METHODS AND COMPOSITIONS FOR TARGETING EFEMP1

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/309,952, filed on February 14, 2022, entitled “METHODS AND COMPOSITIONS FOR TARGETING EFEMP1,” the entire disclosure of which is hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (A13617C)OOOWOOO-SEQ-OMJ.xml; Size: 820,382 bytes; and Date of Creation: February 9, 2023) is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates to methods and compositions involving nucleic acids that are complementary to an EFEMP1 target sequence.

BACKGROUND

There are currently no cures for numerous autosomal dominant or recessive diseases that have a profoundly negative impact on quality of life. Dominant forms of autosomal dominant drusen, retinitis pigmentosa (adRP), cone-rod dystrophies and juvenile macular degenerations are prime examples of dominant autosomal diseases that affect the eye.

One autosomal dominant disease-related gene is EFEMP1. A missense mutation in EFEMP1 (R345W -EFEMP 1) is associated with Autosomal Dominant Drusen, Doyne’s honeycomb retinal dystrophy, and Malattia Leventinese, which are a type of autosomal dominant inherited degenerative diseases that share clinical features with age-related macular degeneration (AMD), such as presence of basal deposits and drusen (Stone, et al (1999) Nat Genet 22: 199; Marmorstein (2004) Ophthalmic Genet 25:219-26). The human EFEMP 1 gene (UniProt Q12805 FBLN3 HUMAN) encodes EGF- containing fibulin-like extracellular matrix protein 1 (also known as Fibulin-3). Human EFEMP 1 is located at chromosome 2pl6 and contains 12 exons that encode a 493 -amino acid protein having a molecular weight of 55 kDa. The EFEMP 1 amino acid sequence comprises a signal peptide, five tandem arrays of cbEGF (calcium binding EGF) domains, a modified cbEGF domain containing an 88-amino acid insert, and a C-terminal fibulin-type module. The EFEMP1 protein is an extracellular glycoprotein expressed at the basement membrane of epithelial and endothelial cells. In human adults EFEMP1 is distributed in many tissues, including the Bruch’s membrane in the retina. EFEMP1 is thought to contribute to the integrity of the basement membrane by anchoring extracellular matrix (ECM) structures.

Autosomal dominant diseases are characterized by the presence of one or more mutations in a gene that result in a defective protein product or overexpression of the encoded protein. Such diseases are not readily amenable to therapies that simply add a normal, healthy gene (so called "gene supplementation" or "gene addition"), since the disease-causing gene is still present.

SUMMARY OF THE DISCLOSURE

Some aspects of the disclosure provide an inhibitory oligonucleotide comprising a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the EFEMP1 target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 2-378 or 756-826.

In some embodiments, the target sequence comprises at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 2-378 or 756-826. In some embodiments, the target sequence comprises 8-20 contiguous nucleotides of the nucleic acid sequences of any one of SEQ ID NOs: 2-378 or 756-826. In some embodiments, the target sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 2-378 or 756-826.

In some embodiments, the nucleic acid sequence comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 379- 755 or 827-897. In some embodiments, the nucleic acid sequence comprises or consists of the nucleic acid sequence of any one of SEQ ID NOs: 379-755 or 827-897.

Some aspects of the disclosure provide an inhibitory oligonucleotide comprising at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 379-755 or 827-897.

Some aspects of the disclosure provide an inhibitory oligonucleotide of 10-50 nucleosides in length, comprising a sequence at least 90% identical to any one of SEQ ID NOs: 379-755 or 827-897.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 463. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 463.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 516. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 139. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 516.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 387. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 387.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 502. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 502.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 517. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 140. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 517.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 400. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 400.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 401. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 401.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 453. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 76. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 453.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 830. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 759. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 830.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 837. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 766. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 837.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 840. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 769. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 840.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 859. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 788. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 859.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 873. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 802. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 873.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 886. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 815. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 886.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 895. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 824. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 895.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 896. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 825. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 896.

In some aspects, the disclosure provides an oligonucleotide comprising a sequence of about 10-50 nucleosides in length, wherein the sequence comprises a region of at least 10 contiguous nucleosides complementary to an EFEMP1 target sequence, wherein an EFEMP1 target sequence is about 10-50 nucleotides and comprises at least 10 contiguous nucleotides of a sequence selected from SEQ ID NOs: 2-378 or 756-826, and wherein the oligonucleotide comprises one or more modified nucleosides. In some aspects, an EFEMP1 target sequence is about 10-50 nucleotides and comprises at least 10 contiguous nucleotides of a sequence selected from SEQ ID NOs: 2-140. In some aspects, an EFEMP1 target sequence is about 10-50 nucleotides and comprises at least 10 contiguous nucleotides of a sequence selected from SEQ ID NOs: 141-378. In some aspects, the region of at least 10 contiguous nucleosides are fully complementary to the EFEMP1 target sequence. In some aspects, the region of at least 10 contiguous nucleosides comprises no more than four mismatches to the EFEMP1 target sequence. In some aspects, the region of at least 10 contiguous nucleosides comprises 1, 2, 3 or 4 mismatches to the EFEMP1 target sequence.

In some aspects, the disclosure provides an oligonucleotide comprising a sequence of about 10-50 nucleosides in length, wherein the sequence comprises a region of at least 10 contiguous nucleosides, wherein at least 90% of the contiguous nucleosides are fully complementary to an EFEMP1 target sequence, wherein the EFEMP1 target sequence comprises a sequence selected from SEQ ID NOs: 2-378 or 756-826, and wherein the oligonucleotide comprises one or more modified nucleosides. In some aspects, the EFEMP1 target sequence comprises a sequence selected from SEQ ID NOs: 2-140. In some aspects, the EFEMP1 target sequence comprises a sequence selected from SEQ ID NOs: 141-378.

In some aspects, the disclosure provides an oligonucleotide comprising a sequence of about 10-50 nucleosides in length, wherein the sequence comprises a formula 5 ' -[Fl ] _n -C-[F2] _n -3 ', wherein C consists of a sequence of 8-10 contiguous nucleosides fully complementary to an EFEMP1 target sequence, wherein an EFEMP1 target sequence is selected from SEQ ID NOs: 2-378 or 756-826, wherein Fl and F2 independently comprise one or more nucleosides, and n is an integer from 1-20, and wherein the oligonucleotide comprises one or more modified nucleosides. In some aspects, n is 1- 19, 1-18, 1-17, 2-15, 2-10, 2-5, 3-15, 3-10, 5-15, or 5-10. In some aspects, an EFEMP1 target sequence is selected from SEQ ID NOs: 2-140. In some aspects, an EFEMP1 target sequence is selected from SEQ ID NOs: 141-378.

In some embodiments, in any of the foregoing or related aspects, an oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 379-755 or 827-897. In some aspects, an oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 379-517. In some aspects, an oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 518-755.

In some aspects, an oligonucleotide is 10-30 nucleosides in length. In some aspects, an oligonucleotide is 20-25 nucleosides in length. In some aspects, an oligonucleotide is 20 nucleosides in length.

In some aspects, the disclosure provides an oligonucleotide comprising a sequence of about 10-50 nucleosides in length, wherein the sequence comprises a region of at least 10 contiguous nucleosides of a sequence selected from SEQ ID Nos: 379-755 or 827-897, and wherein the oligonucleotide comprises one or more modified nucleosides. In some aspects, the sequence is selected from SEQ ID NOs: 379-517. In some aspects, the sequence is selected from SEQ ID NOs: 518-755.

In some aspects, the disclosure provides an oligonucleotide of about 10-50 nucleosides in length, comprising a sequence at least 90% identical to any one of SEQ ID NOs: 379-755 or 827-897, wherein the oligonucleotide comprises one or more modified nucleosides. In some aspects, the sequence is at least 90% identical to any one of SEQ ID NOs: 379-517. In some aspects, the sequence is at least 90% identical to any one of SEQ ID NOs: 518-755.

In some embodiments, in any of the foregoing or related aspects, an oligonucleotide is 10-30 nucleosides in length. In some aspects, an oligonucleotide is 20-25 nucleosides in length. In some aspects, an oligonucleotide is 20 nucleosides in length. In some aspects, an oligonucleotide is fully or partially complementary to an EFEMP1 target sequence selected from SEQ ID NOs: 2-378 or 756- 826. In some aspects, an oligonucleotide is fully or partially complementary to an EFEMP1 target sequence selected from SEQ ID NOs: 2-140. In some aspects, an oligonucleotide is fully or partially complementary to an EFEMP1 target sequence selected from SEQ ID NOs: 141-378.

In some embodiments, in any of the foregoing or related aspects, an oligonucleotide is singlestranded. In some aspects, an oligonucleotide is an antisense oligonucleotide. In some aspects, an oligonucleotide comprises ribonucleosides, deoxyribonucleosides, or a combination thereof. In some aspects, an oligonucleotide comprises a gap segment consisting of linked nucleosides, a 5 '-wing segment consisting of linked nucleosides, and a 3 '-wing segment consisting of linked nucleosides, wherein the gap segment is positions between the 5 '-wing segment and the 3 '-wing segment. In some aspects, the gap segment consists of RNaseH competent nucleosides, and wherein the 5'- and 3 '-wing segments consist of RNaseH resistant nucleosides. In some aspects, the gap segment consists of deoxyribonucleosides, and wherein the 5'- and 3'- wing segments consist of ribonucleosides. In some aspects, the gap segment is 5-15 nucleosides in length. In some aspects, the 5'- and 3'-wing segments are each independently 1-6 nucleosides in length. In some aspects, the 5'- and 3 '-wing segments are the same length. In some aspects, an oligonucleotide reduces or inhibits EFEMP1 expression. In some aspects, an oligonucleotide reduces or inhibits EFEMP1 expression by RNaseHl recruitment and mediated cleavage and degradation. In some aspects, an oligonucleotide reduces or inhibits EFEMP1 expression by steric blocking by the oligonucleotide.

In some embodiments, in any of the foregoing or related aspects, an oligonucleotide is double -stranded. In some aspects, an oligonucleotide that is double-stranded comprises (i) an antisense strand comprising the sequence of an oligonucleotide described herein; and (ii) a sense strand comprising a sequence of 10-50 nucleosides in length, wherein the sense strand forms a duplex region with the antisense strand. In some aspects, the antisense strand comprises a sequence selected from SEQ ID NOs: 379-755 or 827-897. In some aspects, the antisense strand comprises a sequence selected from SEQ ID NOs: 379-517. In some aspects, the antisense strand comprises a sequence selected from SEQ ID NOs: 518-755. In some aspects, an oligonucleotide comprising (i) and (ii) comprises a blunt end. In some aspects, the blunt end comprises the 3 ' end of the sense strand. In some aspects, an oligonucleotide comprising (i) and (ii) comprises an overhang of at least one nucleoside in length. In some aspects, the sense strand comprises an overhang at the 3’ end and the antisense strand comprises an overhang at the 3’ end. In some aspects, the overhang is two nucleosides in length. In some aspects, the sense strand and antisense strand are each independently 15-25 nucleosides in length. In some aspects, the sense and antisense strands are the same length. In some aspects, the duplex region is 15-25 nucleosides in length. In some aspects, an oligonucleotide reduces or inhibits EFEMP1 expression. In some aspects, an oligonucleotide reduces or inhibits EFEMP1 expression by RISC-mediated degradation.

In some aspects, the disclosure provides an antisense oligonucleotide comprising a sequence of about 10-50 nucleosides in length, wherein the sequence comprises a region of at least 10 contiguous nucleosides complementary to an EFEMP1 target sequence, wherein an EFEMP1 target sequence is a sequence of about 10-50 nucleotides in a human EFEMP1 pre-mRNA comprising the nucleotide sequence of SEQ ID NO: 1, and wherein the oligonucleotide comprises one or more modified nucleosides. In some aspects, the EFEMP1 target sequence comprises at least 10 contiguous nucleotides of a sequence in an intron of the human EFEMP1 pre-mRNA. In some aspects, the intron is selected from: intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10 and intron 11. In some aspects, the intron is intron 5. In some aspects, the EFEMP1 target sequence comprises at least 10 contiguous nucleotides of a sequence that spans an adjacent intron and exon of the human EFEMP1 pre-mRNA. In some aspects, the sequence spans an adjacent intron and exon selected from: exon 3-intron 3, intron 3-exon 4, exon 4-intron 4, exon 5-intron 5, intron 5-exon 6, exon 6-intron 6, intron 6-exon 7, exon 8-intron 8, and intron 10-exon 11.

In some aspects, the disclosure provides an antisense oligonucleotide comprising a sequence of 15-25 nucleosides, wherein the sequence comprises 8-12 contiguous nucleosides complementary to an EFEMP1 target sequence selected from SEQ ID NOs: 2-378 or 756-826, and wherein the sequence comprises no more than four mismatches to the EFEMP1 target sequence. In some aspects, the antisense oligonucleotide comprises a sequence of 15-25 nucleosides, wherein the sequence comprises a region of at least 15 contiguous nucleotides complementary to an EFEMP1 target sequence selected from SEQ ID NOs: 2-378 or 756-826, and wherein the sequence comprises no more than four mismatches to the EFEMP1 target sequence. In some aspects, an EFEMP1 target sequence is selected from SEQ ID NOs: 2-140. In some aspects, an EFEMP1 target sequence is selected from SEQ ID NOs: 141-378.

In some aspects, the disclosure provides an antisense oligonucleotide comprising a sequence of 15-25 nucleosides, wherein the sequence is complementary to an EFEMP1 target sequence, and wherein the sequence comprises at least 15 contiguous nucleosides of any one of SEQ ID NOs: 379- 755 or 827-897. In some aspects, the sequence comprises at least 15 contiguous nucleosides of any one of SEQ ID NOs: 379-517. In some aspects, the sequence comprises at least 15 contiguous nucleosides of any one of SEQ ID NOs: 518-755. In some aspects, the sequence is 20 nucleosides and comprises any one of SEQ ID NOs: 379-755 or 827-897. In some aspects, the sequence is 20 nucleosides and comprises any one of SEQ ID NOs: 379-517. In some aspects, the sequence is 20 nucleosides and comprises any one of SEQ ID NOs: 518-755. In some aspects, the sequence of 20 nucleosides in length comprises positions 1-20 numbered 5’ to 3’, wherein no more than two mismatches occur within each of positions 1-4 and 17-20.

In some embodiments, in any of the foregoing or related aspects, the antisense oligonucleotide reduces or inhibits EFEMP1 expression. In some aspects, EFEMP1 expression is reduced or inhibited by RNaseHl recruitment and mediated cleavage and degradation. In some aspects, EFEMP1 expression is reduced or inhibited by steric blocking by the oligonucleotide.

In any of the foregoing or related aspects, an oligonucleotide or an antisense oligonucleotide comprises one or more modified nucleosides. In some aspects, the modification is selected from a modified sugar moiety, a modified intemucleoside linkage, a modified nucleobase, and a combination thereof. In some aspects, an oligonucleotide or an antisense oligonucleotide comprises a modified sugar moiety. In some aspects, the modified sugar moiety is a 2'-ribose modification. In some aspects, the 2 '-ribose modification is selected from one or more of a 2'-O-methoxyethyl modified sugar moiety, a 2 '-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety. In some aspects, the 2'-ribose modification is selected from one or more of 2'-O-methyl (2'OMe), 2'-O-methoxyethyl (2 'MOE), and 2 '-fluoro (2'F). In some aspects, an oligonucleotide or an antisense oligonucleotide comprises one or more modified nucleosides selected from one or more of a 2'-deoxy ribonucleotide, a 2'-O-methyl ribonucleotide, a 2'-fluoro modified ribonucleotide, a 2'-amino modified ribonucleotide, and a 2'-thio modified ribonucleotide. In some aspects, an oligonucleotide or an antisense oligonucleotide comprises a 2'-deoxy ribonucleotide. In some aspects, the 2'-deoxy ribonucleotide is 2'-deoxy adenosine or 2'-deoxy guanosine. In some aspects, an oligonucleotide or an antisense oligonucleotide comprises a 2'-fluoro modified ribonucleotide. In some aspects, the 2'-fluoro modified nucleotide is 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, or 2'-fluoro- guanosine, or wherein the 2'-amino modified ribonucleotide is 2'-amino-cytidine, 2'-amino-uridine, 2'- amino-adenosine, 2'-amino-guanosine or 2'-amino-butyryl-pyrene-uridine. In some aspects, an oligonucleotide or an antisense oligonucleotide comprises one or more modified nucleosides selected from one or more of a 5-bromo-uridine, a 5-iodo-uridine, a 5 -methyl -cytidine, a ribo-thymidine, a 2- aminopurine, a 5 -fluoro-cytidine, a 5 -fluoro-uridine, a 2,6-diaminopurine, a 4-thio-uridine, and a 5- amino-allyl-uridine. In some aspects, an oligonucleotide or an antisense oligonucleotide comprises one or more modified nucleosides, wherein the one or more modified nucleosides is a bridged nucleic acid (BN A). In some aspects, the BNA is a locked nucleic acid (LNA). In some aspects, an oligonucleotide or an antisense oligonucleotide comprises one or more modified intemucleoside linkages. In some aspects, the one or more modified intemucleoside linkages comprise alkylphosphonate, phosphorothioate, ethylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, or carboxymethyl ester, or a combination thereof. In some aspects, the one or more modified intemucleoside linkages is phosphorothioate .

In some embodiments, in any of the foregoing or related aspects, an oligonucleotide or an antisense oligonucleotide comprises at least one conjugate moiety operably linked to the oligonucleotide. In some aspects, the at least one conjugate moiety is a tissue targeting moiety.

In some aspects, the disclosure provides a composition comprising an oligonucleotide or an antisense oligonucleotide described herein, and a pharmaceutically acceptable carrier. In some aspects, the composition is formulated for topical or intravitreal administration. In some aspects, the composition comprises a viral vector comprising an oligonucleotide or antisense oligonucleotide. In some aspects, the viral vector is an adenovirus associated (AAV) vector.

In some aspects, the disclosure provides a method of reducing or inhibiting expression of EFEMP1 in a subject, comprising administering to the subject an oligonucleotide or an antisense oligonucleotide described herein, or a composition described herein. In some aspects, EFEMP1 expression is reduced or inhibited in retinal pigment epithelium (RPE).

In some aspects, the disclosure provides a method of treating a disease, disorder or condition associated with a mutant form of EFEMP1 in a subject in need thereof, comprising administering to the subject an oligonucleotide or an antisense oligonucleotide described herein, or a composition described herein. In some embodiments, in any of the foregoing or related aspects, the subject has a disease, disorder or condition associated with (i) a mutant form of EFEMP 1; (ii) dysfunction of EFEMP 1; (iii) gain of function of EFEMP 1; and/or (iv) overexpression of EFEMP1. In some aspects, an oligonucleotide, antisense oligonucleotide or composition is administered via intravitreal administration.

In some aspects, the disclosure provides a kit comprising an oligonucleotide or an antisense oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with a mutant form of EFEMP1.

In some aspects, the disclosure provides an oligonucleotide or an antisense oligonucleotide described herein, or a composition described herein, for use, or adaptable for use, in the treatment of a disease, disorder or condition associated with a mutant form of EFEMP 1.

In some aspects, the disclosure provides use of an oligonucleotide or an antisense oligonucleotide described herein, or a composition described herein, in the manufacture of a medicament for the treatment of a disease, disorder or condition associated with a mutant form of EFEMP 1.

In any of the foregoing or related aspects, the disease, disorder or condition is selected from: ophthalmic disease, retinal degeneration, Sorsby’s fundus dystrophy, Stargardt disease, bestrophinopathy, retinitis pigmentosa, macular degeneration, macular dystrophy, Doyne honeycomb retinal dystrophy, preventative and acute geographic atrophy, macular edema, intermediate age- related macular degeneration, primary open-angle glaucoma, normal tension glaucoma, and diabetic retinopathy.

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. 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 in this application 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, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The term “a” or “an” refers to one or more of an entity.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

FIG. 1 provides a graph showing the ability of antisense oligonucleotides to inhibit human EFEMP1 mRNA expression in cellulo.

FIG. 2 provides a graph showing the ability of selected antisense oligonucleotides to inhibit human EFEMP1 mRNA expression in cellulo.

FIGs. 3A-3B provide graphs showing the ability of selected antisense oligonucleotides to inhibit human EFEMP1 mRNA expression in cellulo in a dose-dependent manner.

DETAILED DESCRIPTION

The present disclosure provides oligonucleotides for targeting a nucleic acid encoding EFEMP1, e.g., an RNA transcript encoding EFEMP1, for modulating (e.g., reducing or inhibiting) expression of EFEMP 1 in a cell or population thereof. In some embodiments, oligonucleotides modulate (e.g., reduce or inhibit) expression of EFEMP1 by binding to (e.g., by base-pairing to) a target sequence in a nucleic acid encoding EFEMP 1, e.g. , a target sequence in an RNA transcript encoding EFEMP 1. In some embodiments, oligonucleotides modulate (e.g., reduce or inhibit) expression of EFEMP 1 by hybridizing to a target sequence in a nucleic acid encoding EFEMP 1, e.g., a target sequence in an RNA transcript encoding EFEMP 1.

In some embodiments, the disclosure provides oligonucleotides targeting a nucleic acid encoding EFEMP1, e.g., an RNA transcript encoding EFEMP1, for modulating (e.g., reducing or inhibiting) expression of EFEMP 1 in a cell of the retinal pigment epithelium (RPE). The RPE is a single monolayer of cells in the retina that has an essential role in maintaining retinal homeostasis, including supporting the visual cycle, phagocytosis and degradation of the shed photoreceptor outer segments (POS), absorption of damaging light, and control of the flux of ions to and from the choroidal vasculature (see, e.g., Strauss (2005) Physiol Rev. 85:845-881). Impairment of RPE function contributes to pathological manifestation of macular degeneration (AMD), a condition in which cells of the macula, the central part of the retina at the back of the eye, become damaged and result in loss of visual acuity (see, e.g., Galloway, et al (2017) A4S E8214-23). EFEMP1 is expressed by RPE cells. Mutations in EFEMP1 result in deposit of material between Bruch’s membrane (the ECM layer separating the RPE from the underlying layers of capillaries of the choroid) and the RPE and correlates with complement activation in the retina (see, e.g., Fu, et al (2007) Human Mol Genetic 16:2411). EFEMP1 also co-localizes to basal deposits of patients with Doyne honeycomb retinal dystrophy caused by the R345W -EFEMP 1 mutation (see, e.g., Marmorstein, et al (2002) PNAS 99: 13067; Klenotic et al (2004) JBC 279:30469).

In some aspects, the disclosure provides methods for treatment of a disease or disorder in a subject associated with (i) one or more mutations in the EFEMP 1 gene, (ii) an abnormal expression level of a transcriptional and/or translational product of the EFEMP 1 gene, (iii) an abnormal activity of a transcriptional and/or translational product of the EFEMP1 gene, or (iv) a combination of (i)-(iii), comprising administering to the subject one or more oligonucleotides, or compositions thereof, described herein.

In some embodiments, the disease or disorder in the subject is associated with one or more mutations in the EFEMP1 gene that are gain of function mutations (e.g., one or more mutations in the EFEMP1 gene that result in an increased expression of an encoded EFEMP1 polypeptide, e.g., an amplification of the EFEMP1 gene). In some embodiments, the disease or disorder in the subject is associated with an increased or overabundant expression of an EFEMP1 RNA transcript and/or EFEMP1 polypeptide encoded by the EFEMP1 gene. In some embodiments, the disease or disorder in the subject is associated with an increased or abnormal activity of an EFEMP1 RNA transcript and/or EFEMP1 polypeptide encoded by the EFEMP1 gene.

In some embodiments, the disclosure provides methods for treatment of an ocular disease or disorder in a subject, wherein the disease or disorder is associated with (i) one or more mutations (e.g., gain of function mutations) in the EFEMP1 gene, (ii) an abnormal expression level of a transcriptional and/or translational product of the EFEMP1 gene in one or both eyes of the subject, (iii) an abnormal activity of a transcriptional and/or translational product of the EFEMP1 gene in one or both eyes of the subject, or (iv) a combination of (i)-(iii), comprising administering to the subject one or more oligonucleotides, or compositions thereof, described herein.

In some embodiments, one or more mutations in, an abnormal expression of, and/or an abnormal activity of the EFEMP1 gene, or a transcriptional or translational product thereof, results in altered (e.g., increased) production of ECM within the retina (e.g., within Bruch’s membrane). Increased ECM deposits are known to inhibit efflux of metabolic waste produced by the RPE and development of drusen (see, e.g., Crabb, et al (2002) PNAS 99: 14682) (with drusen formation and thickening of the underlying ECM being pathological manifestations of AMD (see, e.g., Galloway, et al (2017) PNAS E8214-23)). Additionally, alterations in the ECM have been shown to result in local activation of complement by the RPE (see, e.g., Fu, et al (2007) Hum. Mol. Genet., 16, 2411-2422; Garland, et al (2014) Hum. Mol. Genet., 23, 52-68; Femandez-Godino, et al (2015) Hum. Mol. Genet., 24, 5555-5569). Furthermore, inflammation induced by aberrant activation of the complement pathway is believed to contribute to the onset and/or pathology of macular degeneration by causing a loss of viability of one or more retinal cell layers (e.g., photoreceptors, RPE, and/or choriocapillaris). In some embodiments, one or more mutations in, an abnormal expression of, and/or an abnormal activity of the EFEMP1 gene, or a transcriptional or translational product thereof, results in altered (e.g., increased) production of ECM within the retina. In some embodiments, one or more mutations in, an abnormal expression of, and/or an abnormal activity of the EFEMP1 gene, or a transcriptional or translational product thereof, results in inflammation within the retina. In some embodiments, one or more mutations in, an abnormal expression of, and/or an abnormal activity of the EFEMP1 gene, or a transcriptional or translational product thereof, results in development of drusen within the retina. Accordingly, the present disclosure provides methods for treatment of an ocular disease or disorder in a subject having one or more mutations in, an abnormal expression of, and/or an abnormal activity of the EFEMP1 gene, or a transcriptional or translational product thereof, comprising administering to the subject one or more oligonucleotides, or compositions thereof, described herein targeting a nucleic acid encoding EFEMP1 (e.g., an RNA transcript encoding EFEMP1) for modulating (e.g., reducing or inhibiting) expression of EFEMP1 in a cell (e.g., RPE cell), wherein modulation of EFEMP1 expression results in decreased ECM deposition, a reduced quantity of components of the complement pathway, and/or a reduced inflammatory response in one or both eyes of the subject, thereby treating the ocular disease or disorder. In some embodiments, the ocular disease or disorder is a retinal disease or disorder. In some embodiments, the retinal disease or disorder is a diabetic retinopathy. In some embodiments the retinal disease or disorder is a macular degeneration. In some embodiments, the macular degeneration is an age-related macular degeneration (AMD). In some embodiments, the AMD is an early-stage AMD or late-stage AMD. In some embodiments, the AMD is a neovascular (wet) AMD or preventative geographic atrophy (dry) AMD. In some embodiments, the macular degeneration is a macular dystrophy.

Oligonucleotides for Modulating EFEMP1 Expression

The present disclosure provides oligonucleotides (e.g., inhibitory nucleic acids) comprising a region of complementarity to a target sequence in a nucleic acid encoding EFEMP1 (e.g., a target sequence in an RNA transcript encoding EFEMP1, such as a pre-mRNA encoding EFEMP1 or an mRNA encoding EFEMP1). In some embodiments, inhibitory oligonucleotides comprising a region of complementarity to a target sequence in a nucleic acid encoding EFEMP1 are referred to herein as “EFEMP1 -targeting oligonucleotides,” “EFEMP1 -targeting inhibitory oligonucleotides,” “EFEMP1- targeting nucleic acids,” “EFEMP1 -targeting inhibitory nucleic acids,” “inhibitory nucleic acids,” or “inhibitory oligonucleotides.”

As used herein, the terms “nucleic acid” and “oligonucleotide,” which are used interchangeably herein, refer to a polymer of nucleosides and/or nucleotides joined together, e.g. , by a phosphodiester linkage between 5' and 3' carbon atoms. A nucleic acid includes, but is not limited to, ribonucleic acid (RNA) nucleobases and/or deoxyribonucleic acid (DNA) nucleobases. A nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. A nucleic acid may comprise a polymer of ribonucleosides, a polymer of deoxyribonucleosides, or a polymer of ribonucleosides and deoxyribonucleosides.

As used herein, the term “nucleoside” refers to a molecule comprising a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. As used herein, the term “nucleotide” refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates.

When referring to a sequence of a nucleic acid, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides using the standard nucleotide nomenclature. “G,” “C,” “A,” “T” and “U” respectively correspond to nucleotides or nucleosides comprising a guanine, cytosine, adenine, thymine and uracil as a base. In some embodiments, the nucleobase is a purine heterocyclic compound found in a naturally occurring nucleic acid, or an analog or derivative thereof. In some embodiments, the nucleobase is a pyrimidine heterocyclic compound found in a naturally occurring nucleic acid, or an analog or derivative thereof. Guanine, cytosine, adenine, thymine and uracil are exemplary nucleobases found in naturally occurring nucleic acids. It should be appreciated that nucleic acids described herein can include nucleosides and/or nucleotides.

As used herein, the terms “EFEMP1 -targeting oligonucleotides,” “EFEMP1 -targeting inhibitory oligonucleotides,” “EFEMP1 -targeting nucleic acids,” “EFEMP1 -targeting inhibitory nucleic acids,” “inhibitory nucleic acids,” or “inhibitory oligonucleotides,” which may be used interchangeably, refer to a molecule that comprises at least one strand of two or more covalently linked nucleosides and/or nucleotides and which is capable of modulating (e.g., reducing or inhibiting) expression of EFEMP 1. The nucleosides and/or nucleotides can be modified or unmodified independent of each other. In some embodiments, an oligonucleotide comprises at least 8 contiguous nucleosides (e.g., at least 10, at least 12, at least 15, or at least 20 nucleosides). In some embodiments, an oligonucleotide comprises at least one strand of 8-20, 10-25, 15-30, or 10-100 covalently linked nucleosides. In some embodiments, an oligonucleotide comprises at least one strand of 8-50 nucleosides. In some embodiments, an oligonucleotide comprises a single-stranded RNA, a singlestranded DNA, a single-stranded modified RNA, a single-stranded modified DNA, or a singlestranded hybrid of RNA, DNA, modified RNA, and/or modified DNA. In some embodiments, the single-strand comprises one or more secondary structures, e.g., a stem -loop structure. In some embodiments, an oligonucleotide comprises two or more strands. In some embodiments, an oligonucleotide is double-stranded. In some embodiments, an oligonucleotide comprises a two- stranded DNA/DNA duplex, a two-stranded DNA/RNA duplex, or a two-stranded RNA/RNA duplex, including modified oligonucleotides thereof.

In some embodiments, an oligonucleotide modulates expression of a target nucleic acid (e.g., target nucleic acid encoding EFEMP 1) by one or more mechanisms or processes including, for example, RNase H mediated cleavage, splicing modulation, RNA and non-coding RNA inhibition, translation arrest, and gene activation or editing. Such oligonucleotides modulate the normal function of the EFEMP 1 gene, by for example, inhibiting, interfering, cleaving, degrading, disrupting, blocking, and/or activating nucleic acid expression. Examples of oligonucleotides and nucleic acids include, but not limited to, an antisense oligonucleotide or an RNA interference (RNAi) oligonucleotide. Exemplary RNAi oligonucleotides include small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a dicer substrate interfering RNA (DisRNA), as further described herein.

Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. In some embodiments, the oligonucleotides described herein are chemically synthesized and purified or isolated.

Within an oligonucleotide structure, the phosphate groups are commonly referred to as forming the intemucleoside linkages of the oligonucleotide. In some embodiments, the phosphate group is covalently bonded to a 5'adjacent nucleoside to form an intemucleoside linkage. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.

In some embodiments, an oligonucleotide comprises ribonucleosides, and optionally, one or more non-ribonucleosides, e.g., a deoxyribonucleoside.

In some embodiments, an oligonucleotide comprises one or more chemically-modified nucleosides (e.g., one or more chemically-modified ribonucleosides or deoxyribonucleosides), as further described herein.

EFEMP1 Target Sequences

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in a nucleic acid encoding EFEMP1 (e.g., an RNA transcript encoding EFEMP1). In some embodiments, an oligonucleotide is a single-stranded oligonucleotide (e.g., an antisense oligonucleotide). In some embodiments, an oligonucleotide is double -stranded (e.g., an shRNA, an siRNA, double-stranded RNA, or DisRNA). In some embodiments, an oligonucleotide binds to or hybridizes to a target sequence, thereby modulating (e.g., inhibiting or reducing) expression of the target nucleic acid encoding EFEMP1.

As used herein, the term “target sequence” refers to a contiguous nucleotide sequence present in a target nucleic acid, (e.g., a nucleic acid encoding EFEMP1). In some embodiments, a target sequence is an “EFEMP1 target sequence.” As used herein, there term “EFEMP1 target sequence” refers to a contiguous nucleotide sequence present in a nucleic acid encoding EFEMP 1 (e.g. , a pre- mRNA or an mRNA encoding EFEMP 1). “Contiguous nucleotides” is intended to mean nucleotides that are covalently linked and immediately adjacent to each other.

In some embodiments, the EFEMP 1 target sequence comprises a contiguous nucleotide sequence present in a nucleic acid encoding EFEMP 1. In some embodiments, the nucleic acid encodes EFEMP 1 of any species. In some embodiments, the nucleic acid encodes a mammalian EFEMP 1. In some embodiments, the nucleic acid encodes human EFEMP 1. In some embodiments, the nucleic acid encoding EFEMP 1 shares homology between species (e.g., encodes human and non- human primate EFEMP1). In some embodiments, the nucleic acid encoding human EFEMP1 is an RNA transcript of the human EFEMP1 gene, e.g. , a pre-mRNA transcript or mRNA transcript of the EFEMP1 human gene. Gene information for human EFEMP1 is available via the ENSEMBLE database under gene ID ENSG00000115380. The genomic coordinates of the human EFEMP1 gene are 55,865,967-55,924,163, according to human reference genome GRCh38.pl3 (with the EFEMP1 gene is located on the negative strand of the reference genome).

In some embodiments, the EFEMP1 target sequence comprises a contiguous nucleotide sequence present in a pre-mRNA encoding a human EFEMP1 protein. An exemplary human EFEMP1 pre-mRNA nucleotide sequence is set forth in SEQ ID NO: 1. The regions of SEQ ID NO: 1 corresponding to exonic and intronic regions of the human EFEMP1 pre-mRNA are provided in

Table 1.

Table 1: Genomic and transcript coordinates for regions in human EFEMP1 pre-mRNA

¹ corresponds to the start site of the indicated region relative to the 5 'end of the human EFEMP1 pre-mRNA nucleotide sequence set forth in SEQ ID NO: 1.

² corresponds to the stop site of the indicated region relative to the 5 'end of the human EFEMP1 pre-mRNA nucleotide sequence set forth in SEQ ID NO: 1.

³ corresponds to the start site of the indicated region in the human genome according to human reference genome GRCh38.pl 3

⁴ corresponds to the stop site of the indicated region in the human genome according to human reference genome GRCh38.pl 3 In some embodiments, the EFEMP1 target sequence comprises a contiguous nucleotide sequence present in an mRNA encoding a human EFEMP1 protein. The nucleotide sequence for human EFEMP1 mRNA is available via the NCBI database under reference sequence NM_001039348.3. Sequence information for the human EFEMP1 protein is available via the UniProtKB database under Q 12805 (FBLN3 HUMAN).

In some embodiments, the human EFEMP1 encoding pre-mRNA comprises a nucleotide sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the human EFEMP1 encoding pre-mRNA comprises SEQ ID NO: 1. In some embodiments, the human EFEMP1 encoding pre- mRNA consists of SEQ ID NO: 1.

In some embodiments, the EFEMP1 target sequence comprises a contiguous nucleotide sequence present in a non-human nucleic acid encoding EFEMP 1. In some embodiments, the nucleic acid encoding non-human EFEMP1 is an RNA transcript of the macaque EFEMP 1 gene, e.g., a pre- mRNA transcript or mRNA transcript of the macaque EFEMP 1 gene. Gene information for macaque EFEMP 1 is available via the ENSEMBLE database under gene ID ENSMFAG00000044235. In some embodiments, the nucleic acid encoding non-human EFEMP 1 is an RNA transcript of the mouse EFEMP 1 gene, e.g., a pre-mRNA transcript or mRNA transcript of the mouse EFEMP 1 gene. Gene information for the mouse EFEMP 1 gene is available via the ENSEMBLE database under gene ID ENSMUSG00000020467. In some embodiments, the nucleic acid encoding non-human EFEMP 1 is an RNA transcript of the rat EFEMP 1 gene, e.g. , a pre-mRNA transcript or mRNA transcript of the rat EFEMP 1 gene. Gene information for the rat EFEMP 1 gene is available via the ENSEMBLE database under gene ID ENSRNOG00000003553.

In some embodiments, an EFEMP 1 target sequence is about 8-100 nucleotides in length. In some embodiments, an EFEMP1 target sequence is about 10-100, 25-100, 50-100, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14- 25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19- 30, 19-25, or 19-20 nucleotides in length. In some embodiments, an EFEMP1 target sequences is, e.g., 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, or 50 contiguous nucleotides present in a nucleic acid encoding EFEMP 1 (e.g., present in the human EFEMP 1 pre-mRNA sequence set forth in SEQ ID NO: 1).

In some embodiments, an EFEMP1 target sequence is a nucleotide sequence of 8-100 (e.g., 10-50) nucleotides in length and is in any one of exons 1-12 of a nucleic acid encoding EFEMP1 (e.g., any one of exons 1-12 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 1 of a nucleic acid encoding EFEMP1 (e.g., in exon 1 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 2 of a nucleic acid encoding EFEMP1 (e.g., in exon 2 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 3 of a nucleic acid encoding EFEMP1 (e.g., in exon 3 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 4 of a nucleic acid encoding EFEMP1 (e.g., in exon 4 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 5 of a nucleic acid encoding EFEMP1 (e.g., in exon 5 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 6 of a nucleic acid encoding EFEMP1 (e.g., in exon 6 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 7 of a nucleic acid encoding EFEMP1 (e.g., in exon 7 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 8 of a nucleic acid encoding EFEMP1 (e.g., in exon 8 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 9 of a nucleic acid encoding EFEMP1 (e.g., in exon 9 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 10 of a nucleic acid encoding EFEMP1 (e.g., in exon 10 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 11 of a nucleic acid encoding EFEMP1 (e.g., in exon 11 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in exon 12 of a nucleic acid encoding EFEMP1 (e.g., in exon 12 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1).

In some embodiments, an EFEMP1 target sequence is a nucleotide sequence of 8-100 (e.g., 10-50) nucleotides in length and is in any one of introns 1-11 of a nucleic acid encoding EFEMP1 (e.g., any one of introns 1-11 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 1 of a nucleic acid encoding EFEMP1 (e.g., in intron 1 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 2 of a nucleic acid encoding EFEMP1 (e.g., in intron 2 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 3 of a nucleic acid encoding EFEMP1 (e.g., in intron 3 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 4 of a nucleic acid encoding EFEMP1 (e.g., in intron 4 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 5 of a nucleic acid encoding EFEMP1 (e.g., in intron 5 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 6 of a nucleic acid encoding EFEMP1 (e.g., in intron 6 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 7 of a nucleic acid encoding EFEMP1 (e.g., in intron 7 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 8 of a nucleic acid encoding EFEMP1 (e.g., in intron 8 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 9 of a nucleic acid encoding EFEMP1 (e.g., in intron 9 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 10 of a nucleic acid encoding EFEMP1 (e.g., in intron 10 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence is in intron 11 of a nucleic acid encoding EFEMP1 (e.g., in intron 11 of the human EFEMP1 pre-mRNA sequence set forth in SEQ ID NO: 1).

In some embodiments, an EFEMP1 target sequence is a nucleotide sequence of 8-100 (e.g., 10-50) nucleotides in length and spans adjacent exon and intron sequences of a nucleic acid encoding EFEMP1 (e.g., spans adjacent exon and intron sequences of a human EFEMP1 pre-mRNA as set forth in SEQ ID NO: 1). In some embodiments, an EFEMP1 target sequence spans exon 1 and intron 1 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 1 and exon 2 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 2 and intron 2 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 2 and exon 3 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 3 and intron 3 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 3 and exon 4 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 4 and intron 4 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 4 and exon 5 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 5 and intron 5 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 5 and exon 6 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 6 and intron 6 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 6 and exon 7 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 7 and intron 7 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 7 and exon 8 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 8 and intron 8 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 8 and exon 9 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 9 and intron 9 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 9 and exon 10 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 10 and intron 10 of a nucleic acid encoding EFEMP1. In some embodiments, an EFEMP1 target sequence spans intron 10 and exon 11 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans exon 11 and intron 11 of a nucleic acid encoding EFEMP 1. In some embodiments, an EFEMP 1 target sequence spans intron 11 and exon 12 of a nucleic acid encoding EFEMP 1.

Oligonucleotide Sequences Complementary to an EFEMP1 Target Sequence

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to an EFEMP 1 target sequence described herein.

As used herein, the term “region of contiguous nucleosides complementary to an EFEMP 1 target sequence” refers to a contiguous nucleoside sequence present in a sequence of an oligonucleotide that is sufficiently complementary to an EFEMP 1 target sequence to enable the oligonucleotide to specifically bind to a target sequence by forming base-pairs.

As used herein, the term “complementary” refers to the ability of a pair of nucleotides or a pair of nucleic acid sequences (e.g., an inhibitory nucleic acid of the disclosure and a target pre- mRNA sequence) to bind to one another (e.g., using hydrogen bond pairing between two nucleotides). Two nucleic acid sequences or nucleic acid strands are “complementary” to one another if they basepair, or bind, to each other to form a double -stranded nucleic acid molecule via Watson-Crick interactions and non-Watson-Crick base pairing (also referred to as hybridization). Binding to form a double -stranded nucleic acid molecule can refer to an association between at least two strands due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions (e.g., under physiological conditions). Non-Watson-Crick base pairing include wobble base pairing and Hoogsteen base pairing. In some embodiments, for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U); cytosine-type bases (C) are complementary to guanosine-type bases (G); and universal bases such as 3 -nitropyrrole, 5 -nitroindole, or inosine (I) can hybridize to and are considered complementary to any A-type, C- type, U-type, G-type, or T-type bases. In some embodiments, two nucleic acid sequences are complementary to one another if at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nucleobases across the length of one of the sequences are base-paired to nucleobases of the other sequence. Thus, in some embodiments, a sequence within an antisense oligonucleotide is complementary to a target sequence if at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nucleobases across the length of said sequence are base-paired to nucleobases of the target sequence. In some embodiments, an antisense oligonucleotide comprises a sequence that is sufficiently complementary to a cognate target nucleotide sequence such that the antisense oligonucleotide is capable of hybridizing to the cognate target nucleotide sequence (e.g., under physiological conditions such as the conditions in a cell). In some embodiments, an antisense oligonucleotide comprises a sequence that contains 1, 2, 3, 4, or 5 nucleobase mismatches with its cognate target nucleotide sequence. For example, an antisense oligonucleotide may comprise a sequence of 15-25 nucleotides that contains 1, 2, 3, 4, or 5 nucleobase mismatches with its cognate target nucleotide sequence.

An oligonucleotide that “specifically binds to” an EFEMP1 target sequence refers to one that will not appreciably bind to a reference sequence, e.g., a nucleic acid lacking an EFEMP1 target sequence. For example, in some embodiments, an oligonucleotide that specifically binds to an EFEMP 1 target sequence described herein will exhibit a substantially higher binding affinity for the EFEMP1 target sequence compared to a reference sequence. As is understood by the skilled artisan, the binding affinity between a first nucleic acid strand and a second nucleic acid strand is measured as the melting temperature (T _m ), which is the temperature at which half the first nucleic acid strand is duplexed with the second nucleic acid strand. The standard state Gibbs free energy (AG°) is also used to represent binding affinity between a first nucleic acid strand and a second nucleic acid strand and is related to the dissociation constant (Kd) of the reaction by AG° = -RTIn(Kd), where R is the gas constant and T is the absolute temperature. A low AG° of the reaction between a first nucleic acid and a second nucleic acid reflects a strong binding. AG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC).

In some embodiments, the region of contiguous nucleosides is complementary to an EFEMP 1 target sequence if it hybridizes to the EFEMP 1 target sequence under conditions suitable for modulating of EFEMP 1 expression using the oligonucleotide, e.g., to inhibit or reduce EFEMP 1 expression in vitro or in vivo. Such conditions can be stringent conditions, e.g., combination of an oligonucleotide and EFEMP 1 target sequence in buffer comprising 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at a temperature of 50°C-70°C for 12-16 hours, followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions include physiologically relevant conditions as can be encountered inside an organism. 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.

In some embodiments, the region of contiguous nucleosides is complementary to an EFEMP 1 target sequence present in a pre-mRNA transcript of the human EFEMP 1 gene. In some embodiments, the region of contiguous nucleosides is complementary to an EFEMP 1 target sequence present in an mRNA transcript of a human EFEMP 1 gene. In some embodiments, the region of contiguous nucleosides is complementary to an EFEMP 1 target sequence present in the human EFEMP 1 gene. In some embodiments, the region of contiguous nucleosides is complementary to an EFEMP 1 target sequence present in a pre-mRNA transcript of a non-human EFEMP 1 gene (e.g. , a macaque, mouse, or rat EFEMP 1 gene). In some embodiments, the region of contiguous nucleosides is complementary to an EFEMP 1 target sequence present in an mRNA transcript of a non-human EFEMP 1 gene (e.g., a macaque, mouse, or rat EFEMP 1 gene). In some embodiments, the region of contiguous nucleosides is complementary to an EFEMP1 target sequence present in a non-human EFEMP1 gene (e.g., a macaque, mouse, or rat EFEMP1 gene).

In some embodiments, an inhibitory oligonucleotide comprises a region of complementarity to a target sequence. As used herein a “region of complementarity” within an oligonucleotide refers to a region of nucleosides and/or nucleotides within the oligonucleotide which is complementarity to a target sequence. In some embodiments, a region of complementarity within an oligonucleotide is sufficient for the oligonucleotide to bind (e.g., hybridize) to a target sequence. In some embodiments, a region of complementarity is 8-20, 10-20, 12-20, 15-20, or 10-15 nucleotides in length. In some embodiments, a region of complementarity is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. An inhibitory oligonucleotide may comprise a region of complementarity to an EFEMP 1 target sequence and a region (or segment) that is not complementary to an EFEMP 1 target sequence.

In some embodiments, the region of contiguous nucleosides complementary to an EFEMP 1 target sequence is at least 10 nucleosides in length. In some embodiments, the region of contiguous nucleosides is not more than 50 nucleosides in length. In some embodiments, the region of contiguous nucleosides is 10-50 nucleosides in length, e.g., 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13- 25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18- 30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 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, or 50 nucleosides in length. In some embodiments, a region of complementarity is 3-20, 5- 20, 8-20, 5-10, 8-10, 8-20, 10-20, 12-20, 15-20, or 10-15 nucleotides in length.

In some embodiments, the region of contiguous nucleosides complementary to an EFEMP 1 target sequence spans the entire length of a nucleic acid strand present in an oligonucleotide, i.e., wherein the entire length of a strand in an oligonucleotide, such as a strand in a single-stranded or double -stranded oligonucleotide, is complementary to the EFEMP 1 target sequence. In some embodiments, the region of contiguous nucleosides complementary to an EFEMP 1 target sequence is less than the full-length of a strand present in an oligonucleotide, i.e., wherein only a portion of a strand in an oligonucleotide, such as a strand in a single-stranded or double -stranded oligonucleotide, is complementary to the EFEMP 1 target sequence. In some embodiments, the portion is a 5 'portion, an internal portion, or a 3 'portion of the strand present in an oligonucleotide.

In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to at least about 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to at least about 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to at least about 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 141-378. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to at least about 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826 coupled to a 5'- flanking nucleotide sequence and/or a 3 '-flanking sequence present in a human EFEMP1 mRNA (e.g., as set forth in SEQ ID NO: 1). In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to at least about 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-140 coupled to a 5'-flanking nucleotide sequence and/or a 3'- flanking sequence present in a human EFEMP1 mRNA (e.g., as set forth in SEQ ID NO: 1). In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to at least about 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 141-378 coupled to a 5 '-flanking nucleotide sequence and/or a 3 '-flanking sequence present in a human EFEMP1 mRNA (e.g., as set forth in SEQ ID NO: 1).

In some embodiments, the nucleotide sequences set forth by SEQ ID NOs: 2-378 or 756-826 identify regions in a nucleic acid encoding EFEMP1 (e.g., EFEMP1 mRNA) that are targeted by oligonucleotides of the disclosure for modulating (e.g., inhibiting or reducing) expression of EFEMP1 (e.g., by antisense inhibition and/or RNA interference). In some embodiments, the disclosure provides oligonucleotides comprising a region of contiguous nucleosides having substantial complementary to an EFEMP1 target sequence comprising a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826 (e.g., there is a sufficient degree of complementarity between an oligonucleotide and the EFEMP1 target sequence so that they bind and induce a desired effect). In some embodiments, the region of contiguous nucleosides has substantial complementary to an EFEMP1 target sequence comprising a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, the region of contiguous nucleosides has substantial complementary to an EFEMP1 target sequence comprising a nucleotide sequence selected from SEQ ID NOs: 141-378. While the sequences identified, for example, in SEQ ID NOs: 2-378 or 756-826 represent effective EFEMP1 target sequences, it is understood by the skilled artisan that additional EFEMP 1 target sequence may be identified by progressively “walking the window” at least one nucleotide (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) upstream or downstream of an initial target sequence (e.g., any one of SEQ ID NOs: 2-378 or 756-826). In some embodiments, the length of a target sequence is a given size, e.g., about 10-50 nucleotides, but shifted upstream or downstream of an initial target sequence (e.g., any one of SEQ ID NOs: 2-378 or 756-826) by at least one nucleotide (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). Thus, in some embodiments, the disclosure provides an EFEMP 1 target sequence of 10- 50 nucleotides in length comprising at least 4-10 contiguous nucleotides of an initial target sequence (e.g., any one of SEQ ID NOs: 2-378 or 756-826) and at least one nucleotide (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) upstream and/or at least one nucleotide (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) downstream of the initial target sequence in a nucleic acid encoding EFEMP 1 (e.g., an EFEMP 1 mRNA). In some embodiments, an EFEMP1 target sequence is about 20 nucleotides in length and comprises about 10 contiguous nucleotides of an initial target sequence (e.g., any one of SEQ ID NOs: 2-378 or 756-826) and about 10 nucleotides upstream the initial target sequence in a nucleic acid encoding EFEMP1 (e.g., an EFEMP1 mRNA). In some embodiments, an EFEMP1 target sequence is about 20 nucleotides in length and comprises about 10 contiguous nucleotides of an initial target sequence (e.g., any one of SEQ ID NOs: 2-378 or 756-826) and about 10 nucleotides downstream the initial target sequence in a nucleic acid encoding EFEMP1 (e.g., an EFEMP1 mRNA). In some embodiments, oligonucleotides directed to EFEMP1 target sequences that overlap and/or flank an initial target sequence provide substantially equivalent or improved modulation of EFEMP1 expression in a cell when evaluated using assays described herein (e.g., in vitro EFEMP1 mRNA knockdown assays) or as known in the art.

Accordingly, in some embodiments, the EFEMP1 target sequence comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826 or a contiguous portion of 4, 5, 6, 7, 8, 9, 10 or more contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756- 826. In some embodiments, the EFEMP1 target sequence comprises a nucleotide sequence selected from SEQ ID NOs: 2-140 or a contiguous portion of 4, 5, 6, 7, 8, 9, 10 or more contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, the EFEMP1 target sequence comprises a nucleotide sequence selected from SEQ ID NOs: 141-378 or a contiguous portion of 4, 5, 6, 7, 8, 9, 10 or more contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 141-378. In some embodiments, the EFEMP1 target sequence comprises at least 4 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756- 826. In some embodiments, the EFEMP1 target sequence comprises at least 5 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the EFEMP 1 target sequence comprises at least 6 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the EFEMP1 target sequence comprises at least 7 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2- 378 or 756-826. In some embodiments, the EFEMP1 target sequence comprises at least 8 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the EFEMP 1 target sequence comprises at least 9 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the EFEMP 1 target sequence comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the EFEMP1 target sequence comprises at least 11 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2- 378 or 756-826. In some embodiments, the EFEMP1 target sequence comprises at least 12 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the EFEMP 1 target sequence comprises at least 13 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the EFEMP1 target sequence comprises at least 14 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the EFEMP1 target sequence comprises at least 15 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2- 378 or 756-826. In some embodiments, the EFEMP1 target sequence comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826.

In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826 or a contiguous portion of 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 2-140 or a contiguous portion of 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 141-378 or a contiguous portion of 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide sequence selected from SEQ ID NOs: 141-378. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 4 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 5 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 6 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 7 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 8 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 9 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 11 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 12 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 13 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 14 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises at least 15 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826.

In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826 or a contiguous portion of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 2-140 or a contiguous portion of a nucleotide sequence selected from SEQ ID NOs: 2- 140. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 141-378 or a contiguous portion of a nucleotide sequence selected from SEQ ID NOs: 141-378. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 4 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 5 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 6 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 7 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 8 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 9 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 11 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 12 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 13 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 14 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises at least 15 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-20 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence of about 10-50 nucleosides in length, wherein the sequence comprises a region of at least 10 contiguous nucleosides complementary to an EFEMP1 target sequence, wherein an EFEMP1 target sequence is 10-50 nucleotides in length and comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826 or at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides in length and comprises a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides in length and comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides in length and comprises a nucleotide sequence selected from SEQ ID NOs: 141-378. In some embodiments, an EFEMP1 target sequence is 10-50 nucleotides in length and comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 141-378.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence of about 10-50 nucleosides in length, wherein the sequence comprises a region of at least 8 contiguous nucleosides fully complementary to an EFEMP1 target sequence, wherein an EFEMP1 target sequence is 10-50 nucleotides in length and comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756- 826 or at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, the region of at least 8 contiguous nucleosides is fully complementary to an EFEMP1 target sequence of 10-50 nucleotides in length and comprising a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, the region of at least 8 contiguous nucleosides is fully complementary to an EFEMP1 target sequence of 10-50 nucleotides in length and comprising at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, the region of at least 8 contiguous nucleosides is fully complementary to an EFEMP1 target sequence of 10-50 nucleotides in length and comprising a nucleotide sequence selected from SEQ ID NOs: 141-378. In some embodiments, the region of at least 8 contiguous nucleosides is fully complementary to an EFEMP1 target sequence of 10-50 nucleotides in length and comprising at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 141-378.

In some embodiments, an oligonucleotide comprises a sequence comprising a region of contiguous nucleosides that is fully complementary to the EFEMP1 target sequence. As used herein, “fully complementary” refers to a first sequence (e.g., the region of contiguous nucleosides) that basepairs to a second sequence (e.g., the EFEMP1 target sequence) over its entire length, wherein the first sequence has 100% complementarity to the second sequence. As used herein, the term “% complementarity” refers to the percentage of nucleobases in a first contiguous nucleotide sequence of a first oligonucleotide that base-pair with nucleobases in a second contiguous nucleotide sequence of a second oligonucleotide. In some embodiments, the percentage is calculated by counting the number of base pairs in the first contiguous nucleotide sequence that base pair with the second contiguous nucleotide sequence (upon alignment of the first contiguous nucleotide sequence and the second contiguous nucleotide sequence in opposing orientations, i.e., the first contiguous nucleotide sequence oriented 5' to 3' and the second contiguous nucleotide sequence oriented 3' to 5'), dividing by the total number of nucleotides/nucleosides in the first contiguous nucleotide sequence, and multiplying by 100. In such a comparison a nucleobase/nucleotide that does not align by forming a base pair are termed a mismatch. Preferably, insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence.

In some embodiments, an oligonucleotide comprises a sequence comprising a region of contiguous nucleosides that is partially complementary to the EFEMP1 target sequence. As used herein, “partially complementary” or “partial complementarity” refers to a first nucleoside sequence (e.g., the region of contiguous nucleosides) that base-pairs to a second nucleoside sequence (e.g., the EFEMP 1 target sequence), wherein the majority (e.g. , at least 80% or more) of the nucleosides present in the first sequence base pair to nucleosides present in the second sequence. In some embodiments, the first nucleoside sequence and the second nucleoside sequence bind to or hybridize with one or more mismatched base pairs (e.g., not more than 5, 4, 3, 2 or 1 mismatched base pairs). In some embodiments, a region of contiguous nucleosides that is partially complementary to an EFEMP 1 target sequence bind to or hybridize with a nucleic acid comprising the EFEMP 1 target sequence under conditions suitable to its application, e.g., antisense inhibition of EFEMP 1 expression in vitro or in vivo.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence of about 10-50 nucleosides in length having partial complementarity to an EFEMP 1 target sequence, wherein the sequence comprises at least 8 contiguous nucleosides having full complementarity to the EFEMP 1 targets sequence, and wherein an EFEMP1 target sequence is not more than 50 nucleotides in length and comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826 or at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826. In some embodiments, at least about 80%, about 85%, about 90%, about 95%, or about 99% of the nucleosides in an oligonucleotide sequence base-pair to a nucleotide in the EFEMP 1 target sequence. In some embodiments, an oligonucleotide sequence comprises one or more mismatches relative to the EFEMP 1 target sequence. In some embodiments, the one or more mismatches are internal, terminal, or a combination thereof. In some embodiments, an oligonucleotide sequence comprises no more than 5 mismatches (e.g. , no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches) when bound to or hybridized with the EFEMP 1 target sequence. In some embodiments, the region of contiguous nucleosides comprises a single mismatch when bound to or hybridized with the EFEMP 1 target sequence. In some embodiments, the region of contiguous nucleosides comprises at least 8-10 contiguous nucleosides fully complementary to the EFEMP 1 target sequence. In some embodiments, the region of contiguous nucleosides comprises at least 8-12 contiguous nucleosides fully complementary to the EFEMP1 target sequence.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides, wherein at least about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the nucleosides are complementary to an EFEMP1 target sequence, wherein an EFEMP1 target sequence is not more than 50 nucleotides in length and comprises a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826 or at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-378 or 756-826.

In any of the foregoing embodiments, the EFEMP1 target sequence not more than 50 nucleotides in length comprises a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, the EFEMP1 target sequence not more than 50 nucleotides in length comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-140. In some embodiments, the EFEMP1 target sequence not more than 50 nucleotides in length comprises a nucleotide sequence selected from SEQ ID NOs: 141-378. In some embodiments, the EFEMP1 target sequence not more than 50 nucleotides in length comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 141-378

In some embodiments, the disclosure provides an oligonucleotide comprising a region of at least 8-10 contiguous nucleosides fully complementary to the EFEMP1 target sequence and one or more “flanking” nucleosides located 5' and/or 3' to the region of contiguous nucleosides. In some embodiments, the one or more flanking nucleosides comprises one or more mismatches to the EFEMP1 target sequence. In some embodiments, an oligonucleotide comprises flanking nucleoside regions adjacent to, e.g., from 1- 20 nucleosides located 5' and/or 3' the region contiguous nucleosides fully complementary to the EFEMP1 target sequence. In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence according to the formula 5 '-[Fl]n-C-[F2]n-3', wherein C consists of a sequence of at least 8-10 contiguous nucleosides fully complementary to an EFEMP1 target sequence or portion thereof, wherein Fl and F2 independently comprise one or more nucleosides, and n is an integer from 1-20. In some embodiments, Fl is fully complementary to the EFEMP1 target sequence. In some embodiments, Fl is partially complementary to the EFEMP1 target sequence, wherein Fl comprises one or more mismatches relative to the EFEMP1 target sequence. In some embodiments, F2 is fully complementary to the EFEMP1 target sequence. In some embodiments, F2 is partially complementary to the EFEMP1 target sequence, wherein F2 comprises one or more mismatches relative to the EFEMP1 target sequence. In some embodiments, n is 1-30, 1-25, 1-20, 1-15, 1-20, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, 25-30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, an oligonucleotide comprises a sequence of about 8-100 nucleosides in length, wherein the sequence comprises at least 10 contiguous nucleosides of a sequence selected from SEQ ID NOs: 379-755 or 827-897. For example, in some embodiments, an oligonucleotide comprises a sequence of about 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 in length.

In some embodiments, an oligonucleotide comprises a sequence of about 10-100, 25-100, 50-100, 8-

40, 8-35, 8-30, 8-25, 8-20, 8-15, 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30, 11- 20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16- 25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, or 19-20 nucleotides in length. In some embodiments, the sequence comprises at least 10 contiguous nucleosides of a sequence selected from SEQ ID NOs: 379-517. In some embodiments, the sequence comprises at least 10 contiguous nucleosides of a sequence selected from SEQ ID NOs: 518-755. In some embodiments, the sequence comprises at least 10 contiguous nucleosides of a sequence selected from SEQ ID NOs: 827-897.

In some embodiments, an oligonucleotide comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 379-755 or 827-897. In some embodiments, an oligonucleotide comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 379-517. In some embodiments, an oligonucleotide comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 518-755.

In some embodiments, an oligonucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 379-755 or 827-897. In some embodiments, an oligonucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 379-517. In some embodiments, an oligonucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 518-755.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 1 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 1 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 1 comprises at least 10 contiguous nucleotides of a sequence extending from position 73 to position 843 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 1 comprises at least 10 contiguous nucleotides of a sequence extending from position 150 to position 300 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 1 comprises at least 10 contiguous nucleotides of a sequence extending from position 150 to position 250 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 1 comprises at least 10 contiguous nucleotides of a sequence extending from position 200 to position 250 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 1 comprises at least 10 contiguous nucleotides of a sequence extending from position 300 to position 400 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 1 comprises at least 10 contiguous nucleotides of a sequence extending from position 500 to position 750 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 1 comprises at least 10 contiguous nucleotides of a sequence extending from position 600 to position 700 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 1 comprises at least 10 contiguous nucleotides of a sequence extending from position 650 to position 700 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 1 is selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, and 151. In some embodiments, a target sequence in intron 1 is selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, and 9. In some embodiments, a target sequence in intron 1 is selected from SEQ ID NOs: 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, and 151. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 1 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 379, 380, 381, 382, 383, 384, 385, 386, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, and 528. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 1 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 379, 380, 381, 382, 383, 384, 385, and 386. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 1 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, and 528. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 1 comprises a nucleotide sequence selected from SEQ ID NOs: 379, 380, 381, 382, 383, 384, 385, and 386. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 1 comprises a nucleotide sequence selected from SEQ ID NOs: 141, 142, 181, 182, 183, 249, 250, and 251. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 1 comprises a nucleotide sequence selected from SEQ ID NOs: 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, and 528.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 2 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 2 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 2 comprises at least 10 contiguous nucleotides of a sequence extending from position 885 to position 1335 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 2 comprises at least 10 contiguous nucleotides of a sequence extending from position 885 to position 1000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 2 comprises at least 10 contiguous nucleotides of a sequence extending from position 1000 to position 1300 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 2 comprises at least 10 contiguous nucleotides of a sequence extending from position 1100 to position 1200 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 2 comprises at least 10 contiguous nucleotides of a sequence extending from position 1200 to position 1300 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 2 is selected from SEQ ID NOs: 152, 153, 154, 155, 156, 157, and 158. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 2 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 529, 530, 531, 532, 533, 534, and 535. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 2 comprises a nucleotide sequence selected from SEQ ID NOs: 529, 530, 531, 532, 533, 534, and 535.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 3 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 3 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 1336 to position 1423 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 1350 to position 1400 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 3 is selected from SEQ ID NOs: 159 and 160. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 3 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 536 and 537. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 3 comprises a nucleotide sequence selected from SEQ ID NOs: 536 and 537.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 3 and intron 3 of a human EFEMP1 pre-mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence that spans exon 3 and intron 3 of a human EFEMP1 pre-mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence that spans exon 3 and intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 1373 to position 1473 of SEQ ID NO: 1. In some embodiments, a target sequence that spans exon 3 and intron 3 is set forth in SEQ ID NO: 161. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 3 and intron 3 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 538. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 3 and intron 3 comprises SEQ ID NO: 538.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 3 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 3 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 1424 to position 5515 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 1600 to position 1900 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 2000 to position 2200 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 2200 to position 2500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 2500 to position 2800 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 2800 to position 3100 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 3100 to position 3400 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 3400 to position 3500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 3500 to position 3800 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 3700 to position 3800 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 3800 to position 3900 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 3900 to position 4000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 4000 to position 4100 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 4100 to position 4400 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 4400 to position 4700 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 4700 to position 5000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 4950 to position 5050 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 5000 to position 5300 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 5100 to position 5300 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 comprises at least 10 contiguous nucleotides of a sequence extending from position 5300 to position 5500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 3 is selected from SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, 18, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, and 175. In some embodiments, a target sequence in intron 3 is selected from SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, and 18. In some embodiments, a target sequence in intron 3 is selected from SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, and 175. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 3 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 387, 388, 389, 390, 391, 392, 393, 394, 395, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, and 552. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 3 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 387, 388, 389, 390, 391, 392, 393, 394, and 395. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 3 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a nucleotide sequence selected from SEQ ID NOs: 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, and 552. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 3 comprises a nucleotide sequence selected from SEQ ID NOs: 387, 388, 389, 390, 391, 392, 393, 394, 395, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, and 552. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 3 comprises a nucleotide sequence selected from SEQ ID NOs: 387, 388, 389, 390, 391, 392, 393, 394, and 395. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 3 comprises a nucleotide sequence selected from SEQ ID NOs: 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, and 552.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 4 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 4 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 4 comprises at least 10 contiguous nucleotides of a sequence extending from position 5516 to position 5564 of SEQ ID NO: 1. In some embodiments, atarget sequence in exon 4 comprises at least 10 contiguous nucleotides of a sequence extending from position 5450 to position 5550 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 4 is SEQ ID NO: 176. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 4 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 553. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 4 comprises SEQ ID NO: 553. In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 4 and intron 4 of a human EFEMP1 pre-mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence that spans exon 4 and intron 4 of a human EFEMP1 pre-mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence that spans exon 4 and intron 4 comprises at least 10 contiguous nucleotides of a sequence extending from position 5514 to position 5614 of SEQ ID NO: 1. In some embodiments, a target sequence that spans exon 4 and intron 4 is selected from SEQ ID NOs: 19, 20, and 177. In some embodiments, a target sequence that spans exon 4 and intron 4 is selected from SEQ ID NOs: 19 and 20. In some embodiments, a target sequence that spans exon 4 and intron 4 is SEQ ID NO: 177. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 4 and intron 4 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 396, 397, and 554. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 4 and intron 4 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 396 and 397. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 4 and intron 4 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 554. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 4 and intron 4 comprises any one of SEQ ID NOs: 396, 397, and 554. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 4 and intron 4 comprises any one of SEQ ID NOs: 396 and 397. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 4 and intron 4 comprises SEQ ID NO: 554.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 5 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 5 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 5732 to position 6118 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 5700 to position 5800 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 5800 to position 5900 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 5900 to position 6000 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 6000 to position 6100 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 5 is selected from SEQ ID NOs: 21, 22, 23, 24, 25, 26, 178, 179, 180, 181, 182, 183, 184, and 185. In some embodiments, a target sequence in exon 5 is selected from SEQ ID NOs: 21, 22, 23, 24, 25, and 26. In some embodiments, a target sequence in exon 5 is selected from SEQ ID NOs: 178, 179, 180, 181, 182, 183, 184, and 185. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 5 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 398, 399, 400, 401, 402, 403, 555, 556, 557, 558, 559, 560,

561, and 562. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 5 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 398, 399, 400, 401, 402, and 403. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 5 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 555, 556, 557, 558, 559, 560, 561, and

562. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 5 comprises any one of SEQ ID NOs: 398, 399, 400, 401, 402, 403, 555, 556, 557, 558, 559, 560, 561, and 562. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 5 comprises any one of SEQ ID NOs: 398, 399, 400, 401, 402, and 403. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 5 comprises any one of SEQ ID NOs: 555, 556, 557, 558, 559, 560, 561, and 562.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 5 and intron 5 of a human EFEMP1 pre-mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence that spans exon 5 and intron 5 of a human EFEMP1 pre-mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence that spans exon 5 and intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 6068 to position 6168 of SEQ ID NO: 1. In some embodiments, a target sequence that spans exon 5 and intron 5 is set forth in SEQ ID NO: 186. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 5 and intron 5 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity SEQ ID NO: 563. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 5 and intron 5 comprises of SEQ ID NO: 563.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 5 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 5 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 5732 to position 42048 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 5800 to position 9000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 7000 to position 8000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 8000 to position 9000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 9000 to position 10000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 7000 to position 10000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 10000 to position 13000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 13000 to position 16000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 15500 to position 16500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 15800 to position 16100 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 16000 to position 16100 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 16000 to position 19000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 17000 to position 18000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 19000 to position 21000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 21000 to position 24000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 22000 to position 23000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 22000 to position 22500 of SEQ ID NO: 1. In some embodiments, atarget sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 22000 to position 22300 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 24000 to position 27000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 24000 to position 25000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 24000 to position 24500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 24000 to position 24100 of SEQ ID NO: 1. In some embodiments, atarget sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 24500 to position 25000 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 24900 to position 25000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 27000 to position 30000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 28000 to position 29000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 28000 to position 28500 of SEQ ID NO: 1. In some embodiments, atarget sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 28400 to position 28500 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 28500 to position 28600 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 28600 to position 28700 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 28700 to position 28800 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 28800 to position 28900 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 30000 to position 33000 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 33000 to position 35000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 34000 to position 35000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 34500 to position 35000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 34600 to position 34700 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 34700 to position 34800 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 34000 to position 37000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 35000 to position 37000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 37000 to position 40000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 37000 to position 38000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 38000 to position 39000 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 39000 to position 40000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 40000 to position 42000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 comprises at least 10 contiguous nucleotides of a sequence extending from position 40000 to position 41000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 5 is selected from SEQ ID NOs: 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 187, 188, 189,

190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,

210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,

230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,

250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,

270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,

290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, and 314. In some embodiments, a target sequence in intron 5 is selected from

SEQ ID NOs: 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, and 119. In some embodiments, a target sequence in intron 5 is selected from SEQ ID NOs: 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,

212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,

252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,

272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,

292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,

312, 313, and 314. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 5 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439,

440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,

460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,

480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 564, 565, 566,

567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586,

587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606,

607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,

627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,

647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666,

667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686,

687, 688, 689, 690, and 691. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 5 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435,

436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455,

456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475,

476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, and 496. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 5 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601,

602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621,

622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641,

642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661,

662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681,

682, 683, 684, 685, 686, 687, 688, 689, 690, and 691. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 5 comprises any one of SEQ ID NOs: 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,

437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456,

457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,

477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,

564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583,

584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603,

604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,

624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643,

644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,

664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,

684, 685, 686, 687, 688, 689, 690, and 691. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 5 comprises any one of SEQ ID NOs: 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,

421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,

441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,

461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,

481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, and 496. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 5 comprises any one of SEQ ID NOs: 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,

591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610,

611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630,

631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,

651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, and 691.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 5 and exon 6 of a human EFEMP1 pre-mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence that spans intron 5 and exon 6 of a human EFEMP1 pre-mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence that spans intron 5 and exon 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 41998 to position 42098 of SEQ ID NO: 1. In some embodiments, a target sequence that spans intron 5 and exon 6 is selected from SEQ ID NOs: 120 and 135. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 5 and exon 6 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity of any one of SEQ ID NOs: 497 and 692. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 5 and exon 6 comprises any one of SEQ ID NOs: 497 and 692.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 6 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 6 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 42049 to position 42171 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 6 is selected from SEQ ID NOs: 316 and 317. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 6 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 693 and 694. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 6 comprises any one of SEQ ID NOs: 693 and 694.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 6 and intron 6 of a human EFEMP1 pre-mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence that spans exon 6 and intron 6 of a human EFEMP1 pre-mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence that spans exon 6 and intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 42121 to position 42221 of SEQ ID NO: 1. In some embodiments, a target sequence that spans exon 6 and intron 6 is SEQ ID NOs: 121. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 6 and intron 6 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity of SEQ ID NO: 498. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 6 and intron 6 comprises SEQ ID NO: 498.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 6 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 6 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 42172 to position 45917 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 42100 to position 43000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 42000 to position 43000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 43000 to position 44000 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 43000 to position 43500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 43100 to position 43200 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 43200 to position 43300 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 44000 to position 45000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 6 comprises at least 10 contiguous nucleotides of a sequence extending from position 45000 to position 45500 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 6 is selected from SEQ ID NOs: 122, 123, 124, 125, 126, 127, 128, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, and 331. In some embodiments, a target sequence in intron 6 is selected from SEQ ID NOs: 122, 123, 124, 125, 126, 127, and 128. In some embodiments, a target sequence in intron 6 is selected from SEQ ID NOs: 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, and 331. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 6 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 499, 500, 501, 502, 503, 504, 505, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, and 708. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 6 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 499, 500,

501, 502, 503, 504, and 505. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 6 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,

707, and 708. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 6 comprises any one of SEQ ID NOs: 499, 500, 501, 502, 503, 504, 505, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, and

708. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 6 comprises any one of SEQ ID NOs: 499, 500, 501,

502, 503, 504, and 505. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 6 comprises any one of SEQ ID NOs: 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, and 708.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 6 and exon 7 of a human EFEMP1 pre-mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence that spans intron 6 and exon 7 of a human EFEMP1 pre-mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence that spans intron 6 and exon 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 45868 to position 45968 of SEQ ID NO: 1. In some embodiments, a target sequence that spans intron 6 and exon 7 is SEQ ID NOs: 332. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 6 and exon 7 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity of SEQ ID NO: 709. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 6 and exon 7 comprises SEQ ID NO: 709.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 7 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 7 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 45918 to position 46037 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 45900 to position 46000 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 45950 to position 46030 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 7 is selected from SEQ ID NOs: 333 and 334. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 7 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 710 and 711. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 7 comprises any one of SEQ ID NOs: 710 and 711.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 7 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 7 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 46038 to position 47040 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 46100 to position 47000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 46400 to position 46500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 46500 to position 46600 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 46600 to position 46700 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 7 comprises at least 10 contiguous nucleotides of a sequence extending from position 46700 to position 46800 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 7 is selected from SEQ ID NOs: 335-338. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 7 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 712-715. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 7 comprises any one of SEQ ID NOs: 712-715.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 8 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 8 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47041 to position 47160 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47060 to position 47150 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 8 is SEQ ID NO: 339. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 8 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 716. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to atarget sequence in exon 8 comprises SEQ ID NO: 716.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 8 and intron 8 of a human EFEMP1 pre-mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence that spans exon 8 and intron 8 of a human EFEMP1 pre-mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence that spans exon 8 and intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47110 to position 47210 of SEQ ID NO: 1. In some embodiments, a target sequence that spans exon 8 and intron 8 is SEQ ID NOs: 129. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 8 and intron 8 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity of SEQ ID NO: 506. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans exon 8 and intron 8 comprises SEQ ID NO: 506.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 8 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 8 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47161 to position 48717 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47200 to position 47400 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47300 to position 47400 of SEQ ID NO: 1. In some embodiments, atarget sequence in intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47300 to position 47700 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47700 to position 47900 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 47900 to position 48200 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 48200 to position 48500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 8 comprises at least 10 contiguous nucleotides of a sequence extending from position 48300 to position 48700 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 8 is selected from SEQ ID NOs: 130, 131, 340, 341, 342, 343, 344, and 345. In some embodiments, atarget sequence in intron 8 is selected from SEQ ID NOs: 130 and 131. In some embodiments, a target sequence in intron 8 is selected from SEQ ID NOs: 340, 341, 342, 343, 344, and 345. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to atarget sequence in intron 8 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 507, 508, 717, 718, 719, 720, 721, and 722. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 8 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 507 and 508. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 8 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 717, 718, 719, 720, 721, and 722. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 8 comprises any one of SEQ ID NOs: 507, 508, 717, 718, 719, 720, 721, and 722. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 8 comprises any one of SEQ ID NOs: 507 and 508. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 8 comprises any one of SEQ ID NOs: 717, 718, 719, 720, 721, and 722.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 9 of a human EFEMP 1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 9 of a human EFEMP 1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 9 comprises at least 10 contiguous nucleotides of a sequence extending from position 48838 to position 52659 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 9 comprises at least 10 contiguous nucleotides of a sequence extending from position 49000 to position 50000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 9 comprises at least 10 contiguous nucleotides of a sequence extending from position 50000 to position 51000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 9 comprises at least 10 contiguous nucleotides of a sequence extending from position 51000 to position 52000 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 9 comprises at least 10 contiguous nucleotides of a sequence extending from position 52000 to position 52600 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 9 is selected from SEQ ID NOs: 132, 133, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, and 358. In some embodiments, a target sequence in intron 9 is selected from SEQ ID NOs: 132 and 133. In some embodiments, a target sequence in intron 9 is selected from SEQ ID NOs: 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, and 358. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 9 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 509, 510, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, and 735. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 9 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 509 and 510. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 9 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 723, 724,

725, 726, 727, 728, 729, 730, 731, 732, 733, 734, and 735. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 9 comprises any one of SEQ ID NOs: 509, 510, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, and 735. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 9 comprises any one of SEQ ID NOs: 509 and 510. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 9 comprises any one of SEQ ID NOs: 723, 724, 725,

726, 727, 728, 729, 730, 731, 732, 733, 734, and 735.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 10 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 10 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 10 comprises at least 10 contiguous nucleotides of a sequence extending from position 52660 to position 52783 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 10 is selected from SEQ ID NOs: 359 and 360. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 10 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 736 and 737. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 10 comprises any one of SEQ ID NOs: 736 and 737.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 10 and exon 11 of a human EFEMP1 pre-mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence that spans intron 10 and exon 11 of a human EFEMP1 pre-mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence that spans intron 10 and exon 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 52817 to position 52917 of SEQ ID NO: 1. In some embodiments, a target sequence that spans intron 10 and exon 11 is SEQ ID NO: 361. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 10 and exon 11 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity of SEQ ID NO: 738. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence that spans intron 10 and exon 11 comprises SEQ ID NO: 738.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 11 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 11 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 52868 to position 53063 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 11 is selected from SEQ ID NOs: 134 and 362. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 11 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 511 and 739. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 11 comprises any one of SEQ ID NOs: 511 and 739.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in intron 11 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in intron 11 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 53064 to position 56548 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 53000 to position 55000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 53000 to position 54000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 53400 to position 55000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 54000 to position 56000 of SEQ ID NO: 1 . In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 55000 to position 56000 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 56000 to position 56500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 56100 to position 56500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 11 comprises at least 10 contiguous nucleotides of a sequence extending from position 56400 to position 56500 of SEQ ID NO: 1. In some embodiments, a target sequence in intron 11 is selected from SEQ ID NOs: 135, 136, 137, 138, 363, 364, 365, 366, 367, 368, 369, 370, 371, and 372. In some embodiments, a target sequence in intron 11 is selected from SEQ ID NOs: 135, 136, 137, and 138. In some embodiments, a target sequence in intron 11 is selected from SEQ ID NOs: 363, 364, 365, 366, 367, 368, 369, 370, 371, and 372. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 11 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 512, 513, 514, 515, 740, 741, 742, 743, 744, 745, 746, 747, 748, and 749. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 11 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 512, 513, 514, and 515. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 11 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 740, 741, 742, 743, 744, 745, 746, 747, 748, and 749. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 11 comprises any one of SEQ ID NOs: 512, 513, 514, 515, 740, 741, 742, 743, 744, 745, 746, 747, 748, and 749. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 11 comprises any one of SEQ ID NOs: 512, 513, 514, and 515. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in intron 11 comprises any one of SEQ ID NOs: 740, 741, 742, 743, 744, 745, 746, 747, 748, and 749.

In some embodiments, the disclosure provides an oligonucleotide (e.g., an antisense oligonucleotide or an RNAi oligonucleotide) comprising a sequence comprising a region of contiguous nucleosides complementary to a target sequence in exon 12 of a human EFEMP1 pre- mRNA of 10-50 nucleotides in length. In some embodiments, an oligonucleotide comprises a region of contiguous nucleosides complementary to a target sequence in exon 12 of a human EFEMP1 pre- mRNA set forth in SEQ ID NO: 1 of 10-50 nucleotides in length. In some embodiments, a target sequence in exon 12 comprises at least 10 contiguous nucleotides of a sequence extending from position 56549 to position 57816 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 12 comprises at least 10 contiguous nucleotides of a sequence extending from position 56500 to position 56900 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 12 comprises at least 10 contiguous nucleotides of a sequence extending from position 56600 to position 57000 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 12 comprises at least 10 contiguous nucleotides of a sequence extending from position 5700 to position 57400 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 12 comprises at least 10 contiguous nucleotides of a sequence extending from position 57400 to position 57800 of SEQ ID NO: 1. In some embodiments, a target sequence in exon 12 is selected from SEQ ID NOs: 139, 140, and 373-378. In some embodiments, a target sequence in exon 12 is selected from SEQ ID NOs: 139 and 140. In some embodiments, a target sequence in exon 12 is selected from SEQ ID NOs: 373-378. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 12 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 516, 517, and 750-755. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 12 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 516 and 517. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 12 comprises a nucleotide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 750-755. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 12 comprises any one of SEQ ID NOs: 516, 517, and 750-755. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 12 comprises any one of SEQ ID NOs: 516 and 517. In some embodiments, an oligonucleotide comprising a region of contiguous nucleosides complementary to a target sequence in exon 12 comprises any one of SEQ ID NOs: 750-755.

It should be appreciated that, while the Examples describe the exemplary nucleic acid sequences provided in Tables 2 and 3 in a gapmer design, these exemplary nucleic acid sequences provided in Table 2 can be used with any of the nucleic acid designs described herein and/or known to one of ordinary skill in the art (e.g., antisense oligonucleotide, siRNA, gapmer, with or without chemical modifications, etc.). Table 2 provides the “start site” for each nucleic acid, which corresponds to the position in the human EFEMP1 pre-mRNA nucleotide sequence that binds to the 5 '-terminal nucleoside of the nucleic acid set forth in the corresponding SEQ ID NO.

It should also be appreciated that the nucleic acid sequences for targeting EFEMP 1 in Table 3 are provided in a DNA format (i.e., using G, C, T, and A). In embodiments that utilize an RNA format, the T’s (e.g., thymine) may be replaced with U’s (e.g., uracil).

Table 2. Exemplary Nucleic Acid Sequences

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 463. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 463.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 516. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 139. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 516.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 387. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 387.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 502. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 502.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 517. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 140. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 517.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 400. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 400.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 401. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 401.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 453. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 76. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 453.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 830. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 759. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 830.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 837. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 766. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 837.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 840. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 769. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 840.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 859. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 788. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 859.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 873. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 802. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 873.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 886. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 815. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 886.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 895. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 824. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 895.

In some embodiments, an oligonucleotide comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 896. In some embodiments, an oligonucleotide comprises a nucleic acid sequence that is complementary to an EFEMP1 target sequence, wherein the target sequence comprises at least 8 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 825. In some embodiments, the nucleic acid sequence of an oligonucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 896.

In some embodiments, the present disclosure provides an oligonucleotide for targeting EFEMP1 that is an antisense oligonucleotide. In some embodiments, the present disclosure provides antisense oligonucleotides and compositions thereof for modulating (e.g., reducing or inhibiting) expression of EFEMP1. In some embodiments, the antisense oligonucleotide modulates (e.g., inhibits or reduces) expression of EFEMP1 in a cell (e.g., an RPE cell) in vitro or in vivo.

As used herein, an “antisense oligonucleotide” refers to a synthetic, single-stranded nucleic acid polymer of about 10-50 nucleosides in length that contains RNA nucleotides, DNA nucleotides, or combinations thereof, and/or modified nucleotides or modified nucleoside linkages, which targets a nucleic acid comprising a target sequence described herein (e.g. , a nucleic acid encoding EFEMP1) and associated with a target sequence in a sequence-specific manner (e.g., binds to or hybridizes with a target sequence). Antisense oligonucleotides as described herein specifically bind to or specifically hybridize with a target nucleic acid molecules (e.g., Watson-Crick hydrogen bonding) and modulate expression of a targeted nucleic acid by one or more processes to inhibit, interfere, disrupt, cleave, degrade, and/or alter, activate or block normal function and/or expression. This interference with or modulation of the function of a target nucleic acid by an antisense oligonucleotide described herein is referred to as “antisense inhibition.” Antisense oligonucleotides as described herein mediate a variety of effects including, for example, bind to a target pre-mRNA molecule (e.g., an EFEMP1 pre-mRNA molecule) and block an activity/effect (e.g., splicing pattern) of the targeted pre-mRNA sequence; interact with a target gene (e.g. , an EFEMP1 gene) to modulate transcriptional inhibition; and interact with a target an RNA transcript (e.g. , an EFEMP 1 pre-mRNA or an EEMP 1 mRNA) for translational inhibition and/or degradation.

In some embodiments, antisense oligonucleotides of the disclosure induce inhibition by an RNase H-mechanism or by steric block. RNase H enzyme RNASEH1 recognizes RNA-DNA heteroduplex substrates when an oligonucleotide comprising DNA binds to or hybridizes with an RNA transcript and recruits RNASEH1 to mediate transcript cleavage and degradation. In some embodiments, the disclosure provides antisense oligonucleotides comprising one or more RNase H competent nucleosides and one or more RNase H resistant nucleosides. As used herein, an “RNase H competent nucleoside” refers to a nucleoside present in an oligonucleotide bound to a target sequence in a target RNA, wherein the nucleoside is a substrate for RNase H. Exemplary RNase H competent nucleosides are known in the art and include, but are not limited to, deoxyribonucleosides or modified deoxyribonucleosides. As used herein, an “RNase H resistant nucleoside” refers to a nucleoside present in an oligonucleotide bound to a target sequence in a target RNA, wherein the nucleoside is not a substrate for RNase H (e.g., does not substantially induce RNase H-mediated recruitment and/or activity). Exemplary RNase H resistant nucleosides are known in the art and include, but are not limited to, ribonucleosides or modified ribonucleosides (e.g., a ribonucleoside comprising a 2 'modification, e.g., a 2'MOE modification). Methods to measure RNase H activity, e.g., using an in vitro RNA cleavage assay, are known in the art. For example, a suitable in vitro assay to measure RNase H-mediated cleavage comprises (i) incubating RNase H with (a) an oligonucleotide comprising one or more RNase H competent nucleosides and one or more RNase H resistant nucleosides and (b) an RNA molecule comprising a target sequence, and (ii) performing analysis of cleavage products by gel electrophoresis.

In some embodiments, the antisense oligonucleotide modulates EFEMP1 expression by RNase H recruitment and RNase H-mediated cleavage and degradation. In some embodiments, the antisense oligonucleotide reduces or inhibits EFEMP1 expression by RNase H recruitment and RNase H-mediated cleavage and degradation. In some embodiments, the RNase H competent nucleosides comprise deoxyribonucleosides or modified deoxyribonucleosides and the RNase H resistant nucleosides comprise ribonucleosides or modified ribonucleosides. In some embodiments, the one or more RNase H competent nucleosides and one or more RNase H resistant nucleosides are arranged in a gapmer motif, as further described herein. A “gapmer” refers to a chimeric antisense oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. In some embodiments, the internal region is referred to as a “gap segment” and the external regions are referred to as “wing segments.” The wing segment at the 5'-end of the gap segment is referred to as the “5 '-wing segment” and the wing segment at the 3 '-end of the gap segment is referred to as the “3'-wing segment.” Antisense oligonucleotides of the disclosure comprising a gapmer pattern are further described herein. In some embodiments, the antisense oligonucleotide specifically binds to an EFEMP1 target sequence present in an EFEMP1 RNA transcript to form a RNA/DNA duplex, thereby recruiting RNase H and inhibiting expression of EFEMP1 via RNase H-mediated cleavage and degradation. In some embodiments, the disclosure provides an antisense oligonucleotide comprising one or more RNase H competent nucleosides and one or more RNase H resistant nucleosides that are arranged in a gapmer motif, wherein the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2. In some embodiments, the antisense oligonucleotide is 20 nucleosides in length and comprises a nucleotide sequence set forth in Table 2. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has a 5'-wing segment, a gap segment, and a 3 '-wing segment.

In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has a 5 '-wing segment comprising RNAse H resistant nucleosides, a gap segment comprising RNAse H competent nucleosides, and a 3 '-wing segment comprising RNAse H resistant nucleosides. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has a 5 '-wing segment of 5-10 RNAse H resistant nucleosides, a gap segment of 10-20 RNAse H competent nucleosides, and a 3 '-wing segment of 5-10 RNAse H resistant nucleosides. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has a 5 '-wing segment of 5 RNAse H resistant nucleosides, a gap segment of 10 RNAse H competent nucleosides, and a 3 '-wing segment of 5 RNAse H resistant nucleosides.

In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has a 5 '-wing segment of 5-10 ribonucleosides (modified or unmodified ribonucleosides), a gap segment of 10-20 RNAse H competent deoxynucleosides (modified or unmodified deoxynucleosides), and a 3'-wing segment of 5-10 ribonucleosides (modified or unmodified ribonucleosides). In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has a 5 '-wing segment of 5 ribonucleosides (modified or unmodified ribonucleosides), a gap segment of 10 RNAse H competent deoxynucleosides (modified or unmodified deoxynucleosides), and a 3 '-wing segment of 5 ribonucleosides (modified or unmodified ribonucleosides).

In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has 5'-wing segment of 5-10 ribonucleosides comprising a 2'-sugar modification (e.g., a 2'-O-methoxyethyl (2'-M0E) modification), a gap segment of 10-20 deoxynucleosides (modified or unmodified deoxynucleosides), and a 3 '-wing segment of 5-10 ribonucleosides comprising a 2'-sugar modification (e.g., a 2'-O-methoxyethyl (2'-M0E) modification), optionally wherein one or more intemucleoside linkages of the 5 '-wing segment, the gap segment, and/or the 3'- wing segment are phosphorothioate linkages. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has 5'-wing segment of 5 ribonucleosides comprising a 2'-sugar modification (e.g., a 2'-O-methoxyethyl (2'-M0E) modification), a gap segment of 10 deoxynucleosides (modified or unmodified deoxynucleosides), and a 3 '-wing segment of 5 ribonucleosides comprising a 2'-sugar modification (e.g., a 2'-O-methoxyethyl (2'-M0E) modification), optionally wherein one or more intemucleoside linkages of the 5 '-wing segment, the gap segment, and/or the 3 '-wing segment are phosphorothioate linkages.

In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has 5'-wing segment of 5-10 ribonucleosides comprising a 2'-M0E modification, a gap segment of 10-20 deoxynucleosides (modified or unmodified deoxynucleosides), and a 3 '-wing segment of 5-10 ribonucleosides comprising a 2'-M0E modification, optionally wherein one or more intemucleoside linkages of the 5 '-wing segment, the gap segment, and/or the 3 '-wing segment are phosphorothioate linkages. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence set forth in Table 2 and has 5 '-wing segment of 5 ribonucleosides comprising a 2'-M0E modification, a gap segment of 10 deoxynucleosides (modified or unmodified deoxynucleosides), and a 3'-wing segment of 5 ribonucleosides comprising a 2'-M0E modification, optionally wherein one or more intemucleoside linkages of the 5 '-wing segment, the gap segment, and/or the 3 '-wing segment are phosphorothioate linkages.

In some embodiments, the disclosure provides antisense oligonucleotides that induce inhibition via steric block lack RNaseH competence in that they are formed from non-deoxyribosides or lack stretches of consecutive deoxyriboside sequences. In some embodiments, antisense oligonucleotides of the disclosure reduce or inhibit expression of a target RNA transcript by masking specific sequences within the RNA transcript that form RNA-RNA and/or RNA-protein interactions necessary for splicing, transportation, and/or translation.

In some embodiments, antisense oligonucleotides of the disclosure reduce or inhibit expression of a target nucleic acid or protein in a cell. The language “inhibiting an expression level” of a target nucleic acid and/or target protein in a cell (e.g. , a cell in a subject) refers to the expression level of a target nucleic acid and or/target protein in the cell in the presence of an antisense oligonucleotide as compared to an expression level of a target nucleic acid and/or target protein in the absence of the antisense oligonucleotide. In some embodiments, the antisense oligonucleotide of the disclosure comprises a gapmer and inhibits translation by base pairing to an RNA transcript and inducing RNase H-mediated degradation. In some embodiments, the antisense oligonucleotide inhibits translation in a stoichiometric manner by base pairing to a target mRNA and physically obstructing the translation machinery (see, e.g., Dias, et al (2002) Mol Cancer Ther 1:347-55).

In some embodiments, the present disclosure provides an oligonucleotide targeting EFEMP1 that is an RNAi oligonucleotide. In some embodiments, the present disclosure provides RNAi oligonucleotides and compositions thereof for modulating (e.g., reducing or inhibiting) expression of EFEMP1. In some embodiments, the RNAi oligonucleotide modulates (e.g., inhibits or reduces) expression of EFEMP1 in a cell (e.g., an RPE cell) in vitro or in vivo. Herein, the term “RNA interference (RNAi) molecule” refers to any molecule inhibiting RNA expression or translation via the RNA reducing silencing complex (RISC) in a cell's cytoplasm, where the RNAi molecule interact with the catalytic RISC component argonaute.

In some embodiments, the EFEMP1 -targeting oligonucleotides described herein are doublestranded RNA. The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where separate RNA molecules, such dsRNA are often referred to as siRNA (“short interfering RNA”) or DsiRNA (“Dicer substrate siRNAs”). Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3 '-end of one strand and the 5' end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”, “short hairpin RNA” or “shRNA”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3 '-end of one strand and the 5 'end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. In addition, as used herein, “dsRNA” may include chemical modifications to ribonucleotides, intemucleoside linkages, end-groups, caps, and conjugated moieties, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA- or DsiRNA-type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.

In some embodiments, an EFEMP1 -targeting oligonucleotide described herein is an siRNA. In some embodiments, a small interfering RNA (siRNA) is a double-stranded RNA complex comprising a passenger (sense) and a guide (antisense) oligonucleotide (strand), which when administered to a cell, results in the incorporation of the guide (antisense) strand into the RISC complex (siRISC) resulting in the RISC-associated inhibition of translation or degradation of complementary RNA target nucleic acids in the cell. The sense strand is also referred to as the passenger strand, and the antisense strand as the guide strand. Each strand comprises RNA, RNA analog(s) or RNA and DNA. In some embodiments, the siRNA has 2 bp overhangs on the 3' ends of each strand such that the duplex region in the siRNA comprises 17-21 nucleotides, or 19 nucleotides. In some embodiments, the siRNA comprises a 2 bp overhang on the 3 ’ end of the antisense strand, and a blunt end comprising the 5’ end of the antisense strand and the 3’ end of the sense strand. In some embodiments, the antisense strand of the siRNA is sufficiently complementary with a target sequence of the EFEMP1 target nucleic acid. In some embodiments, an EFEMP1 -targeting oligonucleotide described herein is an shRNA. A small hairpin RNA (shRNA) is a single nucleic acid molecule which forms a stem loop (hairpin) structure that is able to degrade mRNA via RISC.

RNAi nucleic acid molecules may be synthesized chemically (typical for siRNA complexes) or by in vitro transcription, or expressed from a vector. shRNA molecules are generally between 40 and 70 nucleotides in length, such as between 45 and 65 nucleotides in length, such as 50 and 60 nucleotides in length, and interacts with the endonuclease known as Dicer which is believed to processes dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs which are then incorporated into an RNA-induced silencing complex (RISC). In some embodiments, the guide (antisense) strand of an siRNA (or antisense region of a shRNA) is 17 - 25 nucleotide in length, such as 19 - 23 nucleotides in length and complementary to a target nucleic acid or target sequence. In an siRNA complex, the guide (antisense) strand and passenger (sense) strand form a double stranded duplex, which may comprise 3’ terminal overhangs of e.g., 1- 3 nucleotides (resembles the product produced by Dicer), or may be blunt ended (no overhang at one or both ends of the duplex).

It will be recognized that RNAi may be mediated by longer dsRNA substrates which are processed into siRNAs within the cell (a process which is thought to involve the dsRNA endonuclease DICER). Effective extended forms of Dicer substrates have been described in US 8,349,809 and US 8,513,207, hereby incorporated by reference.

In some embodiments, an EFEMP1 -targeting oligonucleotide described herein is not a double -stranded RNA. In some embodiments, an EFEMP1 -targeting oligonucleotide described herein is not a small interfering RNA (siRNA). In some embodiments, an EFEMP1 -targeting oligonucleotide described herein is not a short hairpin RNA (shRNA).

Chemical Modifications

In some embodiments, an oligonucleotide described herein comprises one or more chemical modifications. In some embodiments, the one or more chemical modifications is a modified nucleotide or modified nucleoside. As used herein, the term “modified nucleotide” or “modified nucleoside” refers to a non-standard nucleotide or nucleoside respectively, including non-naturally occurring ribonucleotides/ribonucleosides or deoxyribonucleotides/deoxyribonucleosides. In some embodiments, the modification is at any position so as to alter certain chemical properties of the nucleotide or nucleoside yet retain the ability of the nucleotide or nucleoside to perform its intended function. In some embodiments, the nucleotide or nucleoside comprises a modified sugar moiety, a modified intemucleoside linkage, and/or modified nucleobase. Thus, the terms “modified nucleotide” and “modified nucleoside” encompass substitutions, additions or removal of, e.g., a functional group or atom, to intemucleoside linkages, sugar moieties, and/or nucleobases. In some embodiments, an oligonucleotide is chemically modified to enhance stability, increase melting temperature (Tm) upon annealing to a target sequence, and/or other beneficial characteristics. In some embodiments, an oligonucleotide is fully modified (i.e., referring to a chemical modification which applies to each nucleotide or linked nucleoside of an oligonucleotide of the disclosure). For example, a fully modified oligonucleotide may comprise a modified nucleobase, a modified sugar, and/or a modified intemucleoside linkage at each nucleotide or linked nucleoside. In some embodiments, the fully modified oligonucleotide is uniformly modified (i.e., referring to each nucleotide and/or linked nucleoside present in an oligonucleotide having the same modification of the nucleobase, sugar, and/or intemucleoside linkage). In some embodiments, the fully modified oligonucleotide is non-uniformly modified (i.e., referring to the nucleotides and/or linked nucleosides present in an oligonucleotide as each having a modification of the nucleobase, sugar, and/or intemucleoside linkage that is independent of one another). In some embodiments, an oligonucleotide is partially modified (i.e., referring to an oligonucleotide comprising a combination of modified and unmodified nucleotides and/or linked nucleosides). In some embodiments, an oligonucleotide is substantially modified, wherein not more than about 5, 4, 3, 2, or 1 nucleosides are unmodified.

In some embodiments, the oligonucleotides described herein are synthesized and/or modified by standard methods known in the art as further discussed below, e.g., solution-phase or solid-phase organic synthesis or both, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. Well-established methods for the synthesis and/or modification of the oligonucleotides described herein are provided by, e.g., “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, (incorporated by reference). Modifications include, for example, end modifications, e.g., 5 '-end modifications (phosphorylation, conjugation, inverted linkages) or 3 '-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g. , at the 2'- position or 4'-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages.

In some embodiments, an oligonucleotide comprises one or more modified nucleosides. Exemplary modified nucleosides include, but are not limited to, nucleosides comprising modified linkages or non-natural linkages in its intemucleoside linkage to a 5' adjacent nucleoside and/or to a 3' adjacent nucleoside. Exemplary non-natural or modified intemucleoside linkages include, but are not limited to, those that do not have a phosphoms atom in the backbone. In some embodiments, the modified nucleoside comprises a phosphoms atom in its intemucleoside linkage to a 5' adjacent nucleoside and/or to a 3' adjacent nucleoside.

In some embodiments, an oligonucleotide comprises one or more modified intemucleoside linkage(s) selected from a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonate, phosphinate, phosphoramidate (e.g., 3 '-amino phosphoramidate and aminoalkylphosphoramidate), thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate. In some embodiments, the one or more modified intemucleoside linkage is a 3'-5' linkage or a 2'-5'-linkage. In some embodiments, the one or more modified intemucleoside linkage has inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Salts, mixed salts and free acid forms of the modified intemucleoside linkage are further encompassed by embodiments described herein.

Representative U.S. patents that describe the preparation of the above phosphoms-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, an oligonucleotide comprises one or more phosphodiester intemucleotide linkages. In some embodiments, an oligonucleotide is modified to comprise one or more modified intemucleotide linkages. A modified intemucleotide linkage may be any intemucleotide linkage other than a phosphodiester intemucleoside linkage. Examples of modified intemucleotide linkages include phosphorothioates (e.g., having Rp or Sp stereochemistry), phosphotriesters, methyl phosphonates, short chain alkyl linkages, cycloalkyl linkages, and heteroatomic or heterocyclic linkages. Additional phosphoms-containing linkages that may be used include phosphorodithioates, aminoalkylphosphotriesters, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. In some embodiments, at least one, two, three, four, five or more of the intemucleotide linkages of an oligonucleotide are modified intemucleotide linkages. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the intemucleotide linkages of an oligonucleotide are modified intemucleotide linkages.

In some embodiments, an antisense oligonucleotide is modified to comprise one or more modified nucleotides comprising modified sugar moieties. In some embodiments, an oligonucleotide comprises a 2 ’-modified nucleotide such as a 2 ’-deoxy, 2 ’-deoxy -2’ -fluoro, 2’-O-methyl, 2’-O- methoxyethyl (2’-0-M0E), 2’-O-aminopropyl (2’-O-AP), 2’-O-dimethylaminoethyl (2’-O-DMAOE), 2 ’-0 -dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), or 2’-0 — N-methylacetamido (2’-0 — NMA) nucleotide. In some embodiments, an oligonucleotide comprises at least one nucleotide having a modification. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nucleotides within an oligonucleotide comprise modification. In some embodiments, all of the nucleotides within an oligonucleotide comprise a modification. In some embodiments, an oligonucleotide comprises at least one nucleotide having a 2’-O-methyl modification. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nucleotides within an oligonucleotide comprise a 2’-O-methyl modification (e.g., 2’ -MOE modification). In some embodiments, all of the nucleotides within an oligonucleotide comprise a 2’-O-methyl modification. In other preferred embodiments, RNA 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 RNA. In some embodiments, an oligonucleotide comprises modified nucleotides in which the sugar moiety comprises a bridging linker connecting two atoms in the ring (e.g., connecting the 2’-0 atom to the 4’-C atom of the sugar).

In some embodiments, an oligonucleotide of the disclosure comprises a modified backbone that does not include a phosphorus atom therein. Exemplary backbones include those 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. Further exemplary modifications include 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 CEE component parts.

Representative U.S. patents that describe preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the disclosure provides oligonucleotides comprising one or more nucleoside mimetics comprising a sugar and one or more intemucleoside linkage replaced with modified and/or non-natural chemical groups. In some embodiments, the nucleoside base is maintained for hybridization with a target sequence. For example, an exemplary RNA mimetic with superior hybridization properties is a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the oligonucleotides described herein are provided in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

In some embodiments, the disclosure provides oligonucleotides comprising one or more intemucleoside linkages comprising heteroatoms, amides, amines, or diamine. For example, the intemucleoside linkage comprising a native phosphodiester backbone (represented as — O — P — O — CH2 — ) is modified to a backbone selected from:

(i) — CH ₂ — NH— CH ₂ — ;

(ii) — CH ₂ — N(CHs) — O — CH ₂ — (i.e., a methylene (methylimino) or MMI backbone);

(iii) — CH ₂ — O— N(CH ₃ )— CH ₂ — ;

(iv) — CH ₂ — N(CH ₃ )— N(CH ₃ )— CH2— ; and

(v) — N(CH ₃ )— CH ₂ — CH ₂ — .

The foregoing linkages are described in U.S. Pat. No. 5,489,677 and U.S. Pat. No. 5,602,240. In some embodiments, the disclosure provides oligonucleotides comprising one or more intemucleoside linkages comprising a morpholino backbone, e.g., as described in U.S. Pat. No. 5,034,506.

In some embodiments, the disclosure provides oligonucleotides comprising one or more modified or substituted sugar moieties.

In some embodiments, the modified or substituted sugar moieties comprises a substitution at the 2'-position.

In some embodiments, the modified or substituted sugar moiety comprises a 2'-position substituent selected from: OH; F; O — , S — , or N-alkyl; O — , S — , or N-alkenyl; O — , S- orN- alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl comprises a substituted or unsubstituted Cl -CIO alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications 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 ) _n ONH ₂ , and O(CH ₂ ) _n ON[(CH ₂ )nc _H3 )] ₂ , where n and m are from 1 to about 10.

In some embodiments, the modified or substituted sugar moiety comprises a 2' position substituent selected from a Cl -CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, and a group for improving pharmacodynamic properties.

In some embodiments, the 2'-position substituent is an alkoxy-alkoxy group. In some embodiments, the alkoxy-alkoxy group is a 2'-methoxyethoxy (2'-0 — CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2 -methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504).

In some embodiments, the 2'-position substituent is a 2'-dimethylaminooxyethoxy group. In some embodiments, the 2 '-dimethylaminooxyethoxy group is a O(CH ₂ ) ₂ ON(CH ₃ ) ₂ group (also known as 2'-DMAOE). In some embodiments, the 2'-dimethylaminooxyethoxy group is a 2'- dimethylaminoethoxyethoxy, i.e., 2'-0 — CH2 — O — CH2 — N(CH2)2 (also known as 2'-O- dimethylaminoethoxyethyl or 2'-DMAE0E).

In some embodiments, the 2'-position substituent is selected from a 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F).

In some embodiments, an oligonucleotide comprises similar modifications at other positions in the pentofuranosyl sugar of a specific nucleoside, e.g., a substituent at the 3' position of the sugar at the 3' terminus.

In some embodiments, an oligonucleotide comprise sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents describing the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920 (incorporated herein by reference).

In some embodiments, the disclosure provides oligonucleotides comprising one or more modified or substituted nucleosides comprising a bicyclic sugar. As used herein, a “bicyclic sugar” or “bicyclic sugar moiety” refers to a fiiranosyl ring modified by bridging of two atoms. As used herein, a “bicyclic nucleoside” or “BNA” refers to a nucleoside comprising a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring.

In some embodiments, the disclosure provides oligonucleotides comprising one or more modified or substituted nucleosides that are a locked nucleic acid. As used herein a “locked nucleic acid” or “LNA” is a nucleoside comprising a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons, e.g., a 4'-CH2 — O-2' bridge. As is understood by the skilled artisan, the bridging structure effectively “locks” the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to oligonucleotides has been shown to increase their stability in serum and to reduce off-target effects (see, e.g., Elmen, J. et al., (2005) Nucleic Acids Research 33(l):439-447; Mook, O R. et al., (2007) Mol Cane Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

In some embodiments, the disclosure provides oligonucleotides comprising one or more bicyclic nucleosides comprising a 4' to 2' bridge. Exemplary bicyclic nucleosides suitable for use in oligonucleotides of the present disclosure include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'- (CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as “constrained ethyl” or “cEt”) and 4'-CH(CH2OCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)- O-2' (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4'-CH2-O — N(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004/0171570); 4'-CH2-N(R) — O-2', wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426, which is hereby incorporated by reference).

Additional representative U.S. Patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the bicyclic nucleoside comprises one or more stereochemical sugar configurations selected from: a-L-ribofuranose and P-D-ribofuranose (see, e.g., WO 99/14226).

In some embodiments, the bicyclic nucleoside is a constrained ethyl nucleotide. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4'-CH(CH3) — O-2' bridge. In some embodiments, a constrained ethyl nucleotide is in an S conformation and is referred to as an “S -constrained ethyl nucleotide” or “S-cEt.”

In some embodiments, the disclosure provides oligonucleotides comprising one or more modified nucleosides comprising a sugar mimetic. For example, in some embodiments, the modified nucleoside is a “modified tetrahydropyran nucleoside” a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleotides (a sugar surrogate). As another example, the modified nucleoside is a “modified THP nucleosides” selected from a hexitol nucleic acid (HNA), anitol nucleic acid (ANA), a manitol nucleic acid (MNA), and a fluoro HNA (F-HNA) (see, e.g., Leumann, Bioorg. Med. Chem., 2002, 10, 841-854).

In some embodiments, the disclosure provides oligonucleotides comprising one or more modified nucleosides comprising a sugar surrogate, wherein the sugar surrogate comprises one or more rings having more than 5 atoms and more than one heteroatom. In some embodiments, the sugar surrogate comprises a morpholino sugar moieties. In some embodiments, the morpholino sugar moiety is modified by addition of one or more substituent groups. Such sugar surrogates are referred to herein as “modified morpholines. ” Use of morpholino sugar moieties in oligomeric compounds is known in the art, see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

Combinations of modifications are also provided without limitation, such as 2'-F-5 '-methyl substituted nucleosides (see US Patent Application US2010/0190837 for other disclosed 5', 2'-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923) or alternatively 5 '-substitution of a bicyclic nucleic acid (see US Patent 7,547,684 wherein a 4'-CH2-O- 2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In some embodiments, the disclosure provides oligonucleotides comprising one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see, e.g., US8604192; Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111- 2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499- 4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; WO 2006/047842; and WO 2001/049687; the text of each is incorporated by reference herein in their entirety).

In some embodiments, the disclosure provides oligonucleotides comprising one or more of the nucleosides that is a hydroxymethyl substituted nucleoside. A “hydroxymethyl substituted nucleoside” is an acyclic 2'-3 '-seco-nucleotide, also referred to as an “unlocked nucleic acid” (“UNA”) modification. Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the disclosure provides oligonucleotides comprising one or more nucleobase modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxy-thymine (dT), 5 -methylcytosine (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 uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-daazaadenine and 3 -deazaguanine and 3 -deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in “Modified Nucleosides in Biochemistry,” Biotechnology and Medicine, Herdewijn, P. ed. Wiley- VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, pages 289-302, Crooke, S. T. and Lebien, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the agents featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

Representative U.S. patents that describe the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the disclosure provides oligonucleotides comprising one or more stabilizing modifications at its 5' and/or 3' terminus. In some embodiments, the one or more stabilizing modifications is selected from N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6- NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2- docosanoyl-uridine-3 "-phosphate, and inverted base dT(idT). Disclosure of this modification can be found in US Patent Publication No. 2012/0142101.

In some embodiments, the disclosure provides oligonucleotides comprising a region of complementarity comprising contiguous nucleosides, wherein at least one of the nucleosides is a modified nucleoside described herein. In some embodiments, the modified nucleoside comprises a modified sugar, a modified nucleobases, a modified 5'intemucleoside linkage, a modified 3'intemucleoside linkage, or a combination thereof. In some embodiments, an oligonucleotide comprises a plurality of modified nucleosides that constitute a pattern or motif. In some embodiment, an oligonucleotide comprises a plurality of nucleosides comprising modifications of sugar moieties, intemucleoside linkages, and/or nucleobases that are each independent of one another.

In some embodiments, an oligonucleotide comprising modified nucleosides arranged in a pattern or motifs as one or more desirable functional properties, e.g., enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by intracellular or extracellular nucleases. For example, in some embodiments, such oligonucleotides comprise at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for a target nucleic acid, and/or increased inhibitory activity. In some embodiments, a second region of optionally functions as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

In some embodiments, the disclosure provides an oligonucleotide having modified nucleosides arranged in pattern or motif, wherein the oligonucleotide is a gapmer. In some embodiments, the gapmer comprises an internal contiguous nucleotide sequence (the gap segment) positioned between a 5 '-flanking contiguous nucleotide sequence (the 5 '-wing segment) and a 3'- flanking contiguous nucleotide sequence (3 '-wing segment), wherein a plurality of nucleosides in the 5 '-wing segment and the 3 '-wing segment are chemically distinct from the nucleosides present in the gap segment. In some embodiments, the gapmer comprises a gap segment comprising a plurality of nucleotides or linked nucleosides that supports RNaseH cleavage positioned between a 5 '-wing segment and a 3 '-wing segment, each comprising a plurality of nucleotides or linked nucleosides that are chemically distinct from the nucleotides or linked nucleosides of the internal region. As is understood by the skilled artisan, the gap segment generally serves as a substrate for endonuclease cleavage when hybridized to a target sequence (e.g., cleavage mediated by an RNase H endonuclease), whereas the wing segments comprise modified nucleotides.

In some embodiments, the gapmer comprises a 5 '-wing segment, a gap segment, and a 3'- wing segment. In some embodiments, the 5 'end of the gap segment is positioned immediately adjacent to the 3 'end of the 5 '-wing segment and the 3 'end of the gap segment is positioned immediately adjacent to the 5'end of the 3'-wing segment (i.e., no intervening nucleotides exist between the 5' wing segment and the gap segment or the gap segment and the 3' wing segment). In some embodiments, the 5 '-wing segment and the 3 '-wing segment are the same. In some embodiments, the 5 '-wing segment and the 3 '-wing segment are different. In some embodiments, the 5 '-wing segment is 1-10 nucleosides in length and the 3 '-wing segment is 1-10 nucleosides in length, wherein the 5 '-wing segment and the 3 '-wing segment are the same. In some embodiments, the 5'- wing segment is 1-10 nucleosides in length and the 3'-wing segment is 1-10 nucleosides in length, wherein the 5 '-wing segment and the 3 '-wing segment are different.

In some embodiments, the length of the 5 '-wing segment of the gapmer is 1 to 6 nucleotides in length, e.g., 2 to 6, 2 to 5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to 4 nucleotides in length, e.g., 1, 2,

3, 4, 5, or 6 nucleotides in length.

In some embodiments, the length of the 3 '-wing segment of the gapmer is 1 to 6 nucleotides in length, e.g., 2 to 6, 2-5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to 4 nucleotides in length, e.g., 1, 2, 3,

4, 5, or 6 nucleotides in length.

In some embodiments, the length of the gap segment of the gapmer is 5 to 14 nucleotides in length, e.g., 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 7 to 8, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 14, 10 to 13, 10 to 12, 10 to 11, 11 to 14, 11 to 13, 11 to 12, 12 to 14, 12 to 13, or 13 to 14 nucleotides in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length.

In some embodiments, the 5'-wing segment consists of 2, 3, 4, 5 or 6 nucleotides, the gap segment consists of 7, 8, 9, 10, 11, or 12 nucleotides, and the 3'-wing segment consists of 2, 3, 4, 5 or 6 nucleotides. In some embodiments, the gapmer comprises a 5 '-wing segment, a gap segment, and a 3'-wing segment having respective lengths selected from: 2-7-2, 2-7-3, 2-7-4, 2-7-5, 2-7-6, 3-7-2, 3-7- 3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6-7-6, 3-7-3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6- 7-6, 2-8-2, 2-8-3, 2-8-4, 2-8-5, 2-8-6, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-8-6, 4-8-3, 4-8-4, 4-8-5, 4-8-6, 5-8- 3, 5-8-4, 5-8-5, 5-8-6, 6-8-3, 6-8-4, 6-8-5, 6-8-6, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-9-6, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-6, 4-9-3, 4-9-4, 4-9-5, 4-9-6, 5-9-3, 5-9-4, 5-9-5, 5-9-6, 6-9-3, 6-9-4, 6-9-5, 6-9-6, 2-10-2,

2-10-3, 2-10-4, 2-10-5, 2-10-6, 3-10-2, 3-10-3, 3-10-4, 3-10-5, 3-10-6, 4-10-3, 4-10-4, 4-10-5, 4-10-6,

5-10-3, 5-10-4, 5-10-5, 5-10-6, 6-10-3, 6-10-4, 6-10-5, 6-10-6, 2-11-2, 2-11-3, 2-11-4, 2-11-5, 2-11-6,

3-11-2, 3-11-3, 3-11-4, 3-11-5, 3-11-6, 4-11-3, 4-11-4, 4-11-5, 4-11-6, 5-11-3, 5-11-4, 5-11-5, 5-11-6,

6-11-3, 6-11-4, 6-11-5, 6-11-6, 2-12-2, 2-12-3, 2-12-4, 2-12-5, 2-12-6, 3-12-2, 3-12-3, 3-12-4, 3-12-5,

3-12-6, 4-12-3, 4-12-4, 4-12-5, 4-12-6, 5-12-3, 5-12-4, 5-12-5, 5-12-6, 6-12-3, 6-12-4, 6-12-5, and 6- 12-6.

In some embodiments, the regions of a gapmer are differentiated by the types of modified nucleotides in the region. In some embodiment, 5 '-wing segment and 3 '-wing segment each independently comprise one or more modified nucleosides that differ from one or more modified nucleosides present in the gap segment. For example, in some embodiments, 1, 2, 3, 4, or 5 modified nucleosides at the 3 'end of the 5 '-wing segment and at the 5 'end of 3 '-wing segment differ from neighboring modified nucleosides of the gap segment, thus defining the boundary between the wing segments and the gap segment. In some embodiments, the gap segment comprises a plurality of nucleosides comprising the same modifications. In some embodiments, the gap segment comprises a plurality of nucleosides comprising different modifications.

In some embodiments, the regions of a gapmer are differentiated by a plurality of nucleosides comprising a modified sugar described herein. In some embodiments, the nucleosides of each distinct region comprise uniform sugar moieties. In some embodiments, the nucleosides of each distinct region comprise different sugar moieties. In some embodiments, the sugar nucleoside modification motifs of the two wings are the same as one another. In some embodiments, the sugar nucleoside modification motifs of the 5 '-wing differs from the sugar nucleoside modification motif of the 3 '- wing.

In some embodiments, the types of modified nucleotides that differentiate the regions of a gapmer are selected from p-D-ribonucleosides, P-D-deoxyribonucleosides, 2'-modified nucleosides (e.g., 2'-M0E, 2'F, or 2'-O-CH3), and bicyclic sugar modified nucleosides (e.g., those having a 4'- (CH ₂ )n-O-2' bridge, where n=l or n=2).

In some embodiments, the 5 '-wing segment and/or the 3 '-wing segment of the gapmer independently comprise 1-6 modified nucleosides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleosides. In some embodiments, the 5 '-wing and/or the 3 '-wing segment of the gapmer comprises at least one modified nucleoside. In some embodiments, the 5 '-wing and/or the 3 '-wing segment of the gapmer comprises at least two modified nucleosides. In some embodiments, the 5 '-wing and/or the 3 '-wing segment of the gapmer comprises at least three modified nucleosides. In some embodiments, the 5'- wing and/or the 3'-wing segment of the gapmer comprises at least four modified nucleosides. In some embodiments, the 5 '-wing and/or the 3 '-wing segment of the gapmer comprises at least five modified nucleosides. In certain embodiments, each nucleoside of the 5'-wing and/or the 3'-wing segment of a gapmer is a modified nucleoside.

In some embodiments, at least one modified nucleoside of the 5 '-wing segment of the gapmer is a bicyclic nucleoside, e.g., a constrained ethyl nucleoside or an LNA. In some embodiments, the 5'- wing segment of the gapmer comprises 2, 3, 4, or 5 bicyclic nucleosides. In some embodiments, each nucleotide of the 5 '-wing of a gapmer is a bicyclic nucleoside. In some embodiments, the 5 '-wing segment of the gapmer comprises at least 1, 2, 3, 4, or 5 constrained ethyl nucleosides. In some embodiments, each nucleoside of the 5 '-wing segment is a constrained ethyl nucleoside. In some embodiments, the 5 '-wing segment of the gapmer comprises at least one LNA. In some embodiments, the 5 '-wing segment of the gapmer comprises 2, 3, 4, or 5 LNAs. In some embodiments, each nucleoside of the 5 '-wing segment is an LNA nucleotide.

In some embodiments, at least one modified nucleoside of the 5 '-wing segment of the gapmer is a non-bicyclic modified nucleotide, e.g., a 2'-substituted nucleotide. In some embodiments, the 2'- substituted nucleoside comprises a 2'-position other than H or OH. In some embodiments, the 2'- substituted nucleoside is a 2'-0Me nucleoside or a 2'-M0E nucleoside. In some embodiments, the 5'- wing segment of the gapmer comprises 2, 3, 4, or 5 2'-substituted nucleosides. In some embodiments, each nucleoside of the 5'-wing segment of the gapmer is a 2'-substituted nucleotide. In some embodiments, the 5 '-wing segment of the gapmer comprises at least one 2'-0Me nucleoside. In some embodiments, the 5 '-wing segment of the gapmer comprises at least 2, 3, 4, or 5 2'-0Me nucleosides. In some embodiments, each of the nucleosides of the 5 '-wing segment of the gapmer comprises a 2'- OMe nucleoside. In some embodiments, the 5 '-wing segment of the gapmer comprises at least one 2'- MOE nucleoside. In some embodiments, the 5 '-wing segment of the gapmer comprises at least 2, 3, 4, or 5 2'-M0E nucleosides. In some embodiments, each of the nucleosides of the 5'-wing segment of the gapmer comprises a 2'-M0E nucleoside.

In some embodiments, the 5 '-wing segment of the gapmer comprises at least one 2'- deoxynucleotide. In some embodiments, each nucleoside of the 5'-wing segment of the gapmer is a 2'- deoxynucleotide. In some embodiments, the 5'-wing segment of the gapmer comprises at least one ribonucleotide. In some embodiments, each nucleoside of the 5 '-wing segment of the gapmer is a ribonucleotide.

In some embodiments, at least one modified nucleoside of the 3 '-wing segment of the gapmer is a bicyclic nucleoside, e.g., a constrained ethyl nucleoside or an LNA. In some embodiments, the 3'- wing segment of the gapmer comprises 2, 3, 4, or 5 bicyclic nucleosides. In some embodiments, each nucleotide of the 5 '-wing of a gapmer is a bicyclic nucleoside. In some embodiments, the 3 '-wing segment of the gapmer comprises at least 1, 2, 3, 4, or 5 constrained ethyl nucleosides. In some embodiments, each nucleoside of the 3 '-wing segment is a constrained ethyl nucleoside. In some embodiments, the 3 '-wing segment of the gapmer comprises at least one LNA. In some embodiments, the 3 '-wing segment of the gapmer comprises 2, 3, 4, or 5 LNAs. In some embodiments, each nucleoside of the 3 '-wing segment is an LNA nucleotide.

In some embodiments, at least one modified nucleoside of the 3 '-wing segment of the gapmer is a non-bicyclic modified nucleotide, e.g., a 2'-substituted nucleotide. In some embodiments, the 2'- substituted nucleoside comprises a 2'-position other than H or OH. In some embodiments, the 2'- substituted nucleoside is a 2'-0Me nucleoside or a 2'-M0E nucleoside. In some embodiments, the 3'- wing segment of the gapmer comprises 2, 3, 4, or 5 2'-substituted nucleosides. In some embodiments, each nucleoside of the 3 '-wing segment of the gapmer is a 2'-substituted nucleotide. In some embodiments, the 3 '-wing segment of the gapmer comprises at least one 2'-0Me nucleoside. In some embodiments, the 3 '-wing segment of the gapmer comprises at least 2, 3, 4, or 5 2'-0Me nucleosides. In some embodiments, each of the nucleosides of the 3 '-wing segment of the gapmer comprises a 2'- OMe nucleoside. In some embodiments, the 3 '-wing segment of the gapmer comprises at least one 2'- MOE nucleoside. In some embodiments, the 3 '-wing segment of the gapmer comprises at least 2, 3, 4, or 5 2'-M0E nucleosides. In some embodiments, each of the nucleosides of the 3'-wing segment of the gapmer comprises a 2'-M0E nucleoside.

In some embodiments, the 3 '-wing segment of the gapmer comprises at least one 2'- deoxynucleotide. In some embodiments, each nucleoside of the 3 '-wing segment of the gapmer is a 2'- deoxynucleotide. In some embodiments, the 3 '-wing segment of the gapmer comprises at least one ribonucleotide. In some embodiments, each nucleoside of the 3 '-wing segment of the gapmer is a ribonucleotide.

In some embodiments, the gap segment of the gapmer comprises 5-14 modified nucleosides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 modified nucleosides. In some embodiments, the gap segment of the gapmer comprises at least one 5 -methylcytosine. In some embodiments, the gap segment of the gapmer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 5 -methylcytosines. In some embodiments, all of the nucleosides of the gap segment of the gapmer are 5-methylcytosines.

In some embodiments, the gap segment of the gapmer comprises at least one 2'- deoxynucleoside. In some embodiments, the gap segment of the gapmer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 2 '-deoxynucleosides. In some embodiments, all of the nucleosides of the gap segment of the gapmer are 2'-deoxynucleosides.

In some embodiments, the gapmer comprises one or more modified intemucleoside linkages. In some embodiments, the gapmer comprises one or more phosphodiester intemucleoside linkages. In some embodiments, the gapmer comprises one or more phosphorothioate intemucleoside linkages.

In some embodiments, each nucleoside of a 5 '-wing segment of the gapmer are linked via a phosphorothioate intemucleoside linkage. In some embodiments, each nucleoside of a 3 '-wing segment of the gapmer are linked via a phosphorothioate intemucleoside linkage. In some embodiments, each nucleoside of the gap segment of the gapmer are linked via a phosphorothioate intemucleoside linkage. In some embodiments, all of the nucleosides in the gapmer are linked via phosphorothioate intemucleoside linkages.

In some embodiments, an oligonucleotide targeting a target sequence described herein comprises a gap segment of 5-15 2'-deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of 1-6 nucleosides and a 3 '-wing segment of 1-6 nucleosides.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5'-wing segment of six nucleosides and a 3 '-wing segment of six nucleosides.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of five nucleosides and a 3'-wing segment of five nucleosides.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of four nucleosides and a 3 '-wing segment of four nucleosides.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of three nucleosides and a 3 '-wing segment of three nucleosides.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of two nucleosides and a 3 '-wing segment of two nucleosides.

In some embodiments, each nucleoside of the 5 '-wing segment comprises a modified nucleoside. In some embodiments, each nucleoside of the 3 '-wing segment comprises a modified nucleoside.

In some embodiments, each of the modified nucleosides in the 5 '-wing segment and each of the modified nucleosides in the 3 '-wing segment comprise a 2'-sugar modification. In some embodiments, the 2 '-sugar modification is a 2'-OMe modification. In some embodiments, the 2'-sugar modification is a 2'-M0E modification. In some embodiments, each of the modified nucleosides in the 5 '-wing segment and each of the modified nucleosides in the 3 '-wing segment comprise a bicyclic nucleoside. In some embodiments, the bicyclic nucleoside is a constrained ethyl nucleoside. In some embodiments, the bicyclic nucleoside is an LNA.

In some embodiments, each cytosine in an oligonucleotide is a 5-methylcytosine.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of 1-6 nucleosides, each comprising a 2'0Me modification, and a 3 '-wing segment of 1-6 nucleosides, each comprising a 2'0Me modification, wherein each intemucleoside linkage is a phosphorothioate linkage. In some embodiments, each cytosine is a 5-methylcytosine. In some embodiments, an oligonucleotide further comprises a ligand.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of 1-6 nucleosides, each comprising a 2'MOE modification, and a 3 '-wing segment of 1-6 nucleosides, each comprising a 2'MOE modification, wherein each intemucleoside linkage of an oligonucleotide is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine. In some embodiments, an oligonucleotide further comprises a ligand.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of 1-6 constrained ethyl nucleosides and a 3 '-wing segment of 1-6 constrained ethyl nucleosides, wherein each intemucleoside linkage of an oligonucleotide is a phosphorothioate linkage. In some embodiments, each cytosine of an oligonucleotide is a 5-methylcytosine.

In some embodiments, an oligonucleotide comprises a gap segment of 5-15 2'- deoxyribonucleosides positioned immediately adjacent to and between a 5 '-wing segment of 1-6 LNA nucleosides and a 3 '-wing segment of 1-6 LNA nucleosides, wherein each intemucleoside linkage of an oligonucleotide is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

Further gapmer designs suitable for use in the oligonucleotides, compositions, and methods of the invention are disclosed in, for example, U.S. Pat. Nos. 7,687,617 and 8,580,756; U.S. Patent Publication Nos. 20060128646, 20090209748, 20140128586, 20140128591, 20100210712, and 20080015162A1; and International Publication No. WO 2013/159108, the entire content of each of which are incorporated herein by reference.

In some embodiments, an oligonucleotide comprises an antisense oligonucleotide having 17 to 25 nucleotides in length comprising at least 12 contiguous nucleobases complementary to a portion of a target EFEMP1 RNA sequence, wherein the antisense oligonucleotide compound comprises a 3' domain and a 5' domain, which is contiguous with the 3' domain, wherein the 3' domain is 10 to 12 nucleotides in length and each nucleotide comprises a deoxyribonucleotide and a phospodiester or phosphothioate intemucleotide linkage or combinations thereof; and wherein the 5' domain is 5 to 15 nucleotides in length, and wherein the 5' domain comprises unmodified deoxyribonucleotides, unmodified ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or combinations thereof, provided that the 5' domain comprises at least 1 modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and/or backbone. Each of the nucleotides of the 3 ' domain comprise a deoxyribonucleotide and a phospodiester or phosphothioate intemucleotide linkage or combinations thereof. The nucleotides of the 3'- domain comprise natural deoxyribose sugar and phosphorothioate, phosphodiester or other phosphorus-based linkages or combinations thereof, which are known to activate RNase H.

The 5'- domain hybridizes to a target RNA but does not allow RNase H to excise the target RNA in this domain. The “5' domain” is generally 2 to 15 nucleotides in length and refers to the 11th through the 25th nucleotides, 12th through the 25th nucleotides, or 13th through the 25th nucleotides of the antisense oligonucleotide as measured from the 3' end depending on the length of the 3' domain. In some embodiments, the “5' domain” is generally 5 to 15 nucleotides in length. The 5' domain comprises nucleotides having non-RNase H activating modifications such as modified sugars and/or modified backbones, which do not activate RNase H. In some embodiments, the 5 ' domain comprises nucleotides comprising a modified sugar. In some embodiments, the 5' domain comprises nucleotides comprising a modified backbone. In some embodiments, the 5' domain comprises nucleotides comprising both a modified sugar and modified backbone. In embodiments, the modified backbone is a nonphosphorus- based backbone.

In some embodiments, the 3' domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3' end. In some embodiments, the 3' domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3' end. In some embodiments, the 3' domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end. In some embodiments, when the 3' domain of the antisense oligonucleotide is 12 nucleotides in length, the antisense oligonucleotides of the disclosure are represented by Formula (I): 5'- N _m Ni4Ni3Ni2NnNioN9N8N7N6N5N4N3N2Ni-3'; wherein N is any nucleotide; NB through N _m comprises the 5' domain; Ni through N12 comprises the 3' domain; and m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

Without wishing to be bound to any particular theory, the 9th position from the 3' end of an antisense oligonucleotide may be important in some embodiments. RNase H makes a major cut of an antisense oligonucleotide-target RNA complex at around the 9th position. RNase H can make a second important cut at around the 17th position. Modification or mismatch of the nucleotide of an antisense oligonucleotide at the 9th position can interfere with RNase H activity. Modification or mismatch of the 8th and/or 10th positions from the 3' end of an antisense oligonucleotide can also interfere with RNase H but to a lesser extent than the 9th position. Additionally, an antisense oligonucleotide 17 nucleotides in length may avoid a non-specific second cut by RNase H. In some embodiments, the design of an inhibitory nucleic acid (e.g., pattern of modifications) is as described in International Patent Publication No. W02020/191177, entitled “Antisense oligonucleotides for allele specificity,” filed March 21, 2019, which is incorporated by reference herein.

In some embodiments, an oligonucleotide comprises an antisense oligonucleotide comprising 14 to 30 linked nucleotides having at least 12 contiguous nucleobases complementary to a portion of a target pre- mRNA comprising a retained intron, wherein the antisense oligonucleotide comprises 1 to 3 regions each region independently comprising from 2 to 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2 ’-substituted, non-ionic or constrained sugar nucleotides, or combinations thereof. In this design, an antisense oligonucleotide has two domains. The first domain is comprised of ribonucleotides (RNA), modified RNA or combinations thereof, which provide affinity to target RNA. The second domain is comprised of phosphodiester or phosphorothioate oligodeoxynucleotide (DNA) which allows recruitment of RNase H but does not allow RNase H to cleave the antisense oligonucleotide-target RNA duplex. The recruitment of RNase H and its binding to the oligonucleotide-target RNA duplex, provides steric hinderance at the duplex site and promotes splicing.

In some embodiments, the design of an inhibitory nucleic acid (e.g., pattern of modifications) is as described in International Patent Publication No. W02021/055011, entitled “Compounds and methods useful for modulating gene splicing,” filed March 19, 2020, which is incorporated by reference herein.

Conjugates comprising Inhibitory Nucleic Acids targeting EFEMP1

In some embodiments, the disclosure provides inhibitory nucleic acids (also referred to as oligonucleotides) operably linked to a cell-targeting moiety (e.g., a non-nucleotide moiety). An inhibitory nucleic acid may be associated directly (e.g., chemical bond between the oligonucleotide and the cell-targeting moiety) or via a linker to a cell-targeting moiety. e.g. Conjugation of an oligonucleotide to one or more cell-targeting moieties is used to improve the pharmacology of the oligonucleotide, e.g., by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments, the conjugate moiety modifies or enhances the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In some embodiments, the conjugate targets an oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue or cell type. At the same time the conjugate may serve to reduce activity of an oligonucleotide in non-target cell types, tissues or organs, e.g. , off target activity or activity in non-target cell types, tissues or organs.

In some embodiments, the non-nucleotide moiety (conjugate moiety) is a carbohydrate, cell surface receptor ligand, drug substance, hormone, lipophilic substance, polymer, protein, peptide, toxin (e.g., bacterial toxin), vitamin, viral protein (e.g. capsid) or a combination thereof. In some embodiments, the cell-targeting agent is a protein, a carbohydrate, a cell surface receptor ligand, a hormone, a cytokine, or a lipophilic polymer. In some embodiments, the non-nucleotide moiety is an antibody or antibody fragment, such as antibody or antibody fragment that facilitates delivery of the oligonucleotide across the blood-retina barrier.

In some embodiments, the cell-targeting moiety is a peptide. In some embodiments, the celltargeting moiety is an antibody that targets or binds to a cell protein (e.g., a cell surface protein).

A linker between a cell -targeting moiety and an oligonucleotide may be any reasonable size. In some embodiments, a linker between the cell-targeting moiety and the oligonucleotide keeps the cell-targeting moiety away from contact with the oligonucleotide (e.g., to reduce possible interference with the function of an oligonucleotide to inhibit EFEMP1 expression). A linker may be a cleavable or non-cleavable linker. In some embodiments, a cleavable linker is a protease -sensitive linker (e.g., comprising an amino acid sequence that can be targeted for degradation by a protease). In some embodiments, a cleavable linker is a pH-sensitive linker (e.g., can be degraded in the presence of a change in pH). In some embodiments, a cleavable linker is a glutathione -sensitive linker (e.g., an amino acid sequence comprising a disulfide bond that can be disrupted in the presence of glutathione). A non-cleavable linker may comprise an alkyl chain or a thioether. In some embodiments, the linker may comprise a functional group that forms a covalent bond between the oligonucleotide and the linker. Exemplary functional groups include, but are not limited to, a maleimide group, an iodoacetamide group, a vinyl sulfone group, an acrylate group, an acrylamide group, an acrylonitrile group, and a methacrylate group.

In some embodiments, an oligonucleotide comprises one or more ligand-conjugated nucleosides. In some embodiments, an oligonucleotide comprising one or more ligand-conjugated nucleosides has one or more improved functional properties (e.g., stability, tissue targeting, and/or pharmacodynamic/pharmacokinetic properties) as compared to the oligonucleotide without the one or more ligand-conjugated nucleosides.

Exemplary ligands include, but are not limited to, a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., dihexadecyl -rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3 -phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In some embodiments, the ligand comprises a carbohydrate (e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide). In some embodiments, the carbohydrate is a GalNAc.

In some embodiments, the carbohydrate moiety is conjugated to a modified subunit of the oligonucleotide. For example, in some embodiments, the ribose sugar of one or more ribonucleosides of an oligonucleotide described herein is replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. In some embodiments, a ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). In some embodiments, a cyclic carrier comprises a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. In some embodiments, the cyclic carrier comprises a monocyclic ring system or two or more rings, e.g. fused rings. In some embodiments, the cyclic carrier comprises a fully saturated ring system or comprises one or more double bonds.

In some embodiments, the ligand is conjugated to an oligonucleotide via a carrier. In some embodiments, the carrier comprises (i) at least one backbone attachment point; and (ii) at least one tethering attachment point. As used herein, a “backbone attachment point” refers to a functional group, (e.g. a hydroxyl group) suitable for incorporation of the carrier into the oligonucleotide backbone (e.g., the phosphate or modified phosphate (e.g., sulfur containing) backbone of an oligonucleotide). As used herein, a “tethering attachment point” refers to a constituent ring atom of the cyclic carrier (e.g., a carbon atom or a heteroatom) that is distinct from an atom which provides a backbone attachment point and that connects a selected moiety. In some embodiments, the moiety is a carbohydrate (e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide). Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

In some embodiments, an oligonucleotide is conjugated to the ligand via a carrier, wherein the carrier is selected from a cyclic group or acyclic group. In some embodiments, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin. In some embodiments, the acyclic group is selected from serinol backbone or diethanolamine backbone. Methods of Delivery

In some aspects, the disclosure provides compositions to mediate delivery of an oligonucleotide described herein to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having an ocular disease). In some embodiments, the delivery is performed by contacting the cell with the oligonucleotide in vitro or in vivo. In some embodiments, in vivo delivery is performed by administering a composition comprising the oligonucleotide to a subject.

The oligonucleotide functions, in some embodiments, by binding (e.g., selectively binding) to an intron of the pre-mRNA and inhibiting (e.g., selectively inhibiting) translation of a EFEMP1 pre- mRNA. In this context, selectivity of binding (and inhibition) means that, in some embodiments, an oligonucleotide binds to a target sequence of a EFEMP1 pre-mRNA preferentially relative to other sequences in the cell or subject (e.g., low levels of off-target effects). In some embodiments, an oligonucleotides bind to a target sequence of a EFEMP1 pre-mRNA with more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% binding affinity relative to any non-target sequences in the cell or subject.

A subject may be a human subject, non-human primate, a veterinary or agricultural animal (e.g. , a cow, horse, pig, goat, sheep, etc.), a rodent (e.g. , a mouse or rat), or a bird (e.g. , a duck or a goose). A subject may include a transgenic organism. In some embodiments, a subject may have or be at risk of having an ocular disease or disorder (e.g., glaucoma). In some embodiments, a subject may have one or more symptoms associated with an ocular disease or disorder (e.g., glaucoma). In some embodiments, a subject having one or more symptoms associated with an ocular disease or disorder has an ocular disease or disorder. In some embodiments, a subject having one or more symptoms associated with an ocular disease or disorder has not been diagnosed with an ocular disease or disorder.

As used herein, an ocular disease or disorder refers to any disease or disorder that impacts the eye (e.g. , performance of the eye, health of the eye or its subject, and/or longevity of the proper function of the eye). In some embodiments, non-limiting examples of ocular diseases or disorders include ophthalmic disease, retinal degeneration, Autosomal Dominant Drusen, Sorsby’s fundus dystrophy, Stargardt disease, bestrophinopathy, retinitis pigmentosa, macular degeneration, macular dystrophy, Doyne honeycomb retinal dystrophy, preventative and acute geographic atrophy, macular edema, intermediate age-related macular degeneration, primary open-angle glaucoma, normal tension glaucoma, and diabetic retinopathy.

Methods for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an oligonucleotide described herein (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5): 139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. In some embodiments, the non-specific effects of oligonucleotide are minimized by local administration, for example, by direct injection or implantation into a target tissue or topical administration of the composition. Local administration to a treatment site maximizes local concentration of the oligonucleotide, limits the exposure of the oligonucleotide to systemic tissues that can otherwise be harmed by the oligonucleotide or that can degrade the oligonucleotide, and permits a lower total dose of the oligonucleotide to be administered.

Several studies have shown successful knockdown of gene products when an antisense oligonucleotide is administered locally. For example, intraocular delivery of a VEGF antisense oligonucleotide by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24: 132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of an antisense oligonucleotide in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11 :267 -274) and can prolong survival of tumorbearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515- 523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3: 18; Shishkina, G T., et al (2004) Neuroscience 129:521- 528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101: 17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279: 10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55).

In some embodiments, methods of administration of an oligonucleotide to a cell involves contacting the cell with a suitable formulation of the oligonucleotide. For example, in some embodiments, an oligonucleotide is formulated for delivery to a cell in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle. In some embodiments, an oligonucleotide is contacted with a cell as a naked oligonucleotide (e.g., formulated in a buffered solution without any additional carriers or excipients). In some embodiments, an oligonucleotide is transfected into a cell (e.g., using a transfection reagent such as Lipofectamine). The cell may be a mammalian cell. In some embodiments, the cell is a human cell, optionally a cell from a human subject having or suspected of having a disease or disorder associated with EFEMP1 dysregulation.

In some embodiments, methods of administration of an oligonucleotide to a subject involve administration by intrathecal injection, intravenous injection, intramuscular injection, inhalation, subcutaneous injection, intracranial injection, topical administration (e.g., topical administration to the eye or eyelid), or intraocular injection. In such methods, an oligonucleotide is formulated in accordance with its mode of administration. In some embodiments, methods of administration of an antisense oligonucleotide to a subject involve direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion. An oligonucleotide can be administered as a bolus injection or a continuous infusion over a period of time.

For administering an oligonucleotide systemically for the treatment of a disease, an oligonucleotide can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the oligonucleotide by endo- and exo-nucleases in vivo. Modification of the agent or the pharmaceutical carrier can also permit targeting of the oligonucleotide composition to the target tissue and avoid undesirable off-target effects. In some embodiments, an oligonucleotide is modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.

In some embodiments, an oligonucleotide is delivered using a drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. In some embodiments, positively charged cationic delivery systems are used to facilitate binding of the oligonucleotide (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of the oligonucleotide by the cell. In some embodiments, cationic lipids, dendrimers, or polymers are bound to the oligonucleotide, or induced to form a vesicle or micelle (see e g, Kim S H., et al (2008) Journal of Controlled Release 129(2): 107-116) that encases the oligonucleotide. In some embodiments, the formation of vesicles or micelles prevents degradation of an oligonucleotide when administered systemically.

Methods for making and administering cationic-oligonucleotide complexes are known to the skilled artisan (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. Cancer Res. 9: 1291-1300; Arnold, A S et al (2007) J. Hypertens. 25: 197-205, which are incorporated herein by reference in their entirety).

Non-limiting examples of drug delivery systems useful for systemic delivery of an oligonucleotides described herein include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441: 111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26: 1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg- Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16: 1799-1804).

In some embodiments, systemic delivery of an oligonucleotide described herein comprises formation of a complex with cyclodextrin. Methods for administration and pharmaceutical compositions of oligonucleotides and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

In some embodiments, an oligonucleotide described herein is delivered using a viral vector. In some embodiments, an adeno-associated virus (AAV) vector comprising an oligonucleotide is used for genetic transformation of a cell in vitro or in vivo. As used herein the term "AAV" has its general meaning in the art and is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all serotypes and variants both naturally occurring and engineered forms. In some embodiments, the AAV is selected from an AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), and AAV type 8 (AAV-8) and AAV type 9 (AAV9). The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_001401 (AAV-2), AF043303 (AAV2), and NC_006152 (AAV-5). In some embodiments, the AAV comprises one or more sequences of an oligonucleotide inserted between a 5' and a 3' AAV inverted terminal repeats, which function to regulate expression of the oligonucleotide in a cell.

In some embodiments, the viral vector (e.g., AAV) comprises one or more regulatory sequences for expression and secretion of an oligonucleotide, such as e.g., a promoter, enhancer, polyadenylation signal, internal ribosome entry sites (IRES), sequences encoding protein transduction domains (PTD), and the like. In this regard, the vector comprises a promoter region, operably linked to one or more sequences of the oligonucleotide, to cause or improve expression of the oligonucleotide in transformed cells. Such a promoter may be ubiquitous, tissue-specific, strong, weak, regulated, chimeric, inducible, etc., to allow efficient and suitable production of the oligonucleotide in transformed tissue. The promoter may be a cellular, viral, fungal, plant or synthetic promoter. Examples of ubiquitous promoters include viral promoters, particularly the CMV promoter, CAG promoter (chicken beta actin promoter with CMV enhancer), the RSV promoter, the SV40 promoter, etc. and cellular promoters such as the PGK (phosphoglycerate kinase) promoter. In some embodiments, the promoter is specific or functional in cells of the retina, in particular in photoreceptor or ganglion cells of the retina or in the RPE, i.e., allows (preferential) expression of the transgene in said cells. For example, suitable photoreceptor-specific regulatory elements include, e.g., a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9: 1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an interphotoreceptor retino id- binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); an IRBP gene promoter (Y okoyama et al. (1992) Exp Eye Res. 55:225).

Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions and formulations comprising one or more oligonucleotides targeting EFEMP1 described herein. In some embodiments, the disclosure provides pharmaceutical compositions comprising one or more oligonucleotides targeting EFEMP 1 described herein, and a pharmaceutically acceptable carrier. In some embodiment, provided herein are pharmaceutical compositions suitable for ocular delivery comprising one or more oligonucleotides targeting EFEMP 1 described herein, as described herein, and a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically-acceptable carrier” as used herein refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm components, such as semm albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

In some embodiments, the disclosure provides pharmaceutical composition comprising one or more oligonucleotides targeting EFEMP 1 described herein for treating a disease or disorder associated with the expression or activity of the EFEMP 1 gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g. , by subcutaneous (SC) or intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the eye, e.g., via intravitreal injection

In some embodiments, the pharmaceutical compositions comprising one or more oligonucleotides targeting EFEMP 1 are useful for treating an ocular disease or disorder associated with the expression or activity of the EFEMP1 gene, e.g., expression of an EFEMP1 gene in the eye of a subject. The pharmaceutical compositions of the invention may be administered in dosages sufficient to modulate (e.g., inhibit or reduce) expression of EFEMP1 in an eye cell (e.g., an RPE cell).

Such compositions typically include one or more oligonucleotides described herein (e.g., antisense oligonucleotides or RNAi oligonucleotides) and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration to an ocular cell (e.g., RPE cell). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.

In some embodiments, the pharmaceutical composition suitable for ocular administration comprise one or more oligonucleotides described herein (e.g., antisense oligonucleotides or RNAi oligonucleotides) and a delivery vehicle described herein. In some embodiments, the delivery vehicle can deliver the one or more oligonucleotides to an ocular cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In some embodiments the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.

In some embodiments, the disclosure provides a pharmaceutical composition comprising one or more oligonucleotides described herein (e.g., antisense oligonucleotides or RNAi oligonucleotides) in an injectable dosage form. In some embodiments, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In some embodiments the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol. In some embodiments, the injectable dosage form is administered directly to the eye by an ocular tissue injection such as periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, anterior or posterior juxtascleral, subretinal, subconjunctival, retrobulbar, or intracanalicular injections; by direct application to the eye using a catheter or other placement device such as a retinal pellet, intraocular insert, suppository or an implant comprising a porous, non-porous, or gelatinous material; by topical ocular drops or ointments; or by a slow release device in the cul-de-sac or implanted adjacent to the sclera (transscleral) or in the sclera (intrascleral) or within the eye. Intracameral injection may be through the cornea into the anterior chamber to allow the agent to reach the trabecular meshwork. Intracanalicular injection may be into the venous collector channels draining Schlemm's canal or into Schlemm's canal.

In some embodiment, the injectable dosage form comprising a pharmaceutical composition described herein is administered into the eye, for example the vitreous chamber of the eye, by intravitreal injection, such as with pre-fdled syringes in ready-to-inject form for use by a medical personnel.

In some embodiments, the disclosure provides a pharmaceutical composition comprising one or more oligonucleotides described herein (e.g., antisense oligonucleotides or RNAi oligonucleotides) in a form that may be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.

In some embodiments, the oligonucleotides described herein (e.g., antisense oligonucleotides or RNAi oligonucleotides), are administered to an ocular cell (e.g., RPE cell) in a pharmaceutical composition by a topical route of administration.

In some embodiments, the pharmaceutical composition suitable for ocular delivery may include one or more oligonucleotides described herein (e.g., antisense oligonucleotides or RNAi oligonucleotides) mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In some embodiments, the liposomes are cationic liposomes.

In some embodiments, the oligonucleotides described herein (e.g., antisense oligonucleotides or RNAi oligonucleotides) are admixed with a topical penetration enhancer. In some embodiments, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1- dodecylazacycloheptan-2- one, an acylcamitine, an acylcholine, or a CMO alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. In some embodiments, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24, 25 -dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof. In some embodiments, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, aN-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof. In some embodiments, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof. In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof. In some embodiments, the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.

For ophthalmic delivery, the pharmaceutical composition comprising one or more oligonucleotides described herein (e.g., antisense oligonucleotides or RNAi oligonucleotides) may further comprise ophthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, or water to form an aqueous, sterile ophthalmic suspension or solution. Solution formulations may be prepared by dissolving the conjugate in a physiologically acceptable isotonic aqueous buffer. Further, the solution may include an acceptable surfactant to assist in dissolving the oligonucleotides. Viscosity building agents, such as hydroxymethyl cellulose, hydroxyethyl cellulose, methylcellulose, polyvinylpyrrolidone, or the like may be added to the pharmaceutical compositions to improve the retention of the oligonucleotides.

To prepare a sterile ophthalmic ointment formulation, the one or more oligonucleotides is combined with a preservative in an appropriate vehicle, such as mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations may be prepared by suspending the double-stranded iRNA agents in a hydrophilic base prepared from the combination of, for example, CARBOPOL®- 940 (BF Goodrich, Charlotte, N.C.), or the like, according to methods known in the art.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to modulate (e.g., inhibit or reduce) expression of EFEMP1. The pharmaceutical composition can be administered once daily, or the pharmaceutical composition can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or delivery through a controlled release formulation. In that case, each sub-dose of the one or more oligonucleotides must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the one or more oligonucleotides over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

In some embodiments, a single dose of the pharmaceutical compositions can be long lasting. In some embodiments, a single dose of the pharmaceutical compositions of the invention is administered bi-monthly. In some embodiments, a single dose of a pharmaceutical composition described herein is administered monthly. In some embodiments, a single dose of a pharmaceutical composition described herein is administered quarterly. In some embodiments, a single dose of a pharmaceutical composition described herein is administered bi- annually. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual oligonucleotides can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Methods for Modulating EFEMP1 Expression

The present disclosure provides methods for modulating expression of EFEMP1 in a cell, e.g., an ocular cell or an RPE cell in the eye, e.g., by inhibiting or reducing expression of EFEMP1. In some embodiments, the methods comprise contacting the cell with an EFEMP1 -targeting oligonucleotide (e.g., an EFEMP1 -targeting antisense oligonucleotide or an EFEMP1 -targeting RNAi oligonucleotide) described herein in an amount effective to modulate (e.g., inhibit or reduce) expression of EFEMP 1.

In some embodiments, contacting the cell (e.g., ocular cell or RPE cell) with an EFEMP 1- targeting oligonucleotide (e.g., an EFEMP 1 -targeting antisense oligonucleotide or an EFEMP 1- targeting RNAi oligonucleotide) described herein is performed in vitro or in vivo. In some embodiments, the contacting of an ocular cell in vivo with the EFEMP 1 -targeting oligonucleotide comprises contacting an ocular cell or group of ocular cells within a subject, e.g., a human subject, with the EFEMP 1 -targeting oligonucleotide. In some embodiments, contacting an ocular cell or a group of ocular cells may be accomplished via one or more targeting ligand conjugated to the EFEMP 1 -targeting oligonucleotide, including any ligand described herein or known in the art. In one embodiment, the targeting ligand is a ligand that directs the EFEMP 1 -targeting oligonucleotide to a site of interest, e.g., the ocular cells of a subject.

As used herein, the term “contacting” refers to introducing the EFEMP 1 -targeting oligonucleotide described herein to a cell by any possible means. In some embodiments, the contacting comprises introducing the EFEMP 1 -targeting oligonucleotide to a cell in vitro or in vivo. Contacting a cell in vitro may be done, for example, by incubating the cell with the EFEMP 1- targeting oligonucleotide. In vitro introduction into a cell also includes methods known in the art such as electroporation and lipofection Contacting a cell in vivo may be done, for example, by injecting the EFEMP 1 -targeting oligonucleotide into or near the tissue where the cell is located, or by injecting the EFEMP 1 -targeting oligonucleotide into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the EFEMP 1 -targeting oligonucleotide may contain and/or be coupled to a ligand that directs the EFEMP 1 -targeting oligonucleotide to a site of interest, e.g., the eye. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an EFEMP1 -targeting oligonucleotide and subsequently transplanted into a subject.

In some embodiments, the contacting comprises facilitating or effecting uptake or absorption of the EFEMP1 -targeting oligonucleotide into the cell. Absorption or uptake of an EFEMP1 -targeting oligonucleotide can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. In some embodiments, the contacting comprises introducing the EFEMP1 -targeting oligonucleotide to a cell directly or indirectly. Thus, for example, the EFEMP1 -targeting oligonucleotide may be put into physical contact with the cell by the individual performing the method, or alternatively, the EFEMP1 -targeting oligonucleotide may be put into a situation that will permit or cause it to subsequently come into contact with the cell (e.g. , following in vivo administration).

As used herein, the term "inhibiting," as used herein, is used interchangeably with "reducing," "silencing," "downregulating", "suppressing", and other similar terms, and includes any level of inhibition. Preferably inhibiting includes a statistically significant or clinically significant inhibition. "Inhibiting expression of the EFEMP1" includes any level of inhibition of EFEMP1, e.g., at least partial suppression of the expression of EFEMP1. The expression of the EFEMP1 may be assessed based on the level, or the change in the level, of any variable associated with EFEMP1 gene expression, e.g., EFEMP1 mRNA level, EFEMP1 protein level, the extent of ECM deposits in the retina. This level may be assessed in an individual ocular cell or in a group of ocular cells, including, for example, a sample derived from a subject.

In some embodiments, a reduction or inhibition of EFEMP 1 in a cell is assessed using analytical methods known in the art (e.g., RT-PCR, Northern blot, expression array, etc.), optionally via comparison of EFEMP1 RNA levels in the presence of the EFEMP 1 -targeting oligonucleotide relative to a control. It is also recognized that levels of EFEMP 1 protein can be assessed and that EFEMP 1 protein levels are, under different conditions, either directly or indirectly related to EFEMP 1 RNA levels and/or the extent to which an EFEMP 1 -targeting oligonucleotide inhibits EFEMP 1 expression, thus art-recognized methods of assessing EFEMP 1 protein levels (e.g., Western blot, immunoprecipitation, other antibody-based methods, etc.) can also be employed to examine the inhibitory effect of an EFEMP 1 -targeting oligonucleotide described herein.

In some embodiments, inhibition is assessed by a decrease in an absolute or relative level of one or more variables that are associated with EFEMP 1 expression (e.g., ocular EFEMP 1 expression) 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, ocular cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control). In some embodiments, EFEMP 1 RNA and/or protein levels in the presence of the EFEMP 1 -targeting oligonucleotide are compared to those observed in the presence of vehicle alone, in the presence of an antisense oligonucleotide or RNAi oligonucleotide directed against an unrelated target RNA, or in the absence of any treatment.

In some embodiments, the methods reduce or inhibit expression of a EFEMP1 in a cell (e.g., an ocular cell or an RPE cell) by 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%, at least about 99%%, or to below the level of detection of the assay. In some embodiments, the inhibition of expression of EFEMP1 results in normalization of the level of EFEMP1 such that the difference between the level before treatment and a normal control level is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,

80%, 85%, 90%, or 95%. In some embodiments, the inhibition is a clinically relevant inhibition.

Methods for Treating or Preventing an EFEMPl-Associated Disease

The present disclosure provides methods for treating, preventing, or ameliorating a disease or disorder associated with one or more mutations in, an abnormal expression of, and/or an abnormal activity of the EFEMP1 gene, or a transcriptional or translational product thereof. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount or prophylactically effective amount of an EFEMP1 -targeting oligonucleotide (e.g., an EFEMP1- targeting antisense oligonucleotide or an EFEMP1 -targeting RNAi oligonucleotide) described herein. In some embodiments, the method comprises inhibiting or reducing expression of EFEMP1 in a tissue or organ of the subject. In some embodiments, the inhibited or reduced expression of EFEMP1 prevents or alleviates one or more symptoms of the disease or disorder. In some embodiments, the tissue or organ is the eye.

In some embodiments, the disease or disorder is associated with a gain of function in the EFEMP1 gene (e.g., an overamplification of the EFEMP1 gene). In some embodiments, the disease or disorder is associated with an abnormal expression (e.g., an overexpression) of the EFEMP1 gene, or a transcriptional or translational product thereof. In some embodiments, the disease or disorder is associated with an abnormal activity (e.g. , a dysfunction) of the EFEMP1 gene, or a transcriptional or translational product thereof. In some embodiments, the disease or disorder is an ocular disease or disorder described herein.

In some embodiments, the subject is a human, such as a human being treated or assessed for an disease, disorder or condition that would benefit from a reduction in EFEMP1 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in EFEMP1 expression; a human having a disease, disorder or condition that would benefit from reduction in EFEMP1 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in EFEMP1 expression.

As used herein, a “therapeutically effective amount” refers to an amount of an EFEMP1- targeting oligonucleotide (e.g., an EFEMP1 -targeting antisense oligonucleotide or an EFEMP1- targeting RNAi oligonucleotide) described herein, that, when administered to the subject having a disease or disorder associated with one or more mutations in an EFEEMP1 gene, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the EFEMP1 -targeting oligonucleotide or composition thereof, how an oligonucleotide or composition is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an EFEMP1 -targeting oligonucleotide (e.g., an EFEMP1 -targeting antisense oligonucleotide or an EFEMP1 -targeting RNAi oligonucleotide) described herein, that, when administered to a subject having a disease or disorder associated with one or more mutations in an EFEEMP1 gene, but not immediately experiencing or displaying symptoms of the disease or disorder, and/or a subject at risk of developing an EFEMP1 -associated disease, is sufficient to prevent or ameliorate the disease or disorder or one or more symptoms thereof. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on EFEMP1 -targeting oligonucleotide or composition thereof, how an oligonucleotide or composition is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an EFEMP1 -targeting oligonucleotide, “effective against” an EFEMP1 -associated disease, or a disease associated with one or more mutations in, an abnormal expression of, and/or an abnormal activity of the EFEMP1 gene, or a transcriptional or translational product thereof, indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the disease and the related causes.

In some embodiments, the efficacy of treatment or prevention of disease is assessed by measuring the level of one or more disease markers. In some embodiments, the efficacy of treatment or prevention of disease is assessed by measuring a marker of complement activation measured in a bodily fluid or tissue obtained from the subject (e.g., in serum obtained from the subject or a vitreous or aqueous fluid obtained from the subject). In some embodiments, the efficacy of treatment or prevention of disease is assessed by measuring the C3/C3d ratio in a bodily fluid or tissue obtained from the subject (e.g. , in serum obtained from the subject or a vitreous or aqueous fluid obtained from the subject). In some embodiments, the efficacy of treatment or prevention of disease is assessed by measuring drusen in one or both eyes of the subject. Methods to measure drusen in a routine dilated eye exam are known in the art.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given EFEMP1 -targeting oligonucleotide or formulation thereof can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

In some embodiments, the disclosure provides methods for treating, preventing, or ameliorating an ocular disease or disorder. In some embodiments, the ocular disease or disorder is associated with one or more mutations in an EFEEMP1 gene. In some embodiments, the one or more mutations in an EFEMP1 gene comprises a gain of function mutation. As used herein, a “gain of function mutation” refers to at least one alteration in a coding or non-coding region of a gene that results in an enhanced phenotype relative to the wild-type allele. In some embodiments, the gain of function mutation results in an increased expression of the protein encoded by a gene comprising the mutation relative to the wild-type allele. In some embodiments, the ocular disease or disorder is associated with a gain of function mutation in an EFEEMP1 gene, wherein the subject has an increased expression of EFEMP1 in the eye compared to a baseline expression of EFEMP1 (e.g., an EFEMP1 expression in the eye of a healthy subject). In some embodiments, the method comprises administering to the subject a therapeutically effective amount or prophylactically effective amount of an EFEMP1 -targeting oligonucleotide (e.g., an EFEMP1 -targeting antisense oligonucleotide or an EFEMP1 -targeting RNAi oligonucleotide) described herein, thereby inhibiting or reducing an expression of EFEMP1 in one or both eyes of the subject. In some embodiments, the ocular disease or disorder that is characterized by or comprises damage to the retina. In some embodiments, the ocular disease or disorder is characterized by damage to cells of the RPE. In some embodiments, the ocular disease or disorder is characterized by damage to photoreceptor cells. In some embodiments, the ocular disease or disorder is characterized by damage to the choriocapillaris. In some embodiments, the ocular disease or disorder is characterized by disruption of Bruch’s membrane. For example, the ocular disease or disorder is a macular dystrophy (also referred to as an inherited macular degeneration that includes, but is not limited to, Sorsby’s fundus dystrophy, Stargardt disease, Doyne honeycomb retinal dystrophy, and bestrophinopathy), an early-stage age-related macular degeneration (AMD), a late-stage AMD (e.g., neovascular (wet) AMD, preventative geographic atrophy (dry) AMD, or acute dry AMD), and diabetic retinopathy

In some embodiments, the disclosure provides a method for preventing, treating, or reversing an ocular disease or disorder associated with one or more mutations in an EFEMP1 gene, comprising administering to a subject a therapeutically effective amount or prophylactically effective amount of an EFEMP1 -targeting oligonucleotide described herein (e.g., an EFEMP1 -targeting antisense oligonucleotide or an EFEMP1 -targeting RNAi oligonucleotide). In some embodiments, the ocular disease or disorder is a macular degeneration. As used herein, the term “Macular Degeneration" or “MD” refers to a variety of diseases characterized by a progressive loss of central vision and comprise abnormalities of Bruch's membrane and/or the RPE in the retina. These disorders include very common conditions that affect older patients (age related macular degeneration or “AMD”) as well as rarer, earlier-onset macular dystrophies that in some cases can be detected in the first few decades of life.

AMD is a multi-factorial disease and shares a similar phenotype and clinical characteristics to inherited macular dystrophies (see, e.g., Capon, et al (1989) Ophthalmology 96;1769-77; Marmorstein, et al (2002) PNAS 99: 13067-72; Green (1999) Mol Vis 5:27; Young (1987) Surv Ophthalmol 31:291-306). The pathology in these maculopathies occurs at the retinal pigment epithelium (RPE)-ECM interface within the retina and involves multiple retinal cell layers (see, e.g., Green (1999) Mol Vis 5:27; Davis, et al (2005) Arch Ophthalmol 123: 1484-98; Curcio, et al (1996) Invest Ophthalmol Vis Sci 37: 1236-49; Dorey, et al (1989) Invest Ophthalmol Vis Sci 30: 1691-99; Bhutto, et al (2012) Mol Aspects Med 33:295-17). These layers include the choriocapillaris, which is a layer of capillaries found in the choroid (a highly vascularized layer that supplies oxygen and nutrition to the outer retina) and the metabolically active retinal pigment epithelium (RPE). The choriocapillaris is separated from the RPE by the Bruch’s membrane (BrM), a thin (2-4 pm), acellular sheet of extracellular matrix. The RPE comprises a single monolayer of cells that resides between the light-sensitive photoreceptors and the BrM, where it functions to support the photoreceptor cells and maintain retinal homeostasis (see, e.g., Strauss (2005) Physiol Rev. 85:845-881). Disease presentation for AMD and related MDs appears at the interface between the RPE and ECM in the retina and results in drusen formation, ECM protein accumulation, thickening of Bruch’s membrane, development of choroidal neovascularization, RPE atrophy, and loss of vision. Two forms of advanced AMD are distinguished. Neovascular (wet) AMD is characterized by infdtration of abnormal blood vessels into the retina. These newly formed vessels are fragile and when they break, the leakage of blood constituents in the retina leads to sudden vision loss. Geographic atrophy (dry) AMD is the result of gradual degeneration of the RPE and photoreceptors cells. Wet AMD is responsible for the majority of vision loss associated with AMD. Treatment options for macular degeneration are limited. Vascular endothelial growth factor (VEGF) is involved in neovascularization and excess secretion of VEGF has been demonstrated in certain patients with wet AMD. Treatments for wet AMD include drugs targeting VEGF (e.g., brolucizumab, aflibercept, ranibizumab, pegaptanib sodium, and bevacizumab). However, the benefits of such treatments are limited in that they are effective in only a subset of patients and require frequent intravitreal injections (e.g., every 4-12 weeks) to achieve a response. Reduction in the frequency of administration required for a therapeutic effect is desirable, as are treatment options to prevent progression of early stages of the disease. Accordingly, there remains a need for therapeutic advances to prevent AMD and to mitigate the severity of symptoms following disease onset.

In some embodiments, the method for preventing, treating, or reversing a ocular disease or disorder associated with one or more mutations in an EFEMP1 gene comprises administering to the subject a therapeutically effective amount or prophylactically effective amount of an EFEMP1- targeting oligonucleotide described herein (e.g., an EFEMP1 -targeting antisense oligonucleotide or an EFEMP1 -targeting RNAi oligonucleotide), or pharmaceutical composition thereof, by parenteral delivery (e.g., by subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, or intrathecal injection or infusion), by direct delivery into one or both eyes (e.g., by intravitreal injection), or by topical application to one or both eyes (e.g., as an ointment, spray, and/or aerosol). In some embodiments, the subject is administered at least one EFEMP1 -targeting oligonucleotide described herein. In some embodiments, the subject is administered a combination of EFEMP1 -targeting oligonucleotide described herein. In some embodiments, the one or more EFEMP1 -targeting oligonucleotide is formulated as a pharmaceutical composition as described herein, such as a pharmaceutical composition for ocular delivery. In some embodiments, the subject is administered a single dose of at least one EFEMP1 -targeting oligonucleotide described herein. In some embodiments, the subject is administered repeat doses of at least one EFEMP1 -targeting oligonucleotide described herein.

In some embodiments, the method comprises administering to the subject a therapeutically effective amount or prophylactically effective amount of an EFEMP1 -targeting oligonucleotide described herein (e.g., an EFEMP1 -targeting antisense oligonucleotide or an EFEMP1 -targeting RNAi oligonucleotide), or pharmaceutical composition thereof, in combination with one or more additional agents. The term “in combination with” encompasses administration of the EFEMP1- targeting oligonucleotide at substantially the same time as administration of the one or more additional agents (e.g., simultaneously in the same or different formulations or sequentially in different formulations) or at different times (e.g., prior to or following administration of the one or more additional agents). In some embodiments, the one or more additional agents is a therapeutic agent known in the art for treatment of the ocular disease (e.g., an anti-VEGF therapy).

Kits

The present disclosure provides kits for performing the methods described herein. In some embodiments, the kit comprises one or more oligonucleotides described herein (e.g., an EFEMP1- targeting antisense oligonucleotide or an EFEMP1 -targeting RNAi oligonucleotide), or pharmaceutical compositions thereof, and a package insert comprising instructions for administering the one or more oligonucleotides to a subject having, or at risk of developing, a disease or disorder associated with a mutation in an EFEMP1 gene. In some embodiments, the kit comprises a package insert comprising instructions for administering the one or more oligonucleotides), or pharmaceutical compositions thereof, to a subject having, or at risk of developing, an overexpression EFEMP1 in a tissue or organ. In some embodiments, the kit comprises a package insert comprising instructions for administering a the one or more oligonucleotides), or pharmaceutical compositions thereof, to a subject having, or at risk of developing, an ocular disease or disorder, e.g., an ocular disease or disorder associated with a mutation in an EFEMP1 gene. In some embodiments, the kit comprises a package insert comprising instructions for administering the one or more oligonucleotides), or pharmaceutical compositions thereof, to a subject having, or at risk of developing, an overexpression of EFEMP1 in one or both eyes. In some embodiments, the package insert further comprises instructions for administering the one or more oligonucleotides, or pharmaceutical compositions thereof, in combination with one or more additional agents.

In some embodiments, the kit comprises a container comprising the one or more oligonucleotides, optionally formulated for delivery as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the kit comprises a medicament comprising the one or more oligonucleotides, optionally formulated for delivery as described herein, and optionally a pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament to a subject having, or at risk of developing, a disease or disorder associated with a mutation in an EFEMP1 gene. In some embodiments, the package insert further comprises instructions for administering the medicament prior to, current, with, or subsequent to administration of a second medicament for treating, prevent, or ameliorating a disease or disorder associated with the mutation in the EFEMP1 gene. In some embodiments, the kit further comprises means for administering the one or more oligonucleotides to the subject (e.g., an injection device or an infusion pump). In some embodiments, the kit further comprises a means for measuring a reduced or inhibited expression of EFEMP1 following administration in a tissue sample obtained from the subject (e.g., means for measuring the inhibition of EFEMP1 mRNA or EFEMP1 protein). In some embodiments, the means for measuring a reduced or inhibited expression EFEMP 1 comprises a means for obtaining the tissue sample from a subject.

EXAMPLES

Example 1: Antisense Oligonucleotides Targeting EFEMP1

A library of nucleic acids targeting human and mouse EFEMP 1 was designed in silico. Given the length of the human EFEMP 1 gene (approximately 60kb), there exists an enormous number of possible target sequences that can be selected for targeting by an inhibitory oligonucleotide (e.g., about 60,000 possibilities). Thus, criteria were designed to select target sequences identified in Table 2. The criteria were based on proprietary information and art-recognized methods for selecting nucleic acid target sequences, and included, for example, selecting target sequences predicted to result in minimal off-target specificity within the transcriptome and identified in regions of human EFEMP 1 pre -mRNA predicted to have minimal secondary structure.

Nucleic acids selected for further validation were synthesized using standard automated solid phase oligonucleotide synthesis. Sequences of nucleic acids targeting human EFEMP1 are set forth in SEQ ID NOs: 379-755 and 827-897. The EFEMP 1 target sequences used to generate the nucleic acids are set forth in SEQ ID NOs: 2-378 or 756-826. Table 2 provides the “start site” for each nucleic acid, which corresponds to the position in the human EFEMP 1 pre-mRNA nucleotide sequence that binds to the 5'-terminal nucleoside of the nucleic acid set forth in the corresponding SEQ ID NO.

The 20mer nucleic acids set forth in Table 2 were synthesized as antisense gapmers having from 5' to 3': (i) a 5 '-wing segment of 5 nucleosides; (ii) a gap segment of 10 nucleosides; and (iii) a 3'-wing segment of 5 nucleosides. The nucleosides in the 5'-wing segment and 3'-wing segment have a sugar with a 2'-O-methoxyethyl (2'-M0E) modification and the nucleosides in the gap segment are deoxyribonucleosides. The intemucleoside linkages of the full-length gapmer are phosphorodithioate linkages.

Example 2: In vitro screening of EFEMP1 mRNA knockdown for EFEMP 1 -targetin ASOs

Selections of the antisense oligonucleotides (ASOs) described in Example 1 were evaluated in an in vitro mRNA knockdown assay in a human cell line expressing EFEMP 1 (ARPE-19 human retinal pigment epithelia (RPE) cell line). A library of ASOs was screened for EFEMP 1 targeting activity. Cells were plated in 96-well plates and each well was transfected with one of the antisense oligonucleotides described in Example 1 using lipofectamine. Each ASO was transfected at two concentrations (5nM and 20nM), and each concentration was transfected in four independent wells for biological quadruplicates. Cells treated with lipofectamine (but no antisense oligonucleotide) were used as a negative control, and cells treated with a short interfering RNA (siRNA) were used as a positive control for EFEMP1 expression measurement and downregulation. After a 48-hour incubation period, transfected cells were assayed for gene expression. Levels of EFEMP1 mRNA were determined using a TaqMan qPCR assay.

Reduction of EFEMP1 expression was determined across all ASOs tested (FIGs. 1 and 2). Table 3 provides the average expression of EFEMP1 in the two ASO concentration experiments for each tested ASO. These data demonstrate that targeting EFEMP1 across the length of the gene can result in reduction in EFEMP1 expression in a cellular model system.

Table 3. Exemplary ASOs for targeting EFEMP1

Selected ASOs from the ASO library described above were further characterized for their efficacy and potency in a multi-point dose-response analysis. Cells were plated in 96-well plates and transfected with one of the selected ASOs. Initially, each ASO was transfected at eight concentrations with a five-fold dilution factor (40nM, 8nM, 1.6nM, 0.32nM, 0.064nM, 0.128nM, 0.0026nM, and 0.0005 InM), and each concentration was transfected in three independent wells for biological triplicates. Four of these ASOs were also tested at seven concentrations with a three-fold dilution factor (25nM, 8.33nM, 2.78nM, 0.926nM, 0.309nM, 0.103nM, and 0.034nM). Cells treated with lipofectamine (but no antisense oligonucleotide) were used as a negative control, and cells treated with a short interfering RNA (siRNA) were used as a positive control for EFEMP1 expression measurement and downregulation. After a 48-hour incubation period, transfected cells were assayed for gene expression levels of EFEMP1 mRNA using a TaqMan qPCR assay.

FIGs. 3A-3B provide dose-response curves for each of these ASOs. Table 4 provides the maximum mRNA knockdown (as percent of control) and half maximal inhibitory concentrations (IC- 50) for each of these selected ASOs. These data demonstrate that the tested ASOs exhibit dosedependent inhibition of EFEMP1 in a cellular model system.

Table 4. IC-50 values of selected ASOs

Table 5: Sequences Associated With The Disclosure EQUIVALENTS

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 described here. Such equivalents are intended to be encompassed by the following claims. All references, including patent documents, are incorporated by reference in their entirety.