# US 2021/0340568 A1 · Regulated gene editing system

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- Number: US 2021/0340568 A1
- Document ID: US20210340568A1
- Status: Published application
- Kind: A1
- Application number: 17/283,322
- Filed: 2019-10-09
- Published: 2021-11-04
- Earliest claimed: 2018-10-09
- Assignees: The University of North Carolina at Chapel Hill
- Applicants: The University of North Carolina at Chapel Hill
- Inventors: Richard Jude Samulski
- Web page: https://patentgrep.com/patent/US20210340568A1

## CPC

- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- 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)}
- C12N 2310/11: Antisense
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2320/33: Alteration of splicing
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides a gene editing system having reduced off target effects comprising (a) a vector comprising a nucleic acid sequence encoding a nuclease, wherein the nucleic acid encoding the nuclease contains within its sequence a regulatory nucleic acid sequence having a first and second set of splice elements defining a first and second intron, wherein the first and second intron flank a sequence encoding a non-naturally occurring exon sequence containing an in-frame stop codon sequence, and wherein the first and second intron are spliced from the mRNA message to produce an mRNA encoding a non-functional nuclease that contains an amino acid sequence encoded by the non-naturally occurring exon; and (b) an oligonucleotide that binds to the regulatory sequence. Further provided are methods of using the gene editing system of this invention to regulate transgene expression.

## Claims

### Claim 1

A system for editing a gene (e.g., altering expression of at least one gene product) having reduced off target effects comprising introducing into a cell having a target gene sequence
a) a vector comprising a nucleic acid sequence encoding a nuclease, wherein the nucleic acid encoding the nuclease contains within its sequence a regulatory nucleic acid sequence having a first and second set of splice elements defining a first and second intron, wherein the first and second intron flank a sequence encoding a non-naturally occurring exon sequence containing an in-frame stop codon sequence, and wherein the first and second intron are spliced from the pre-mRNA message to produce an mRNA encoding a non-functional nuclease that contains an amino acid sequence encoded by the non-naturally occurring exon; and
b) an oligonucleotide that binds to the regulatory nucleic acid sequence, wherein within the cell the oligonucleotide prevents splicing of the second set of splice elements from the mRNA, thereby producing an mRNA that lacks the exon and encodes a nuclease that is functional for gene editing of a target gene.

### Claim 2 (depends on 1)

The system of claim 1, wherein the nuclease is selected from the group consisting of a CRISPR-associated nuclease, a meganuclease, a zinc finger nuclease, and a transcription activator-like effector nuclease.

### Claim 3 (depends on 1)

The system of claim 1, wherein the nuclease is an endonuclease or an exonuclease.

### Claim 4 (depends on 1)

The system of claim 1, wherein component (a) further comprises a gRNA that binds to the sequence of the target gene.

### Claim 5 (depends on 1)

The system of claim 1, wherein the regulatory nucleic acid sequence is a beta-globin mutant intron.

### Claim 6

(canceled)

### Claim 7 (depends on 1)

The system of claim 1, wherein the regulatory nucleic acid sequence comprises a sequence selected from the group consisting of: SEQ ID NO: 18 (IVS2-654 intron C-T), SEQ ID NO:50 (IVS2-654 intron with 564CT mutation), SEQ ID NO:51 (IVS2-654 intron with 657G mutation), SEQ ID NO:52 (IVS2-654 intron with 658T mutation), SEQ ID NO:20 (IVS2-654 intron with 657GT mutation), SEQ ID NO:53 (IVS2-654 intron with 200 by deletion), SEQ ID NO:68 (IVS2-654 intron with only 197 bp), SEQ ID NO:55 (IVS2-654 intron with 6A mutation), SEQ ID NO:56 (IVS2-654 intron with 564C mutation), SEQ ID NO:57 (IVS2-654 intron with 841A mutation), SEQ ID NO:59 (IVS2-705 intron with 564CT mutation), SEQ ID NO:60 (IVS2-705 intron with 657G mutation), SEQ ID NO:61 (IVS2-705 intron with 658T mutation), SEQ ID NO:62 (IVS2-705 intron with 657GT mutation), SEQ ID NO:63 (IVS2-705 intron with 200 by deletion), SEQ ID NO:64 (IVS2-705 intron with 425 by deletion), SEQ ID NO:65 (IVS2-705 intron with 6A mutation), SEQ ID NO:66 (IVS2-705 intron with 564C mutation), SEQ ID NO:67 (IVS2-705 intron with 841A mutation), SEQ ID NO: 74, SEQ ID NO:75, SEQ ID NO; 76, SEQ ID NO: 77, SEQ ID NO:78, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148; and in any combination thereof, including singly.

### Claim 8 (depends on 1)

The system of claim 1, wherein the oligonucleotide that binds to the regulatory sequence comprises a sequence selected from the group consisting of: SEQ ID NO:37 (oligo for IVS2-654 CT), SEQ ID NO:38 (oligo for IVS2-654 with 657GT mutation), SEQ ID NO:39 (oligo for 6A mutation in IVS2-654), SEQ ID NO:40 (oligo for 564C mutation in IVS2-654), SEQ ID NO:41 (oligo for 564CT mutation in IVS2-654), SEQ ID NO:43 (oligo for 841A mutation in IVS2-654), SEQ ID NO:44 (oligo for 657G mutation in IVS2-654), SEQ ID NO:45 (oligo for 658T mutation in IVS2-654), SEQ ID NO:42 (oligo for 705G mutation in IVS2-705), SEQ ID NO:49 (oligo for IVS2-705), SEQ ID NO:76 (Antisense exon 23 skipping inducing oligo) respectively, and SEQ ID NO 138 (Oligo for LUC-AON1), SEQ ID NO: 139 (oligo for LUC-AON2), SEQ ID NO: 140 (Oligo for LUC-AON3), SEQ ID NO: 141 (Oligo for LUC-AON4), SEQ ID NO: 142 (Oligo for IVS2(S0)-654, LUC-654) and SEQ ID NO: 149 (Oligo for WT regulatory).

### Claim 9

(canceled)

### Claim 10 (depends on 1)

The system of claim 1, wherein the off-target effects are reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

### Claim 11 (depends on 1)

The system of claim 1, wherein components (a) and (b) are located on same or different vectors.

### Claim 12 (depends on 1)

The system of claim 1, wherein component (b) is introduced to cell as naked DNA, as a lipid formulation, or as a nanoparticle.

### Claim 13

(canceled)

### Claim 14

(canceled)

### Claim 15 (depends on 1)

The system of claim 1, wherein component (b) is administered at a time point following the administration of (a), or components (a) and (b) are administered at substantially the same time.

### Claim 16

(canceled)

### Claim 17 (depends on 1)

The system of claim 1, wherein the expression of (a) is not detected in the cell in the absence of (b), or absence of expression of (b).

### Claim 18

(canceled)

### Claim 19 (depends on 1)

The system of claim 1, wherein component (b) controls an “ON” and/or “OFF” status of the system.

### Claim 20

(canceled)

### Claim 21

(canceled)

### Claim 22 (depends on 1)

The system of claim 1, wherein the vector is a viral vector or a non-viral vector.

### Claim 23 (depends on 22)

The system of claim 22, wherein the viral vector is selected form the group consisting of: from the group consisting of an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector, a baculovirus vector and a chimeric virus vector.

### Claim 24

(canceled)

### Claim 25

(canceled)

### Claim 26 (depends on 2)

The system of claim 2, wherein the CRISPR-associated nuclease
a) creates double stand breaks for gene editing and wherein the CRISPR-associated nuclease is selected from the group consisting of Cpf1, C2c1, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c;
b) is a Cas9 variant selected from Staphylococcus aureus (SaCas9), Streptococcus thermophilus (StCas9), Neisseria meningitidis (NmCas9), Francisella novicida (FnCas9), and Campylobacter jejuni (Cj Cas9); or
c) has been modified for gene-editing without double strand DNA breaks (such as CRISPRi or CRISPRa) and is selected from the group consisting of dCas, nCas, and Cas 13.

### Claim 27

(canceled)

### Claim 28

(canceled)

### Claim 29 (depends on 2)

The system of claim 2, wherein the CRISPR-associated nuclease is codon optimized for expression in the eukaryotic cell.

### Claim 30 (depends on 1)

The system of claim 1, wherein the gene editing is decreasing or increasing the expression of one or more gene products.

### Claim 31

(canceled)

### Claim 32

(canceled)

### Claim 33 (depends on 1)

The system of claim 1, wherein the cell is in vivo or in vitro.

### Claim 34

(canceled)

### Claim 35

(canceled)

### Claim 36 (depends on 1)

A method for editing a gene in a subject, the method comprising administering the system of claim 1 to a subject in need of gene editing.