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US-12616757-B2 - Compositions and methods for correction of heritable ocular disease

US12616757B2US 12616757 B2US12616757 B2US 12616757B2US-12616757-B2

Abstract

A nucleic acid trans-splicing molecule is provided that can replace an exon in a targeted mammalian ocular gene carrying a defect or mutation causing an ocular disease with an exon having the naturally-occurring sequence without the defect or mutation. A method of treating an ocular disease, e.g., Stargardt's Disease, caused by a defect or mutation in a target gene, e.g., ABCA4 comprising: administering to the ocular cells of a subject having an ocular disease a composition comprising a recombinant AAV comprising a nucleic acid trans-splicing molecule as described above.

Inventors

  • Jean Bennett
  • Jeannette BENNICELLI
  • Scott J. Dooley
  • Lloyd G. Mitchell

Assignees

  • THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA

Dates

Publication Date
20260505
Application Date
20210414

Claims (14)

  1. 1 . A nucleic acid trans-splicing molecule comprising (a) a binding domain (BD) that binds a target intron of a mammalian MYO7A pre-mRNA carrying a defect or mutation causing an ocular disease, (b) a splice site, and (c) an exon sequence that encodes one more exons of an MYO7A gene without the defect or mutation.
  2. 2 . The nucleic acid trans-splicing molecule according to claim 1 , wherein the nucleic acid trans-splicing molecule can replace an exon sequence in a targeted mammalian MYO7A gene carrying a defect or mutation causing Ushers Syndrome with Exons 1-18 or Exons 33-49 having the naturally-occurring sequence without the defect or mutation.
  3. 3 . The nucleic acid trans-splicing molecule according to claim 1 , wherein the exon sequence is Exons 1-18.
  4. 4 . The nucleic acid trans-splicing molecule according to claim 1 , wherein the exon sequence is Exons 33-49.
  5. 5 . The nucleic acid trans-splicing molecule according to claim 1 , comprising: (a) a binding domain (BD) that targets a selected sequence of the MYO7A gene and which binds to the target 5′ to the mutation or defect in the target pre-mRNA; (b) an optional spacer; (c) a 3′ splice site, and (d) a sequence that encodes one or all exons of the MYO7A gene that are 3′ to the binding of the BD to the target, said sequence correcting the defects or mutations in the target gene.
  6. 6 . The nucleic acid trans-splicing molecule according to claim 5 , wherein the binding domain binds to a portion of Intron 32 and the coding sequence encodes Exons 33-49.
  7. 7 . The nucleic acid trans-splicing molecule according to claim 1 , comprising in sequential order: (a) a sequence that encodes all exons of the ocular gene that are 5′ to the binding of the BD to the target, said sequence correcting the defects or mutations in the target gene; (b) a 5′ splice site; (c) an optional spacer; and (d) a binding domain (BD) sequence that targets a selected sequence of an ocular gene and which binds to the target 3′ to the mutation or defect in the target pre-mRNA.
  8. 8 . The nucleic acid trans-splicing molecule according to claim 7 , wherein the binding domain binds to a portion of Intron 18 and the coding sequence encodes Exons 1-18.
  9. 9 . The nucleic acid trans-splicing molecule according to claim 1 , which is a nucleic acid sequence of up to 3000 bp in length.
  10. 10 . A proviral plasmid comprising the nucleic acid trans-splicing molecule according to claim 1 .
  11. 11 . A proviral plasmid comprising a wildtype 5′ adeno-associated virus (AAV) ITR sequence, a promoter comprising an ocular cell-specific promoter/enhancer, a multi-cloning polylinker sequence having inserted therein the nucleic acid trans-splicing molecule of claim 1 operatively linked to, and under the regulatory control of, the promoter; and a wildtype 3′ AAV ITR sequence.
  12. 12 . The proviral plasmid according to claim 11 , wherein the plasmid is a p618 plasmid.
  13. 13 . A recombinant adeno-associated virus (AAV) comprising the nucleic acid trans-splicing molecule according to claim 1 .
  14. 14 . A pharmaceutical preparation comprising the recombinant AAV of claim 13 and a pharmaceutically acceptable carrier.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Nonprovisional patent application Ser. No. 15/776,663, filed May 16, 2018, which is a national stage entry of International Patent Application No. PCT/US2016/062941, filed Nov. 18, 2016, which claims the benefit of the priority of U.S. Provisional Patent Application No. 62/257,500, filed Nov. 19, 2015, which applications are incorporated herein by reference. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled “UPN-15-7313PCT_ST25.txt”. BACKGROUND A number of inherited retinal diseases are caused by mutations, generally multiple mutations, located throughout portions of large ocular genes. As one example, Stargardt disease, also known as Stargardt 1 (STGD1), is an autosomal recessive form of retinal dystrophy that is usually characterized by a progressive loss of central vision. Worldwide prevalence of STGD1 is estimated at 1/8,000-1/10,000. The disease typically presents within the first two decades of life. Although disease progression and severity varies widely, STGD1 is usually characterized by a progressive loss of central vision causing blurry vision and, occasionally, an increasing difficulty to adapt in the dark. STGD1 may progress rapidly over a few months or gradually over several years leading to a severe decrease in visual acuity. Most affected individuals also have impaired color vision or photophobia. There is no treatment currently available for STGD1. STGD1 has been linked to mutations in the ABCA4 gene, which has a sequence of 6822 nucleotides that encodes an adenosine triphosphate (ATP)-binding cassette transporter (ABCR) of sub-family A number 4, which is expressed specifically in the cones and rods of the retina. Defects in ABCR function cause the accumulation of all-trans-retinal and its cytotoxic derivatives (e.g., diretinoid-pyridinium-ethanolamine) (lipofuscin pigments) in photoreceptors and retinal pigment epithelial (RPE) cells, ultimately causing RPE cell death and the subsequent loss of photoreceptors. Mutations in ABCA4 have been linked to a spectrum of phenotypes ranging from STGD1, to a juvenile onset macular degeneration, fundus flavimaculatus, to cone-rod dystrophy, and a form of retinitis pigmentosa. ABCA4 mutations also contribute to age-related macular degeneration (AMD) and severe early-onset retinal dystrophy. Similar retinal diseases are caused by defects in other large ocular genes, including CEP290 (7440 nucleotides) which defects or mutations cause Leber's congenital amaurosis, among other ocular disorders, and MYO7A (7465 nucleotides), which defects or mutations cause Usher's disease. The occurrences and locations of multiple mutations in such large ocular genes has made strategies for repairing the mutations very challenging. There remains a need for effective compositions and therapeutic methods for treating such ocular disorders. SUMMARY In one aspect, a composition comprises a pre-RNA trans-splicing molecule (RTM) that can replace an exon or multiple exons in a targeted mammalian ocular gene carrying a defect or mutation causing an ocular disease with an exon(s) having the naturally-occurring sequence without the defect or mutation. In another aspect, a recombinant nucleic acid molecule and vectors capable of expressing the RTMs described herein are provided. In still another aspect, ocular cells expressing the RTM are provided for use in ex vivo repair and reimplantation to the subject from which the ocular cells were extracted. In another aspect, a proviral plasmid comprises a modular recombinant AAV genome comprising in operative association comprising a 5′ AAV2 ITR sequence, a suitable promoter operative in a mammalian ocular cell, an RNA trans-splicing molecule that can replace an exon in a targeted mammalian ocular gene carrying a defect or mutation causing an ocular disease with an exon having the naturally-occurring sequence without the defect or mutation, wherein the RTM is operatively linked to, and under the regulatory control of, the promoter; and a 3′ AAV2 ITR sequence. The modular AAV genome is present in a plasmid backbone comprising the elements necessary for replication in a host cell. In yet another aspect, a cell culture comprises bacterial or mammalian host cells transfected with the plasmids or nucleic acid constructs described herein. In another aspect, a recombinant AAV infectious particle comprises an RTM or nucleic acid construct described herein. In another embodiment, a recombinant AAV infectious particle is produced by culturing a packaging cell carrying a proviral plasmid as described herein and carrying an RTM in the presence of sufficient viral sequences to permit packaging of the ocular gene nucleic acid sequence expression cassette viral genome into an infectious AAV envelope or capsid. In one aspect, a ki