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 . An oligonucleotide conjugate comprising an anti-transferrin receptor antibody or antigen binding fragment thereof conjugated to an oligonucleotide hybridizing to an internal region within an exon of a DMD gene; wherein the oligonucleotide induces exon skipping in a pre-mRNA transcript of the DMD gene to generate an mRNA transcript encoding a truncated DMD protein.
2 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide targets a region at least 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt from the 5′end of an exon of DMD gene.
3 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO) or an antisense oligonucleotide (ASO).
4 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide is delivered into a muscle cell.
5 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide induces skipping of exon 8, 23, or 35 of the DMD gene.
6 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide induces skipping of exon 43, 44, or 45 of the DMD gene.
7 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide induces skipping of exon 50, 51, 52, 53, or 55 of the DMD gene.
8 . The oligonucleotide conjugate of claim 1 , wherein the 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.
9 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide comprises at least from about 10 to about 30 nucleotides in length.
10 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide is conjugated to the antibody or antigen binding fragment thereof via a linker.
11 . The oligonucleotide conjugate of claim 10 , wherein the linker is a cleavable linker.
12 . The oligonucleotide conjugate of claim 10 , wherein the linker is a non-cleavable linker.
13 . The oligonucleotide conjugate of claim 10 , 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 C 1 -C 6 alkyl A group, and a combination thereof.
14 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide conjugate has a oligonucleotide to antibody ratio of about 1:1, 2:1, 3:1, or 4:1.
15 . The oligonucleotide conjugate of claim 1 , wherein the oligonucleotide conjugate is formulated for parenteral administration.
16 . A method of treating muscular dystrophy in a subject in need thereof, wherein the method comprises administering to the subject an appropriate amount of an oligonucleotide conjugate comprising an anti-transferrin receptor antibody or antigen binding fragment thereof conjugated to an oligonucleotide hybridizing to of an internal region within an exon of a DMD gene; wherein the oligonucleotide induces exon skipping in a pre-mRNA transcript of the DMD gene to generate an mRNA transcript encoding a truncated DMD protein.
17 . The method of claim 16 , wherein the truncated DMD protein modulates muscular dystrophy.
18 . The method of claim 17 , wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.
19 . A method of inducing exon skipping in a targeted pre-mRNA transcript of a DMD gene, comprising:
a) contacting a muscle cell with an oligonucleotide conjugate comprising an anti-transferrin receptor antibody or antigen binding fragment thereof conjugated to an oligonucleotide hybridizing to an internal region within an exon of the DMD gene; wherein the oligonucleotide induces exon skipping in a pre-mRNA transcript of the DMD gene to generate an mRNA transcript encoding a truncated DMD protein;
b) hybridizing the oligonucleotide conjugate 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 DMD protein. =
20 . The method of claim 19 , the oligonucleotide induces skipping of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.