# US 11,993,776 B2 · Trans-splicing molecules

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,993,776 B2
- Document ID: US11993776B2
- Status: Granted
- Kind: B2
- Application number: 17/047,496
- Filed: 2019-04-17
- Published: 2021-05-27
- Granted: 2024-05-28
- Earliest claimed: 2018-04-17
- Assignees: Ascidian Therapeutics, Inc.; The Trustees of the University of Pennsylvania
- Applicants: Ascidian Therapeutics, Inc.; The Trustees of the University of Pennsylvania
- Inventors: Philip R. Johnson; Bruce C. Schnepp; Jean Bennett; Scott J. Dooley; Krishna Jawaharlal Fisher; Junwei Sun
- Web page: https://patentgrep.com/patent/US11993776B2

## CPC

- C12N 15/1138: {against receptors or cell surface proteins}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/71: containing domain for transcriptional activaation, e.g. VP16
- C07K 2319/85: containing an RNA binding domain
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/33: Alteration of splicing
- C12N 2320/34: Allele or polymorphism specific uses
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

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.

## Claims

### Claim 1

A nucleic acid trans-splicing molecule comprising, operatively linked in a 3′-to-5′ 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 880 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 1-22;
wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with the functional ABCA4 exons 1-22.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

The nucleic acid trans-splicing molecule of claim 1, wherein the binding domain comprises six or more consecutive nucleotides that are complementary to six or more nucleotides of the binding site.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

The nucleic acid trans-splicing molecule of claim 1, wherein the binding domain is 80% to 100% complementary to the binding site.

### Claim 6 (depends on 1)

The nucleic acid trans-splicing molecule of claim 1, wherein the binding domain comprises six or more consecutive nucleotides that are complementary to six or more nucleotides of the binding site, wherein the binding domain is configured to bind ABCA4 intron 22 at a binding site comprising:
(a) six or more of nucleotides 1041 to 1190 of SEQ ID NO: 28;
(b) six or more of nucleotides 1171 to 1320 of SEQ ID NO: 28; or
(c) six or more of nucleotides 1201 to 1350 of SEQ ID NO: 28.

### Claim 7 (depends on 1)

The nucleic acid trans-splicing molecule of claim 1, wherein:
(i) the coding domain is a cDNA sequence;
(ii) the coding domain comprises a naturally-occurring sequence;
(iii) the coding domain comprises a codon-optimized sequence;
(iv) an artificial intron comprises a spacer sequence;
(v) the trans-splicing molecule is from 3,000 to 4,000 nucleotides in length;
(vi) the mutation in the ABCA4 gene is associated with Stargardt Disease; and/or
(vii) the mutation is expressed in a photoreceptor cell.

### Claim 8 (depends on 1)

A proviral plasmid comprising the nucleic acid trans-splicing molecule of claim 1.

### Claim 9 (depends on 1)

An adeno-associated virus (AAV) comprising the nucleic acid molecule of claim 1.

### Claim 10 (depends on 9)

The AAV of claim 9, wherein the AAV preferentially targets a photoreceptor cell and/or wherein the AAV comprises an AAV5 capsid protein, an AAV8 capsid protein, an AAV8(b) capsid protein, or an AAV9 capsid protein.

### Claim 11 (depends on 1)

An adeno-associated virus (AAV) comprising an assembled capsid having packaged therein a vector genome comprising an AAV 5′ ITR, the nucleic acid molecule of claim 1 under the operative control of regulatory sequences and an AAV3′ ITR.

### Claim 12 (depends on 1)

A method of correcting a mutation in any one or more of ABCA4 exons 1-22 in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the nucleic acid trans-splicing molecule of claim 1.

### Claim 13 (depends on 1)

A method of treating a subject having a disorder associated with a mutation in any one or more of ABCA4 exons 1-22, the method comprising administering to the subject a pharmaceutical composition comprising the nucleic acid trans-splicing molecule of claim 1.

### Claim 14 (depends on 1)

A pharmaceutical composition comprising the nucleic acid trans-splicing molecule claim 1, or a proviral plasmid or AAV plasmid thereof.

### Claim 15 (depends on 14)

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 14.

### Claim 16 (depends on 14)

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 14.

### Claim 17 (depends on 16)

The method of claim 16, wherein:
(a) the subject has Stargardt Disease;
(b) the composition is administered by subretinal injection, intravitreal injection, or intravenous injection; and/or
(c) the subject exhibits at least 10% increase in ABCA4 protein expression after administration.

### Claim 18

A nucleic acid trans-splicing molecule comprising, operatively linked in a 3′-to-5′ direction:
(A) (a) a binding domain configured to bind ABCA4 intron 22 at a binding site comprising six or more of nucleotides 1041 to 1190 of SEQ ID NO: 28, 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;
(b) a splicing domain; and
(c) a coding domain comprising functional ABCA4 exons 1-22;
wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with the functional ABCA4 exons 1-22;
(B) (a) a binding domain configured to bind ABCA4 intron 22 at a binding site comprising six or more of nucleotides 1171 to 1320 of SEQ ID NO: 28, 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;
(b) a splicing domain; and
(c) a coding domain comprising functional ABCA4 exons 1-22;
wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with the functional ABCA4 exons 1-22; or
(C) (a) a binding domain configured to bind ABCA4 intron 22 at a binding site comprising six or more of nucleotides 1201 to 1350 of SEQ ID NO: 28, 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;
(b) a splicing domain; and
(c) a coding domain comprising functional ABCA4 exons 1-22;
wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with the functional ABCA4 exons 1-22.

### Claim 19 (depends on 18)

The nucleic acid trans-splicing molecule of claim 18, wherein the binding domain comprises a sequence ranging from 50-75 nucleotides in length, 75-100 nucleotides in length, 125-150 nucleotides in length, or 150 nucleotides in length.

### Claim 20 (depends on 18)

The nucleic acid trans-splicing molecule of claim 18, 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.

### Claim 21 (depends on 19)

The nucleic acid trans-splicing molecule of claim 19, wherein the binding domain is 80% to 100% complementary to the binding site.