs. For all structures in Figure 4A-F, n = 1-100, R = H, alkyl, aryl, heteroaryl, alkoxy, and all stereogenic centers could be either in the R or S configuration.

[0038] Figure 5A-F illustrates linkers that covalently connect the cobalt atom apical position

(Figure 1B, Co-X) to the 3’-phosphate group of an siRNA sense strand [siRNAJs The group Q could represent O, NH, or two hydrogen atoms (i.e. , as would be attached to carbon creating a methylene). In addition, Q’ could represent O, NH, or methylene. For all structures in Figure 5A-F, n = 1-100, R = H, alkyl, aryl, heteroaryl, alkoxy, and all stereogenic centers could be either in the R or S configuration.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The definitions of certain terms are set out below. It is understood that in the event a specific term is not defined herein below, that term shall have a meaning within its typical use within context by those of ordinary skill in the art.

[0040] It is to be noted that as used herein and in the appended claims, the singular forms

"a," "an" and "the" include plural references unless the context clearly dictates otherwise.

[0041] As used in this document, the terms vitamin B12 and cobalamin (Cbl) refer to chemical compounds described in Figure 1 A, including naturally occurring and synthetic forms, wherein the substituent X may represent CN, Me, 5’-adenosyl, S-glutathionyl, alkoxy, lower alkyl, aryl, substituted aryl, and heteroaryl. It is also understood that the adenosyl ligand on the alpha face of cobalamin may in some circumstances not be coordinated to cobalt.

[0042] As defined in the present invention, a small interfering RNA (siRNA) is a double stranded RNA interference (RNAi) agent comprising a double-stranded ribonucleic acid (dsRNA), which is 16-30 nucleotide pairs in length, for inhibiting the expression of a gene, wherein the first dsRNA comprises a sense (or passenger) strand and the second is an antisense (or guide) strand forming a duplex containing complementary or mostly complementary Watson-Crick base pairs. The antisense strand of the siRNA is largely complementary to a sequence in messenger RNA (mRNA) encoding a targeted gene. [0043] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene of interest for example a CD320 gene or an LRP2 gene, including mRNA that is a product of RNA processing of a primary transcription product.

[0044] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

[0045] "G,” "C," "A" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. "T” and "dT" are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine, 2'- deoxythymidine or thymidine. However, it will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.

[0046] In one embodiment, at least one strand of the RNA molecule has a 3' overhang from about 1 to about 6 nucleotides {e.g., pyrimidine nucleotides, purine nucleotides) in length. In other embodiments, the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length or, for example, the overhang can be up to 14 nucleotides if the guide strand were a 27-mer. In one embodiment the RNA molecule is double stranded, one strand has a 3' overhang and the other strand can be blunt-ended or have an overhang. In the embodiment in which the RNA molecule is double stranded and both strands comprise an overhang, the length of the overhangs may be the same or different for each strand. In a particular embodiment, the RNA of the present invention comprises 21-27 nucleotide strands which are Watson-Crick paired and which have overhangs of from about 1 to about 3, particularly about 2, nucleotides on both 3' ends of the RNA. In order to further enhance the stability of the RNA of the present invention, the 3' overhangs can be stabilized against degradation. In one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by unnatural nucleotides, e.g., substitution of uridine 2 nucleotide 3' overhangs by 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi. The absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium. The 3'- overhangs can be further stabilized by introduction of phosphorothioate groups in place of the phosphodiesters.

[0047] In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, an "RNAi agent" may include ribonucleotides with chemical modifications; ⁵³ an RNAi agent may include substantial modifications at multiple nucleotides or at a single nucleotide. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by "RNAi agent" for the purposes of this specification and claims. Examples of such modifications would include but not be limited to modifications to the ribose moieties of the nucleotides such as: 2'-deoxy, 2'- deoxyfluoro, 2'-methoxy (2'-0-methyl), ⁵⁴ ' ⁵⁵ and 2'-methoxyethyl, wherein it is understood that the stereochemistry of the 2'- substituent could be in the ribo- or arabino- orientation. Another modification could be 2'- trifluoromethoxy. Other modifications to the ribose moieties could include bridging modifications such that the 2'-carbon of the sugar moiety is covalently linked to the 4'-carbon of the sugar moiety by a methylene or methoxymethylene group to afford bridged nucleotides described in the art as LNA and (S)-cET, respectively. ⁵³ In addition, the sugar moiety could be modified by removal of the bond between carbons C2' and C3' to afford "open" chain nucleotides analogous to those described in WO 2011/139843 A2. The ribose moiety of the RNA nucleotides could also be replaced by a morpholino group to afford PMO nucleotides. Modifications to the phosphate diester moieties of the nucleotides are also possible and could include but not be limited to replacement of the phosphodiester group by phosphorothioate and thio phosphoramidate. ⁵⁶ The ends of the sense and antisense strands could be modified with 2'-deoxynucleotides such as dT and, further, the dT nucleotides could be modified by phosphorothioate groups in place of diphosphate esters.

[0048] The term "sense strand," as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0049] As used herein, the term "cleavage region" refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11 , 12 and 13.

[0050] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C. or 70 °C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. For example, a complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0051] Sequences can be "fully complementary" with respect to each when there is basepairing of the nucleotides of the first nucleotide sequence with the nucleotides of the second nucleotide sequence over the entire length of the first and second nucleotide sequences. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary" for the purposes described herein.

[0052] "Complementary" sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non- Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0053] The terms "complementary," "fully complementary" and "substantially complementary" herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

[0054] As used herein, a polynucleotide that is "substantially complementary to at least part of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a gene) including a 5' UTR, an open reading frame (ORF), or a 3' UTR. For example, a polynucleotide is complementary to at least a part of a mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a gene.

[0055] The term "inhibiting," as used herein, is used interchangeably with "reducing,"

"silencing," "downregulating," "suppressing" and other similar terms, and includes any level of inhibition.

[0056] The phrase "inhibiting expression of a CD320," "inhibiting expression of a LRP2" as used herein, includes inhibition of expression of any CD320 or LRP2 gene (such as the identified gene from, e.g., a mouse, a rat, a monkey, or a human) as well as variants, (e.g., naturally occurring variants), or mutants of the identified gene. Thus, the gene may be a wild-type gene, a mutant gene, or a transgenic gene in the context of a genetically manipulated cell, group of cells, or organism.

[0057] "Inhibiting expression of a gene" includes any level of inhibition of a gene, e.g., at least partial suppression of the expression of a gene, such as an inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In a preferred embodiment the inhibition is assessed by expressing the level of protein in treated cells as a percentage of the level of mRNA in control cells.

[0058] Normalized protein level for treated cells/Normalized protein level for control cells.

The control cells are the negative control siRNA. Normalized means the protein level is normalized to the level of a housekeeping protein.

[0059] The expression of a gene may be assessed based on the level of any variable associated with gene expression, e.g., mRNA level, protein level. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

[0060] The term “conjugate” as used herein describes a molecule consisting two individually defined molecules which are conjoined through a covalent bond (e.g., a molecule consisting of a cobalamin molecule covalently modified with an siRNA molecule). Conjugates may entail direct covalent bonding of two molecules to one another or, alternatively, a “linker” or “spacer” moiety may be introduced between the two molecules in such a manner that each molecule has a covalent bond to the linker. [0061] The term “linker” is used throughout the specification within context to describe a covalent moiety that attaches two molecules that comprise a conjugate. Linkers may vary in size, chemical functionality, and biochemical characteristics. ⁵⁷ For example, a linker with two identical functional groups at each of its termini is referred to as “homobifunctional” while one with two different functional groups is referred to as “heterobifunctional.” Moreover, a linker may be designed to undergo cleavage in a certain biological setting or to be stable under such conditions (e.g., the acidic conditions of the endosome). ⁵⁰

[0062] Without being limited by theory, the term "siRNA" refers to a compound, cocktail, composition or agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via the RISC/AGO (RNA-induced silencing complex) complex, whereby the guide strand of the siRNA hybridizes with its complementary mRNA molecule. ⁵⁸ The mRNA is degraded by the catalytic RISC/AGO complex, which has RNAse cleave activity, resulting in mRNA degradation and the protein encoded by the mRNA is not produced or is produced at a reduced level as compared to untreated cell. This causes the "knockdown" effect or reduced protein levels of the gene targeted by the siRNA compared to control treated or untreated cells.

[0063] By “oncogenic protein” is meant a protein, normally or abnormally expressed (e.g., containing mutations, abnormal protein fusions, abnormal level of expression), or functionally which exhibits abnormal location or function (expressed at a level or with a localization that is different from a normal cell (displaying abnormal levels of catalytic or signaling activity, abnormal association with other proteins, post-translational modification or other abnormality leading to structural aberrations such as misfolding) such that it’s presence and function contributes solely, or in combination with other factors, to the a cell displaying the hallmarks of cancer. ⁵⁹

[0064] One embodiment of present invention provides a method for drug delivery of one or more therapeutic siRNA molecules as covalent conjugates to cobalamin (Cbl) in humans or animals.

[0065] An aspect of an embodiment of the invention is a method for administration of siRNA molecules to humans or animals comprising the covalent conjugation of the passenger (sense) strand of such siRNA molecules to cobalamin. The conjugation may be direct or through a covalent linker.

[0066] In a particular embodiment of the invention, we describe a method the treatment of diseases characterized by aberrant cell proliferation (e.g, neoplastic diseases, cancer) by administration to the patient of a cobalamin siRNA conjugate wherein the siRNA targets the mRNA of an oncogenic protein.

[0067] Another aspect of an embodiment is a method for delivery of siRNA molecules to cells in vitro as covalent conjugates wherein the siRNA molecule is covalently conjugated to cobalamin on the passenger strand. [0068] Another embodiment of the invention is a method for the administration of siRNA molecules to humans or animals as covalent conjugates to cobalamin which are further complexed with a cobalamin plasma chaperone protein such as transcobalamin I, transcobalamin II, or transcobalamin III.

[0069] An aspect of an embodiment of the invention is conjugate compositions, illustrated in

Formula B, where a cobalamin molecule is covalently modified on the carboxy group of a side chain (Figure 1 B, positions a-f), with a covalent linker (L _a -L _f ) and siRNA molecule (T _a -T _f ), and moreover, where the group X in Formula B may represent a combination of linkers (L _x ) and siRNA molecules T _x .

[0070] In a more specific embodiment, we describe compositions wherein the linkers are described as those in Figure 3A-F, also see Figure 1B, connecting one or more of the carboxyl functional groups of cobalamin to the 3’-phosphate of the sense strand of an siRNA [siRNAjs.

[0071] In another specific embodiment, we describe compositions, wherein the linkers described are those in Figure 4A-F, also see Figure 1 B, connecting the ribose oxygen of cobalamin to the 3’-phosphate of the sense strand of an siRNA [siRNAjs.

[0072] In yet another specific embodiment, we describe compositions, wherein the linkers described are those in Figure 5A-F, also see Figure 1 B, connecting the cobalt atom of cobalamin, at the apical position (Figure 1B, X) to the 3’-phosphate of the sense strand of an siRNA [siRNAjs.

[0073] Another aspect of an embodiment is a method for the treatment of diseases characterized by abnormal cell proliferation, such as cancer, by administration of siRNA molecules covalently conjugated to cobalamin at the passenger strand. Tablel provides for examples.

[0074] TABLE1 ^a Patisiran package insert: world wide web address: accessdata.fda.gov/drugsatfda_docs/label/2018/210922s000lbl. pdf. ^b Givosiran package insert: world wide web address: alnylam.com/wp- content/uploads/pdfs/GI VLAARI-Prescribing-lnformation.pdf. ^c GalNac conjugate ^d Inclisiran structure reference: world wide web address: cliniexpert.com/attachment/20200818/fa437c9f424149f1 b7606220d107aeb7.jpg. ^e https://images.rxlist.com/images/rxlist/oxlumo1 .gif

Abbreviations

* indicates phosphorothioate m indicates 2’-methoxy f indicates 2’-fluoro d indicates 2’-deoxy

[0075] Another aspect of an embodiment is a method for targeted siRNA drug delivery to the liver by administration of one or more siRNA molecules covalently conjugated to cobalamin and further complexed to transcobalamin I. For example, the siRNA sequence or sequences that are identified is Table 2, Table 3, Table 6 but limited thereto as the drug delivery vehicle can be conjugated to another siRNA than those identified herein.

[0076] TABLE 2 CD320

OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5' TO 3')

OSC1A UCUUAUCCCUGCGCACGCGCA [dT][dT](SEQ OSC1S UGCGCGUGCGCAGGGAUAAGA[dT][dT]

ID NO 1) (SEQ ID NO: 94) OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5' TO 3')

OSC2A UCUCUUAUCCCUGCGCACGCG [dT][dT] (SEQ OSC2S CGCGUGCGCAGGGAUAAGAGA[dT][dT] ID NO 2) (SEQ ID NO: 95)

OSC3A AUGCUGUCCCCACAGCGGCGC[dT][dT] (SEQ OSC3S GCGCCGCUGUGGGGACAGCAU[dT][dT] ID NO 3) (SEQ ID NO: 96)

OSC4A AUCCAACCGCCGCUCAUGCUG[dT][dT] (SEQ OSC4S CAGCAUGAGCGGCGGUUGGAU[dT][dT] ID NO 4) (SEQ ID NO: 97)

OSC5A UGGAAAGCGGGCUCGCGGCGG[dT][dT] (SEQ OSC5S CCGCCGCGAGCCCGCUUUCCA[dT][dT] ID NO 5) (SEQ ID NO: 98)

OSC6A AACUUGGUGGGUGGGCACGAG[dT][dT] (SEQ OSC6S CU CG U GCCCACCCACCAAG U U [dT] [dT] ID NO 6) (SEQ ID NO: 99)

OSC7A UGGAACUUGGUGGGUGGGCAC[dT][dT] (SEQ OSC7S GUGCCCACCCACCAAGUUCCA[dT][dT] ID NO 7) (SEQ ID NO: 100)

OSC8A ACUGGAACUUGGUGGGUGGGC[dT][dT] (SEQ OSC8S GCCCACCCACCAAG U U CCAG U [dT] [dT] ID NO 8) (SEQ ID NO: 101)

OSC9A UAAGCCACUGGUGCGGCACUG[dT][dT] (SEQ OSC9S CAGUGCCGCACCAGUGGCUUA[dT][dT] ID NO 9) (SEQ ID NO: 102)

OSCIOA ACG CAU AAG CCACU G G U GCG G [dT] [dT] (SEQ OSCIOS CCGCACCAGUGGCUUAUGCGU[dT][dT] ID NO 10) (SEQ ID NO: 103)

OSC11A UCCAAGUCCCUGUCGCAGCGC[dT][dT] (SEQ OSC11S GCGCUGCGACAGGGACUUGGA[dT][dT] ID NO 11) (SEQ ID NO: 104)

OSC12A UCCUCAUCGCUGCCAUCGCUG[dT][dT] (SEQ OSC12S CAGCGAUGGCAGCGAUGAGGA[dT][dT] ID NO 12) (SEQ ID NO: 105)

OSC13A UCACUGACGCCGGUGCAGGGG[dT][dT] (SEQ OSC13S CCCCUGCACCGGCGUCAGUGA[dT][dT] ID NO 13) (SEQ ID NO: 106)

OSC14A UUGUCAGUUCCCCCAGAGCAG[dT][dT] (SEQ OSC14S CUGCUCUGGGGGAACUGACAA[dT][dT] ID NO 14) (SEQ ID NO: 107)

OSC15A UUCUUGUCAGUUCCCCCAGAG[dT][dT] (SEQ OSC15S CUCUGGGGGAACUGACAAGAA[dT][dT] ID NO 15) (SEQ ID NO: 108)

OSC16A AGUUUCUUGU CAG U U CCCCCA[dT] [dT] (SEQ OSC16S UGGGGGAACUGACAAGAAACU[dT][dT] ID NO 16) (SEQ ID NO: 109)

OSC17A- CAGUUGCGCAGUUUCUUGUCAGUUC[dT][dT OSC17S GAACU G ACAAG AAACU GCG CAACU G

1 ] (SEQ ID NO 17) -1 [dT][dT] (SEQ ID NO: 110)

OSC17A- CAGUUGCGCAGUUUCUUGUCAGUUC[dT]*[d OSC17S

2 T] (SEQ ID NO 18) -2 GAACU G ACAAG AAACU GCG CAACU G [dT] *

[dT] (SEQ ID NO: 111)

OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] OSC17S [mG][mA][mA][mC][mU][mG][mA][mC][

3 [mA][mG][mU][mU][mU][mC][mU][mU][mG -3 mA][mA][mG][mA][mA][mA][mC][mU][m

][mU][mC][mA][mG][mU][mU][mC][dT]*[dT] G][mC][mG][mC][mA][mA][mC][mU][mG]

] (SEQ ID NO 19) [dT] * [dT] (SEQ ID NO: 112) OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5' TO 3')

OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] OSC17S

4 [mA][mG][mU][mU][mU][mC][mU][mU][mG -4 [mG][mA][mA][mC][mU][mG][mA][mC][ ][mU][mC][mA][mG][mU][mU][mC][dT]*[dT] mA] [mA] [mG] [mA] [mA] [mA] [mC] [mU] [m ] (SEQ ID NO 20) G][mC][mG][mC][mA][mA][mC][mU][mG] (SEQ ID NO: 113)

OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] OSC17S [mG][mA][mA][mC][mU][mG][mA][mC][

5 [mA][mG][mU][mU][mU][mC][mU][mU][mG -5 mA][mA][mG][mA][mA][mA][mC][mll][m ][mU][mC][mA][mG][mU][mU][mC] ] (SEQ ID G][mC][mG][mC][mA][mA][mC][mll][mG] NO 21) [dT]*[dT] (SEQ ID NO: 114)

OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] OSC17S [mG][mA][mA][mC][mll][mG][mA][mC][

6 [mA][mG][mU][mU][mU][mC][mU][mU][mG -6 mA][mA][mG][mA][mA][mA][mC][mll][m ][mU][mC][mA][mG][mU][mU][mC] ] (SEQ ID G][mC][mG][mC][mA][mA][mC][mll][mG] NO 22) (SEQ ID NO: 115)

OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] OSC17S

7 [mA][mG][mU][mU][mU][mC][mU][mU][mG -7 GAACU G ACAAG AAACU GCG CAACUG [dT] * ][mU][mC][mA][mG][mU][mU][mC][dT]*[dT] [dT] (SEQ ID NO: 116) ] (SEQ ID NO 23)

OSC17A- [mC] [2f A] [mG] [2fU] [mU] [2fG] [mC] [2fG] [mC] OSC17S [2fG] [m A] [2f A] [mC] [2f U] [mG] [2f A] [mC] [2

8 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -8 fA] [m A] [2fG] [mA] [2f A] [mA] [2fC] [mU] [2f ][mU][2fC][mA][2fG][mU][2fU][mC][dT]*[dT] G] [mC] [2fG] [mC] [2fA] [mA] [2fC] [mU] [2fG (SEQ ID NO 24) ] [dT] * [dT] (SEQ ID NO: 117)

OSC17A- [mC] [2f A] [mG] [2fU] [mU] [2fG] [mC] [2fG] [mC] OSC17S [2fG] [m A] [2f A] [mC] [2f U] [mG] [2f A] [mC] [2

9 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -9 fA] [m A] [2fG] [mA] [2f A] [mA] [2fC] [mil] [2f ][mU][2fC][mA][2fG][mU][2fU][mC] (SEQ ID G] [mC] [2fG] [mC] [2fA] [mA] [2fC] [mil] [2fG NO 25) ] [dT] * [dT] (SEQ ID NO: 118)

OSC17A- [mC] [2f A] [ G] [2fU] [mU] [2fG] [mC] [2fG] [mC] OSC17S [2fG] [m A] [2f A] [mC] [2f U] [mG] [2f A] [mC] [2

10 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -10 fA] [m A] [2fG] [mA] [2f A] [mA] [2fC] [mil] [2f ][mU][2fC][mA][2fG][mU][2fU][mC][dT]*[dT] G] [mC] [2fG] [mC] [2fA] [mA] [2fC] [mil] [2fG (SEQ ID NO 26) ] (SEQ ID NO: 119)

OSC17A- [mC] [2f A] [mG] [2fU] [mU] [2fG] [mC] [2fG] [mC] OSC17S

11 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -11 [2fG] [m A] [2f A] [mC] [2fU] [mG] [2f A] [mC] [2 ][mU][2fC][mA][2fG][mU][2fU][mC] (SEQ ID fA] [m A] [2fG] [mA] [2f A] [mA] [2fC] [mil] [2f NO 27) G] [mC] [2fG] [mC] [2fA] [mA] [2fC] [mil] [2fG ] (SEQ ID NO: 120)

OSC17A- [2fC] [mA] [2fG] [mU] [2fU] [mG] [2fC] [mG] [2fC] OSC17S

12 [mA] [2fG] [m U] [2fU] [mU] [2fC] [m U] [2f U] [mG -12 [mG] [2fA] [mA] [2fC] [mil] [2fG] [mA][2fC] [

][2fU][mC][2fA][mG][2fU][mU][2fC][dT]*[dT] mA] [2f A] [mG] [2fA] [mA] [2f A] [mC] [2fU] [m (SEQ ID NO 28) G] [2fC] [mG] [2fC] [mA] [2f A] [mC] [2fU] [mG ] [dT] * [dT] (SEQ ID NO: 121)

OSC17A- [2fC] [m A] [2fG] [mU] [2fU] [mG] [2fC] [mG] [2fC] OSC17S [mG] [2fA] [mA] [2fC] [mU] [2fG] [mA][2fC] [

13 [mA] [2fG] [m U] [2fU] [mU] [2fC] [m U] [2f U] [mG -13 mA] [2f A] [mG] [2fA] [mA] [2f A] [mC] [

][2fU][mC][2fA][mG][2fU][mU][2fC] (SEQ ID 2fU][mG] [2fC] [mG] [2fC] [mA] [2fA] [mC][2f NO 29) U][mG] (SEQ ID NO: 122) OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5' TO 3')

OSC17A- [mC] [2fA] [2fG] [2fU][2fU] [2fG] [2fC] [2fG][2fC] OSC17S [2f U] [mG] [2f A] [mC] [2fA] [mA] [2fG] [m A] [2

14 [2fA] [2fG] [2fU][2fU][2fU] [2fC] [2fU] [2fU][2fG -14 fA] [m A] [2fC] [mU] [2fG] [mC] [2fG] [mC] [2f A ] [2fU ] [2fC] [2f A] [2f G] [2f U ] [2fU ] [2fC] [dT] * [dT] ][mA] [2fC][mU] [2fG] [dT]* [dT] (SEQ ID (SEQ ID NO 30) NO: 123)

OSC17A- [mC] [2fA] [2fG] [2fU][2fU] [2fG] [2fC] [2fG][2fC] OSC17S

15 [2fA] [2fG] [2fU][2fU][2fU] [2fC] [2fU] [2fU][2fG -15 [2fG] [m A] [2f A] [mA] [2fC] [mU] [2fG] [mC] [2 ] [2fU ] [2fC] [2f A] [2f G] [2f U ] [2fU ] [2fC] [dT] * [dT] fG] [mC] [2fA] [mA] [2fC] [mU] [2fG] [dT] * [dT (SEQ ID NO 31) ] (SEQ ID NO: 124)

OSC17A- [mC] [2f A] [mG] [2fU] [mU] [2fG] [mC] [2fG] [mC] OSC17S

16 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -16 [2fG] [m A] [2f A] [mA] [2fC] [mU] [2fG] [mC] [2 ][mU][2fC][mA][2fG][mU][2fU][mC][dT]*[dT] fG] [mC] [2fA] [mA] [2fC] [mU] [2fG] [dT] * [dT (SEQ ID NO 32) ] (SEQ ID NO: 125)

OSC17A- [mC] [2f A] [mG] [2fU] [mU] [2fG] [mC] [2fG] [mC] OSC17S [2fG] [m A] [2f A] [mA] [2fC] [mU] [2fG] [mC] [2

17 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -17 fG] [mC] [2fA] [mA] [2fC] [mU] [2fG] (SEQ ID ][mU][2fC][mA][2fG][mU] (SEQ ID NO 33) NO: 126)

OSC17A- [mC] [2f A] [ G] [2fU] [mU] [2fG] [mC] [2fG] [mC] OSC17S [2fG] [m A] [2f A] [mA] [2fC] [mU] [2fG] [mC] [2

18 [2fA][mG][2fU][mU][2fU][mC][2fU][2fU][2fG -18 fG] [mC] [2fA] [mA] [2fC] [mU] [2fG] (SEQ ID ] [2fU ] [2fC] [2f A] [2fG] [2fU ] (SEQ ID NO 34) NO: 127)

OSC18A AUGCAGUCAUCGCUCAGCGUG[dT][dT] (SEQ OSC18S CACGCUGAGCGAUGACUGCAU [dT] [dT]

ID NO 35) (SEQ ID NO: 128)

OSC19A AAUG CAG U CAU CG CU CAGCG U [dT] [dT] (SEQ OSC19S ACGCUGAGCGAUGACUGCAUU[dT][dT]

ID NO 36) (SEQ ID NO: 129)

OSC20A UGGAAUGCAGUCAUCGCUCAG[dT][dT] (SEQ OSC20S CUGAGCGAUGACUGCAUUCCA[dT][dT]

ID NO 37) (SEQ ID NO: 130)

OSC21A AGUGGAAUGCAGUCAUCGCUC[dT][dT] (SEQ OSC21S GAGCGAUGACUGCAUUCCACU[dT][dT]

ID NO 38) (SEQ ID NO: 131)

OSC22A ACAGUCUGGGUGGCCGUCGCA[dT][dT] (SEQ OSC22S UGCGACGGCCACCCAGACUGU[dT][dT]

ID NO 39) (SEQ ID NO: 132)

OSC23A AUUGGUUCCACAGCCGAGCUC[dT][dT] (SEQ OSC23S GAGCUCGGCUGUGGAACCAAU[dT][dT]

ID NO 40) (SEQ ID NO: 133)

OSC24A UCUCAUUGGUUCCACAGCCGA[dT][dT] (SEQ OSC24S UCGGCUGUGGAACCAAUGAGA[dT][dT]

ID NO 41) (SEQ ID NO: 134)

OSC25A AUCUCAUUGGUUCCACAGCCG[dT][dT] (SEQ OSC25S CGGCUGUGGAACCAAUGAGAU[dT][dT]

ID NO 42) (SEQ ID NO: 135)

OSC26A AGGAUCUCAUUGGUUCCACAG[dT][dT] (SEQ OSC26S CUGUGGAACCAAUGAGAUCCU[dT][dT]

ID NO 43) (SEQ ID NO: 136)

OSC27A UGAGAGAGGUGACACUCUCCA[dT][dT] (SEQ OSC27S UGGAGAGUGUCACCUCUCUCA[dT][dT]

ID NO 44) (SEQ ID NO: 137) OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5' TO 3')

OSC28A UGGUUGUGGCAUUCCUGAGAG[dT][dT] OSC28S CUCUCAGGAAUGCCACAACCA[dT][dT] (SEQ ID NO 45) (SEQ ID NO: 138)

OSC29A UGGCAUUCCCGACAGAGGGGA[dT][dT] (SEQ OSC29S UCCCCUCUGUCGGGAAUGCCA[dT][dT] ID NO 46) (SEQ ID NO: 139)

OSC30A AGGAUGUGGCAUUCCCGACAG[dT][dT] (SEQ OSC30S CUGUCGGGAAUGCCACAUCCU[dT][dT] ID NO 47) (SEQ ID NO: 140)

OSC31A UUCCAGACUGGUCUCCGGCAG[dT][dT] (SEQ OSC31S CUGCCGGAGACCAGUCUGGAA[dT][dT] ID NO 48) (SEQ ID NO: 141)

OSC32A AUAACCCCAUAGGCAGUUGGG[dT][dT] (SEQ OSC32S CCCAACUGCCUAUGGGGUUAU[dT][dT] ID NO 49) (SEQ ID NO: 142)

OSC33A UUGCACUGAGCACCGCAGCAG[dT][dT] (SEQ OSC33S CUGCUGCGGUGCUCAGUGCAA[dT][dT] ID NO 50) (SEQ ID NO: 143)

OSC34A AAAAGGAGGAGGGUGGCGGUG[dT][dT] (SEQ OSC34S CACCGCCACCCUCCUCCUUUU[dT][dT] ID NO 51) (SEQ ID NO: 144)

OSC35A ACAAAAGGAGGAGGGUGGCGG[dT][dT] (SEQ OSC35S CCGCCACCCUCCUCCUUUUGU[dT][dT] ID NO 52) (SEQ ID NO: 145)

OSC36A ACCAGUAACCCCAGUGGGCGG[dT][dT] (SEQ OSC36S CCGCCCACUGGGGUUACUGGU[dT][dT] ID NO 53) (SEQ ID NO: 146)

OSC37A U U CAU GG CCACCAG U AACCCC[dT] [dT] (SEQ OSC37S GGGGUUACUGGUGGCCAUGAA[dT][dT] ID NO 54) (SEQ ID NO: 147)

OSC38A ACUCCUUCAUGGCCACCAGUA[dT][dT] (SEQ OSC38S UACUGGUGGCCAUGAAGGAGU[dT][dT] ID NO 55) (SEQ ID NO: 148)

OSC39A UUCUGACAGCAGCAGGGACUC[dT][dT] (SEQ OSC39S GAGUCCCUGCUGCUGUCAGAA[dT][dT] ID NO 56) (SEQ ID NO: 149)

OSC40A UCUGUUCUGACAGCAGCAGGG[dT][dT] (SEQ OSC40S GGCUGCUGCUGUCAGAACAGA[dT][dT] ID NO 57) (SEQ ID NO: 150)

OSC41A UCUUCUGUUCUGACAGCAGCA[dT][dT] (SEQ OSC41S UGCUGCUGUCAGAACAGAAGA[dT][dT] ID NO 58) (SEQ ID NO: 151)

OSC42A AGGUCUUCUGUUCUGACAGCA[dT][dT] (SEQ OSC42S UGCUGUCAGAACAGAAGACCU[dT][dT] ID NO 59) (SEQ ID NO: 152)

OSC43A UUGUCCUCAGGGCAGCGAGGU[dT][dT] (SEQ OSC43S ACCUCGCUGCCCUG AG G ACAA [dT] [dT] ID NO 60) (SEQ ID NO: 153)

OSC44A AAG U GCU U G U CCU CAG GGCAG [dT] [dT] (SEQ OSC44S CU G CCCU G AGG ACAAGCACU U [dT] [dT] ID NO 61) (SEQ ID NO: 154)

OSC45A UACCCAUCCGCAUCACUGCUC[dT][dT] (SEQ OSC45S GAGCAGUGAUGCGGAUGGGUA[dT][dT] ID NO 62) (SEQ ID NO: 155)

OSC46A UCUCUGAGGGCUGGUGUGCCC[dT][dT] (SEQ OSC46S GGGCACACCAGCCCUCAGAGA[dT][dT] ID NO 63) (SEQ ID NO: 156) OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5' TO 3')

OSC47A- AAGAGCUCAGGUCUCUGAGGG[dT][dT] (SEQ OSC47S CCCUCAGAGACCUGAGCUCUU [dT][dT]

1 ID NO 64) -1 (SEQ ID NO: 157)

OSC47A- AAGAGCUCAGGUCUCUGAGGG[dT]*[dT] OSC47S CCCUCAGAGACCUGAGCUCUU[dT]*[dT]

2 (SEQ ID NO 65) -2 (SEQ ID NO: 158)

OSC47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] OSC47S

3 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -3 [mC][mC][mC][mU][mC][mA][mG][mA][ [mG][mG][mG][dT]*[dT] (SEQ ID NO 66) mG][mA][mC][mC][mU][mG][mA][mG][m C][mU][mC][mU][mU][dT]*[dT] (SEQ ID NO: 159)

OSC47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] OSC47S [mC][mC][mC][mU][mC][mA][mG][mA][

4 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -4 mG][mA][mC][mC][mU][mG][mA][mG][m [mG][mG][mG][dT]*[dT] (SEQ ID NO 67) C][mU][mC][mU][mU] (SEQ ID NO: 160)

OSC47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] OSC47S [mC][mC][mC][mU][mC][mA][mG][mA][

5 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -5 mG][mA][mC][mC][mU][mG][mA][mG][m [mG][mG][mG] (SEQ ID NO 68) C][mU][mC][mU][mU][dT]*[dT] (SEQ ID NO: 161)

OSC47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] OSC47S [mC][mC][mC][mU][mC][mA][mG][mA][

6 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -6 mG][mA][mC][mC][mU][mG][mA][mG][m [mG][mG][mG] (SEQ ID NO 69) C][mU][mC][mU][mU] (SEQ ID NO: 162)

OSC47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] OSC47S CCCUCAGAGACCUGAGCUCUU[dT]*[dT]

7 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -7 (SEQ ID NO: 163) [mG][mG][mG][dT]*[dT] (SEQ ID NO 70)

OSC47A- [mA] [2fA] [mG] [2f A] [mG] [2fC] [mU] [2fC] [mA] OSC47S [2fC] [mC] [2fC] [mU] [2fC] [mA] [2fG] [mA] [2

8 [2fG] [mG] [2fU] [mC] [2fU] [ C] [2fU] [ G] [2f A] -8 fG] [mA] [2fC] [mC] [2fU] [mG] [2fA] [mG] [2f [mG][2fG][mG][dT]*[dT] (SEQ ID NO 71) C][mU][2fC][mll][2fU][dT]*[dT] (SEQ ID NO: 164)

OSC47A- [mA] [2fA] [mG] [2f A] [mG] [2fC] [mU] [2fC] [mA] OSC47S [2fC] [mC] [2fC] [mU] [2fC] [mA] [2fG] [mA] [2

9 [2fG] [mG] [2fU] [mC] [2fU] [mC] [2fU] [mG] [2f A] -9 fG] [mA] [2fC] [mC] [2fU] [mG] [2fA] [mG] [2f

[mG][2fG][mG] (SEQ ID NO 72) C][mU][2fC][mll][2fU][dT]*[dT] (SEQ ID NO: 165)

OSC47A- [mA] [2fA] [mG] [2f A] [mG] [2fC] [mU] [2fC] [mA] OSC47S [2fC] [mC] [2fC] [mU] [2fC] [mA] [2fG] [mA] [2

10 [2fG] [mG] [2fU] [mC] [2fU] [mC] [2fU] [mG] [2f A] -10 fG] [mA] [2fC] [mC] [2fU] [mG] [2fA] [mG] [2f [mG][2fG][mG][dT]*[dT] (SEQ ID NO 73) C][mU][2fC][mU][2fU] (SEQ ID NO: 166)

OSC47A- [mA] [2fA] [mG] [2f A] [mG] [2fC] [mU] [2fC] [mA] OSC47S [2fC] [mC] [2fC] [mU] [2fC] [mA] [2fG]][mA] [

11 [2fG] [mG] [2fU][mC] [2fU] [mC] [2fU] [mG] [2f A] -11 2fG [mA] [2fC] [mC] [2fU] [mG] [2f A] [mG] [2f [mG][2fG][mG] (SEQ ID NO 74) C][mU][2fC][mU][2fU] (SEQ ID NO: 167)

OSC47A- [2fA] [mA] [2fG] [mA] [2fG] [mC] [2fU] [mC] [2f A] OSC47S

12 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -12 [mC] [2fC] [mC] [2fU] [mC] [2fA][mG][2fA] [ [2fG][mG][2fG][dT]*[dT] (SEQ ID NO 75) mG][2fA][mC][2fC][mll][2fG][mA][2fG][m C][2fU][mC][2fU][mU][dT][dT]* (SEQ ID NO: 168) OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5' TO 3')

OSC47A- [2fA] [mA] [2fG] [mA] [2fG] [mC] [2f U] [mC] [2f A] OSC47S

13 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -13 [2fC] [mC] [2fC] [mU] [2fC] [mA] [2fG] [mA] [2

[2fG][mG][2fG] (SEQ ID NO 76) fG] [mA] [2fC] [mC] [2fU] [mG] [2fA] [mG] [2f C][mU][2fC][mU][2fU]-LIG-LINKER (SEQ ID NO: 169)

OSC47A- [mA] [2fA] [2fG] [ A] [2fG] [mC] [2f U] [mC] [2f A] OSC47S

14 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -14 [2fC] [mC] [2fC] [mU] [2fC] [mA] [2fG] [mA] [2

[2fG][mG][2fG] (SEQ ID NO 77) fG] [mA] [2fC] [mC] [2fU] [mG] [2fA] [mG] [2f C][mU][2fC][mU][2fU][dT]*[dT] (SEQ ID NO: 170)

OSC47A- [mA] [2fA] [2f G] [mA] [2fG] [mC] [2f U] [mC] [2f A] OSC47S

15 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -15 [2fG] [m A] [2fG] [mA] [2fC] [mC] [2f U] [mG] [ [2fG][mG][2fG][dT]*[dT] (SEQ ID NO 78) 2fA][mG][2fC][mU][2fC][mU][2fU][dT]*[d T] (SEQ ID NO: 171)

OSC47A- [2fA] [mA] [2fG] [mA] [2fG] [mC] [2f U] [mC] [2f A] OSC47S [2fG][mA][2fG][mA][2fC][mC][2fU][mG][

16 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -16 2fA][mG][2fC][mU][2fC][mll][2fU][dT]*[d [2fG][mG][2fG][dT]*[dT] (SEQ ID NO 79) T] (SEQ ID NO: 172)

OSC47A- [2fA] [mA] [2fG] [mA] [2fG] [mC] [2f U] [mC] [2f A] OSC47S

17 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -17 [2fG] [m A] [2fG] [mA] [2fC] [mC] [2f U] [mG] [ [2fG][mG][2fG][dT]*[dT] (SEQ ID NO 80) 2fA] [mG] [2fC] [m U] [2fC] [m U] [2f U] (SEQ ID NO: 173)

OSC47A- [2fA] [mA] [2fG] [mA] [2fG] [mC] [2f U] [mC] [2f A] OSC47S [2fG] [m A] [2fG] [mA] [2fC] [mC] [2f U] [mG] [

18 [mG] [2fG] [mU] [2fC] [mU] [2fC] [2f U] [2fG] [2fA] -18 2f A] [mG] [2fC] [m U] [2fC] [m U] [2fU] (SEQ [2fG][2fG][2fG] (SEQ ID NO 81) ID NO: 174)

OSC48A AAGAGCUCAGGUCUCUGAGGG[dT][dT] (SEQ OSC48S CCCUCAGAGACCUGAGCUCUU[dT][dT] ID NO 82) (SEQ ID NO: 175)

OSC49A AGAAGAGCUCAGGUCUCUGAG[dT][dT] (SEQ OSC49S CUCAGAGACCUGAGCUCUUCU[dT][dT] ID NO: 83) (SEQ ID NO: 176)

OSC50A AUAGGGAGUGUCCAGGGACCC[dT][dT] (SEQ OSC50S GGGUCCCUGGACACUCCCUAU[dT][dT] ID NO: 84) (SEQ ID NO: 177)

OSC51A UCCAUAGGGAGUGUCCAGGGA[dT][dT] (SEQ OSC51S UCCCUGGACACUCCCUAUGGA[dT][dT] ID NO: 85) (SEQ ID NO: 178)

OSC52A AUCUCCAUAGGGAGUGUCCAG[dT][dT] (SEQ OSC52S CUGGACACUCCCUAUGGAGAU[dT][dT] ID NO: 86) (SEQ ID NO: 179)

OSC53A UCAGUUCUGGCUGUGGCAGGU[dT][dT] (SEQ OSC53S ACCUGCCACAGCCAGAACUGA[dT][dT] ID NO: 87) (SEQ ID NO: 180)

OSC54A UUCUACCCCCUGGGAGCUGCC[dT][dT] (SEQ OSC54S GGCAGCUCCCAGGGGGUAGAA[dT][dT] ID NO: 88) (SEQ ID NO: 181)

OSC55A AAGCACAGGGCCGUUCUACCC[dT][dT] (SEQ OSC55S GGGUAGAACGGCCCUGUGCUU[dT][dT] ID NO: 89) (SEQ ID NO: 182)

OSC56A UGUCUUAAGCACAGGGCCGUU[dT][dT] (SEQ OSC56S AACGGCCCUGUGCUUAAGACA[dT][dT] ID NO: 90) (SEQ ID NO: 183) OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5' TO 3')

OSC57A AGUGUCUUAAGCACAGGGCCG[dT][dT] (SEQ OSC57S CGGCCCUGUGCUUAAGACACU[dT][dT] ID NO: 91) (SEQ ID NO: 184)

OSC58A UUUUUUGAGGAUGUGAAGCAA[dT][dT] OSC58S UUGCUUCACAUCCUCAAAAAA[dT][dT] (SEQ ID NO: 92) (SEQ ID NO: 185)

OSC59A UUUUUUUGAGGAUGUGAAGCA[dT][dT] OSC59S UGCUUCACAUCCU CAAAAAA A [dT] [dT] (SEQ ID NO: 93) (SEQ ID NO: 186)

[0077] TABLE 3 LRP2

[0078] TABLE 4 CD320 ANTISENSE TARGET

[0079] TABLE 5 LRP2 ANTISENSE TARGET

Table 6

[0080] Additional table of siRNA sequences

[0081] Key to modifications

[dT] = DNA base (T) within RNA oligo; [mA], [mG], [mC], [mU] “m” = 2’O-Methyl RNA wherein, mA, mC, mG, and mil are 2'-0-methyl adenosine, cytidine, guanosine, or uridine, respectively; 2fA, 2fC, 2fG, and 2fU are 2'-fluoro adenosine, cytidine, guanosine, or uridine, respectively; and * is a phosphorothioate linkage; and for example the sense strand is at least substantially complementary to the antisense strand.

[0082] In some embodiments, the antisense strand (identified with “A” in the OS ID name) and/or the sense strand (identified with “S” in the OS ID name) of an RNAi agent comprises or consists of a nucleobase sequence, for example, OSC17A-1”

CAGUUGCGCAGUUUCUUGUCAGUUC[dT][dT] (SEQ ID NO: 17), and the nucleobase sequence may include at least one or more nucleotides as a modified nucleotide, and wherein SEQ ID NO: 17 is located at positions 1 to 25 (5'- 3') of the antisense strand and forms a duplex with the corresponding sense strand (identified as OSC17S-1. In some embodiments, the antisense strand of an RNAi agent comprises or consists of a nucleobase sequence for example

CAGUUGCGCAGUUUCUUGUCAGUUC[dT][dT] (SEQ ID NO: 17), wherein all or substantially all or 1 , 2, 3, 4 or 5 of the nucleotides are modified nucleotides (see for example SEQ ID NO. 24), and wherein SEQ ID NO: 24 is located at positions 1 to 27 (5' -> 3') of the antisense strand. For any antisense or sense strand disclosed herein, in some embodiments, the antisense strand of an RNAi agent comprises or consists of the sequence (5 3') wherein * is a phosphorothioate linkage between deoxy thymine [dT]; and/or wherein mC, mA, mG, mil are 2’-0-methyl cytidine, 2’-0-methyl adenine, 2’-0-methyl guanosine, 2’-0-methyl uridine respectively; and/or wherein 2fA, 2fU, 2fG, 2fC are 2’-fluoro adenine, 2’-fluoro uridine, 2’-fluoro guanosine, and 2’-fluoro cytosine respectively. The antisense target on the mRNA is identified with the same name but without the notation of “A” or “S” after the name. An antisense sequence with the same name, for example OSC17A-1 through OSC17A-18 binds to the same nucleotide target sequence. [0083] Another aspect of an embodiment is a method for targeted drug delivery the kidney by administration of one or more siRNA molecules covalently conjugated to cobalamin and further complexed to transcobalamin II.

[0084] Note that in the specification and claims, “about” or “approximately” means within twenty percent (20%) of the numerical amount cited; and “a”, “the” and “an” means one or more unless the context clearly indicates otherwise. Although the invention has been described in detail with particular reference to these embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above are hereby incorporated by reference.

[0085] A protein or polypeptide is "substantially pure," "substantially homogeneous," or

"substantially purified" when at least about 60% to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using FIPLC or other means well known in the art for purification.

[0086] The terms "label" or "labeled" as used herein refers to incorporation of another molecule in the sREC. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y,

"To, ⁱ n in, ⁱ²⁵ l, ¹³¹ 1), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, b-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In various embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0087] The term "heterologous" as used herein refers to a composition or state that is not native or naturally found, for example, that may be achieved by replacing an existing natural composition or state with one that is derived from another source. Similarly, the expression of a protein in an organism other than the organism in which that protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0088] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0089] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the disclosure described herein include "consisting" and/or "consisting essentially of aspects and variations.

[0090] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") as well as nucleic acid analogs. Nucleic acid analogs include those which include non- naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

[0091] Conventional notation is used herein to describe polynucleotide sequences: the left- hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0092] "Complementary" refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.

[0093] A "host cell" is a cell that can be used to express a polynucleotide of the disclosure. A host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single- celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0094] The term "isolated molecule" (where the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that by virtue of its origin or source of derivation (1 ) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be "isolated" from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using FIPLC or other means well known in the art for purification. [0095] A protein or polypeptide is "substantially pure," "substantially homogeneous," or

"substantially purified" when at least about 60% to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using FIPLC or other means well known in the art for purification.

[0096] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. “Pharmacologically effective amount" refers to that amount of an agent effective to produce the intended pharmacological result. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21 st Ed. 2005, Mack Publishing Co, Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines. The term "salt" shall mean any salt consistent with the use of the compounds according to the present invention. In the case where the compounds are used in pharmaceutical indications, including the treatment of prostate cancer, including metastatic prostate cancer, the term "salt" shall mean a pharmaceutically acceptable salt, consistent with the use of the compounds as pharmaceutical agents.

[0097] As used herein, “treatment” (and grammatical variations thereof such as “treat” or

“treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. As used herein, to "alleviate" a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to "treatment" include references to curative, palliative and prophylactic treatment.

[0098] The term "effective amount" or “therapeutically effective amount” as used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In reference to cancers or other unwanted cell proliferation, an effective amount comprises an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e. , slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. An effective amount can be administered in one or more administrations. As used herein, “specific binding” is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an receptor to bind to a specific target can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, Radioisotope assays are commonly used wherein there is a competition between immunoglobulin binding to a specific region of a receptor protein vs. a natural ligand.

[0099] The terms “affinity” or “binding affinity” as used herein refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (koff and kon, respectively). A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

[00100] The term “reduced binding”, as used herein refers to a decrease in affinity for the respective interaction, as measured for example by SPR. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

[00101] The phrase “administering” or "cause to be administered" refers to the actions taken by a medical professional {e.g., a physician), or a person controlling medical care of a patient, that control and/or permit the administration of the agent(s)/compound(s) at issue to the patient. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic regimen, and/or prescribing particular agent(s)/compounds for a patient. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like. Where administration is described herein, "causing to be administered" is also contemplated.

[00102] The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, among others. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally (including via intubation through the mouth or nose into the stomach), intraperitoneally or intravenously. [00103] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals {e.g., canine or feline), laboratory mammals {e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals {e.g., equine, bovine, porcine, ovine). In various embodiments, the patient can be a human {e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context. In various embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system including, but not limited to patients having primary immune deficiency, AIDS; cancer and transplant patients who are taking certain immunosuppressive drugs; and those with inherited diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including, but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to. Without being bound by theory, all cells, and cancer cells in particular, require vitamin B12 and thus all cancers express CD320 and/or LRP2. The exception might be hepatocellular carcinoma, which is a strong expressor of asialoglycoprotein receptors 1 and 2, which uptake vitamin B12 as its complex with the plasma chaperone protein TCN1 . (Cancer cells with higher metabolic activity would be expected to respond most to this type of therapy).

[00104] The term "compound" is used herein to describe any specific compound or bioactive agent disclosed herein, including any and all stereoisomers (including diastereomers, individual optical isomers/enantiomers or racemic mixtures and geometric isomers), pharmaceutically acceptable salts and prodrug forms. The term compound herein refers to stable compounds. Within its use in context, the term compound may refer to a single compound or a mixture of compounds as otherwise described herein. It is understood that the choice of substituents or bonds within a Markush or other group of substituents or bonds is provided to form a stable compound from those choices within that Markush or other group.

[00105] Note that in the specification and claims, “about” or “approximately” means within twenty percent (20%) of the numerical amount cited; and “a”, “the” and “an” means one or more unless the context clearly indicates otherwise. Although the invention has been described in detail with particular reference to these embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. For example, the linker selected from La-Lf may be independently selected and be the same or different at each position and/or an siRNA molecule selected from Ta-Tf may be independently selected and be the same or different at each position. The entire disclosures of all references, applications, patents, and publications cited above are hereby incorporated by reference. BIBLIOGRAPHY

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