Trans-splicing molecules
The present invention features nucleic acid trans-splicing molecules (e.g., pre-mRNA trans-splicing molecules (RTMs)) capable of correcting one or more mutations in the ABCA4 gene or the CEP290 gene. Such molecules are useful in the treatment of disorders associated with mutations in ABCA4, such as Stargardt Disease (e.g., Stargardt Disease 1) and disorders associated with a mutation in CEP290, such as Leber congenital amourosis 10 (LCA 10). Also provided by the invention described herein are methods of using the nucleic acid trans-splicing molecules for correcting mutations in ABCA4 and CEP290 and for treating disorders associated with mutations in ABCA4 and CEP290, such as Stargardt Disease and LCA 10.
1. A nucleic acid trans-splicing molecule comprising, operatively linked in a 5′-to-3′ direction:
(a) a binding domain, wherein the binding domain comprises a sequence ranging from 50-300 nucleotides in length and is configured to bind ABCA4 intron 22 at a binding site within nucleotides 60 to 570, 600 to 800, or 900 to 1,350 of SEQ ID NO: 28;
(b) a splicing domain configured to mediate trans-splicing; and
(c) a coding domain comprising functional ABCA4 exons 23-50;
wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 22, thereby replacing endogenous ABCA4 exons 23-50 with the functional ABCA4 exons 23-50.
2. The nucleic acid trans-splicing molecule of claim 1 , wherein the binding site is within nucleotides 60 to 570 of SEQ ID NO: 28.
3. The nucleic acid trans-splicing molecule of claim 2 , wherein the binding site is within nucleotides 70-250 of SEQ ID NO: 28.
4. The nucleic acid trans-splicing molecule of claim 1 , wherein the binding site is within nucleotides 900 to 1,350 of SEQ ID NO: 28.
5. The nucleic acid trans-splicing molecule of claim 1 , wherein the binding site comprises:
(a) six or more of nucleotides 60 to 570 of SEQ ID NO: 28;
(b) six or more of nucleotides 600 to 800 of SEQ ID NO: 28; or
(c) six or more of nucleotides 900 to 1,350 of SEQ ID NO: 28.
6. The nucleic acid trans-splicing molecule of claim 5 , wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of the binding site.
7. The nucleic acid trans-splicing molecule of claim 1 , wherein the binding domain comprises a sequence ranging from 50-75 nucleotides in length, 75-100 nucleotides in length, 125-150 nucleotides in length, 150-175 nucleotides in length, 175-200 nucleotides in length, 200-250 nucleotides in length, 100-200 nucleotides in length, or 150 nucleotides in length.
8. The nucleic acid trans-splicing molecule of claim 1 , wherein the binding domain comprises at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 consecutive nucleotides that are complementary to the binding site.
9. The nucleic acid trans-splicing molecule of claim 1 , wherein the binding domain is 80% to 100% complementary to the binding site.
10. The nucleic acid trans-splicing molecule of claim 1 , wherein:
(i) the binding domain is 100-200 nucleotides in length;
(ii) the coding domain is a cDNA sequence;
(iii) the coding domain comprises a naturally-occurring sequence;
(iv) the coding domain comprises a codon-optimized sequence;
(v) an artificial intron comprises a spacer sequence;
(vi) the trans-splicing molecule is from 3,000 to 4,000 nucleotides in length;
(vii) the mutation in the ABCA4 gene is associated with Stargardt Disease; and/or
(viii) the mutation is expressed in a photoreceptor cell.
11. A proviral plasmid comprising the nucleic acid trans-splicing molecule of claim 1 .
12. An adeno-associated virus (AAV) comprising the nucleic acid trans-splicing molecule of claim 1 .
13. The AAV of claim 12 , wherein the AAV preferentially targets a photoreceptor cell.
14. The AAV of claim 12 , wherein the AAV comprises an AAV5 capsid protein, an AAV8 capsid protein, an AAV8 (b) capsid protein, or an AAV9 capsid protein.
15. The AAV of claim 12 , comprising an assembled capsid having packaged therein a vector genome comprising an AAV 5′ ITR, the nucleic acid trans-splicing molecule under the operative control of regulatory sequences, and an AAV 3′ ITR.
16. A pharmaceutical composition comprising the nucleic acid trans-splicing molecule of claim 1 , or a proviral plasmid thereof, or an AAV plasmid thereof.
17. A method of correcting a mutation in an ABCA4 gene in a target cell of a subject, the method comprising administering to the subject the pharmaceutical composition of claim 16 .
18. A method of correcting a mutation in any one or more of ABCA4 exons 23-50 in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 16 .
19. The method of claim 17 , wherein the subject has Stargardt Disease.
20. The method of claim 17 , wherein the composition is administered by subretinal injection, intravitreal injection, or intravenous injection.
21. A method of treating a subject having a disorder associated with a mutation in ABCA4, the method comprising administering to the subject the pharmaceutical composition of claim 16 .