NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND METHODS OF INDUCING EXON SKIPPING
Disclosed herein are molecules and pharmaceutical compositions that induce an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion. Also described herein include methods for treating a disease or disorder that comprises a molecule or a pharmaceutical composition that induces an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion.
1 . A single stranded oligonucleotide conjugate comprising an anti-transferrin receptor binding moiety conjugated to a single stranded oligonucleotide, wherein the single stranded oligonucleotide hybridizes to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript of a DMD gene, or hybridizes to a site near the acceptor splice site, the donor splice site, or the exonic splice enhancer element; wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate an mRNA transcript encoding a truncated dystrophin protein.
2 . The single stranded oligonucleotide conjugate of claim 1 , wherein the single stranded oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO) or an antisense oligonucleotide (ASO).
3 . The single stranded oligonucleotide conjugate of claim 2 , wherein the anti-transferrin receptor binding moiety is an anti-transferrin receptor antibody or an antigen binding fragment thereof.
4 . The single stranded oligonucleotide conjugate of claim 1 , wherein the single stranded oligonucleotide induces skipping of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.
5 . The single stranded oligonucleotide conjugate of claim 3 , wherein the anti-transferrin receptor antibody or antigen binding fragment thereof comprises a humanized antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab′, divalent Fab2, single chain variable fragment (scFv), diabody, minibody, nanobody, single domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof.
6 . The single stranded oligonucleotide conjugate of claim 1 , wherein the single stranded oligonucleotide comprises at least from about 10 to about 30 nucleotides in length.
7 . The single stranded oligonucleotide conjugate of claim 1 , wherein the single stranded oligonucleotide is conjugated to the antibody or the antigen binding fragment thereof via a linker.
8 . The single stranded oligonucleotide conjugate of claim 7 , wherein the linker is a cleavable linker.
9 . The single stranded oligonucleotide conjugate of claim 7 , wherein the linker is a non-cleavable linker.
10 . The single stranded oligonucleotide conjugate of claim 7 , wherein the linker is selected from the group consisting of a heterobifunctional linker, a homobifunctional linker, a maleimide group, a dipeptide moiety, a benzoic acid group or derivatives thereof, a C1-C6 alkyl A group, and a combination thereof.
11 . The single stranded oligonucleotide conjugate of claim 1 , wherein the single stranded oligonucleotide conjugate has a single stranded oligonucleotide to antibody ratio of about 1:1, 2:1, 3:1, or 4:1.
12 . A method of treating muscular dystrophy in a subject in need thereof, comprising:
administering to the subject a single stranded oligonucleotide conjugate comprising an anti-transferrin receptor binding moiety conjugated to a single stranded oligonucleotide, wherein the single stranded oligonucleotide hybridizes to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript of a DMD gene, or hybridizes to a site near the acceptor splice site, the donor splice site, or the exonic splice enhancer element;
wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate an mRNA transcript encoding a truncated dystrophin protein, thereby treating the muscular dystrophy in the subject.
13 . The method of claim 12 , wherein the single stranded oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO) or an antisense oligonucleotide (ASO).
14 . The method of claim 13 , wherein the anti-transferrin receptor binding moiety is an anti-transferrin receptor antibody or antigen binding fragment thereof.
15 . A method of inducing exon skipping in a targeted pre-mRNA transcript of a DMD gene, comprising:
a) contacting a muscle cell with a single stranded oligonucleotide conjugate comprising an anti-transferrin receptor binding moiety conjugated to a single stranded oligonucleotide, wherein the single stranded oligonucleotide hybridizes to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript of a DMD gene, or hybridizes to a site near the acceptor splice site, the donor splice site, or the exonic splice enhancer element; wherein the single stranded oligonucleotide induces exon skipping in the targeted pre-mRNA transcript, and wherein the single stranded oligonucleotide conjugate is preferentially delivered into the muscle cell;
b) hybridizing the single stranded oligonucleotide to the targeted pre-mRNA transcript to induce exon skipping in the targeted pre-mRNA transcript; and
c) translating an mRNA transcript produced from the targeted pre-mRNA transcript processed in step b) in the muscle cell to generate a truncated dystrophin protein.
16 . The method of claim 15 , wherein the anti-transferrin binding moiety is an anti-transferrin receptor antibody or an antigen binding fragment thereof.