# US 11,993,770 B2 · Methods for targeted insertion of DNA in genes

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- Number: US 11,993,770 B2
- Document ID: US11993770B2
- Status: Granted
- Kind: B2
- Application number: 18/526,745
- Filed: 2023-12-01
- Published: 2024-05-02
- Granted: 2024-05-28
- Earliest claimed: 2018-10-16
- Assignees: Blueallele Corporation
- Applicants: Blueallele Corporation
- Inventors: Nicholas J. Baltes
- Web page: https://patentgrep.com/patent/US11993770B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/86: Viral vectors
- C12N 15/902: {using homologous recombination}
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N 2840/44: being a specific part of the splice mechanism, e.g. donor, acceptor
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods and compositions for modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes.

## Claims

### Claim 1

A recombinant nucleic acid comprising,
(i) a transgene, the transgene comprising from 5′ to 3′ orientation:
a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement, wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, and the second coding sequence is operably linked to the second splice acceptor and second terminator,
wherein the first and second coding sequences differ in nucleic acid sequence but encode the same amino acids, wherein said amino acids encoded by the first and second coding sequences correspond to amino acids encoded by an endogenous gene, and
wherein the transgene is equal to or less than 4.7 kb, and
(ii) adeno-associated virus inverted terminal repeats flanking the transgene, and wherein the recombinant nucleic acid is in a circular conformation.

### Claim 2 (depends on 1)

The recombinant nucleic acid of claim 1, wherein the first terminator is selected from the group consisting of an SV40 poly(A) and a BGH poly(A).

### Claim 3 (depends on 2)

The recombinant nucleic acid of claim 2, wherein the second terminator is selected from the group consisting of an SV40 poly(A) and a BGH poly(A).

### Claim 4 (depends on 1)

The recombinant nucleic acid of claim 1, wherein the transgene does not comprise homology arms.

### Claim 5 (depends on 1)

The recombinant nucleic acid of claim 1, wherein the endogenous gene is selected from the group consisting of Factor VIII, Factor IX, GBA, GLA, IDS, IDUA, ATXN3, USH2A, and G6PT.

### Claim 6 (depends on 1)

The recombinant nucleic acid of claim 1, wherein the endogenous gene encodes alpha-1 antitrypsin.