# US 11,298,410 B2 · Methods and compositions for RNA-guided treatment of HIV infection

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- Number: US 11,298,410 B2
- Document ID: US11298410B2
- Status: Granted
- Kind: B2
- Application number: 15/578,372
- Filed: 2016-06-01
- Published: 2018-10-18
- Granted: 2022-04-12
- Earliest claimed: 2015-06-01
- Assignees: Temple University of the Commonwealth System of Higher Education
- Applicants: Temple University—of the Commonwealth System of Higher Education
- Inventors: Kamel Khalili; Wenhui Hu; Yonggang Zhang
- Web page: https://patentgrep.com/patent/US11298410B2

## CPC

- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61P 31/18: for HIV
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/1132: {against retroviridae, e.g. HIV}
- C12N 15/907: {in mammalian cells}
- C12N 2310/10: Type of nucleic acid
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/11: for the determination of target sites, i.e. of active nucleic acids
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Compositions for specifically cleaving target sequences in retroviruses include nucleic acids encoding a Clustered Regularly Interspace Short Palindromic Repeat (CRISPR) associated endonuclease and a guide RNA sequence complementary to one or more target nucleic acid sequences in a retrovirus genome.

## Claims

### Claim 1

A composition comprising:
(a) a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas9 endonuclease;
(b) a second nucleic acid sequence encoding a first guide RNA (gRNA), the first gRNA being complementary to a first target sequence within a 5′ long terminal repeat (LTR) or a 3′ LTR of a retroviral sequence;
(c) a third nucleic acid sequence encoding a second gRNA, the second gRNA being complementary to a second target sequence within a GagD region of the retroviral sequence; and
(d) an excipient.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the retroviral sequence is a human immunodeficiency virus (HIV) sequence integrated into a mammalian genome.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the first gRNA comprises a nucleic acid sequence having a sequence identity of 100% to any one of SEQ ID NOS: 1-8.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the second gRNA comprises a nucleic acid sequence having a sequence identity of 100% to SEQ ID NO: 47 or SEQ ID NO: 48.

### Claim 5

A pharmaceutical composition comprising:
(a) an expression vector comprising:
(i) a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas9 endonuclease;
(ii) a second nucleic acid sequence encoding a first guide RNA (gRNA), the first gRNA being complementary to a first target sequence within a 5′ long terminal repeat (LTR) or a 3′ LTR of a retroviral sequence; and
(iii) a third nucleic sequence encoding a second gRNA, the second gRNA being complementary to a second target sequence within a GagD region of the retroviral sequence; and
(b) a pharmaceutically acceptable excipient.

### Claim 6 (depends on 5)

The pharmaceutical composition of claim 5, wherein the retroviral sequence is a human immunodeficiency virus (HIV) sequence integrated into a mammalian genome.

### Claim 7 (depends on 5)

The pharmaceutical composition of claim 5, wherein the first gRNA comprises a nucleic acid sequence having a sequence identity of 100% to any one of SEQ ID NOS: 1-8.

### Claim 8 (depends on 5)

The pharmaceutical composition of claim 5, wherein the second gRNA comprises a nucleic acid sequence having a sequence identity of 100% to SEQ ID NO: 47 or SEQ ID NO: 48.

### Claim 9

An expression vector comprising:
(a) a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas9 endonuclease;
(b) a second acid sequence encoding a first guide RNA (gRNA), the first gRNA being complementary to a first target sequence within a 5′ long terminal repeat (LTR) or a 3′ LTR of a retroviral sequence;
(c) a nucleic acid sequence encoding a second gRNA, the second gRNA being complementary to a second target sequence within a GagD region of the retroviral sequence; and
(d) a regulatory region.

### Claim 10 (depends on 9)

The expression vector of claim 9, wherein the retroviral sequence is a human immunodeficiency virus (HIV) sequence integrated into a mammalian genome.

### Claim 11 (depends on 9)

The expression vector of claim 9, wherein the first gRNA comprises a nucleic acid sequence having a sequence identity of 100% to any one of SEQ ID NOS: 1-8.

### Claim 12 (depends on 9)

The expression vector of claim 9, wherein the second gRNA comprises a nucleic acid sequence having a sequence identity of 100% to SEQ ID NO: 47 or SEQ ID NO: 48.

### Claim 13

A polynucleotide encoding:
(a) a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas9 endonuclease;
(b) a second nucleic acid sequence encoding a first guide RNA (gRNA), the first gRNA being complementary to a first target sequence within a 5′ long terminal repeat (LTR) or a 3′ LTR of a retroviral sequence; and
(c) a third nucleic acid sequence encoding a second gRNA, the second gRNA being complementary to a second target sequence within a GagD region of the retroviral sequence.

### Claim 14 (depends on 13)

The polynucleotide of claim 13, wherein the retroviral sequence is a human immunodeficiency virus (HIV) sequence integrated into a mammalian genome.

### Claim 15 (depends on 13)

The polynucleotide of claim 13, wherein the first gRNA comprises a nucleic acid sequence having a sequence identity of 100% to any one of SEQ ID NOS: 1-8.

### Claim 16 (depends on 13)

The polynucleotide of claim 13, wherein the second gRNA comprises a nucleic acid sequence having a sequence identity of 100% to SEQ ID NO: 47 or SEQ ID NO: 48.