# US 7,612,195 B2 · Retroviral vectors for delivery of interfering RNA

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- Number: US 7,612,195 B2
- Document ID: US7612195B2
- Status: Granted
- Kind: B2
- Application number: 10/574,416
- Filed: 2004-10-21
- Published: 2006-11-09
- Granted: 2009-11-03
- Earliest claimed: 2003-10-22
- Assignees: Aventis Pharmaceuticals Inc.
- Inventors: Dorre Grueneberg; Gerard Bain; Nayantara Kothari
- Web page: https://patentgrep.com/patent/US7612195B2

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/86: Viral vectors
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2330/30: chemically synthesised
- C12N 2740/15043: 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

Provided herein are retroviral vectors for delivering interfering RNA into cells.

## Claims

### Claim 1

A retroviral vector for carrying a target gene specific insert into a cell in order to modify the expression of a target gene having a sense strand and an antisense strand, comprising:
(a) a U6 promoter having a sequence of:
(SEQ ID NO:7)
ttcccatgattccttcatatttgcatatacgatacaaggctgttagagag
ataattagaattaatttgactgtaaacacaaagatattagtacaaaatac
gtgacgtagaaagtaataatttcttgggtagtttgcagtttttaaaatta
tgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcga
tttcttgcctttatatatcttgtggaaaggacgaaacaccg;
and
(b) a polylinker region comprising a nucleotide sequence of gatcc gctgggactcctttgcatg ttcaagaga catgcaaaggagtcccagc ttttt ggaa a (SEQ ID NO:4)
(c) a target gene specific insert comprising double stranded RNA, wherein said double stranded RNA comprises a sense portion that is complementary to a portion of the antisense strand of the target gene, and an antisense portion that is complementary to the sense portion, so that the sense portion and antisense portion anneal, and the double stranded RNA folds back upon itself.

### Claim 2 (depends on 1)

The retroviral vector of claim 1, wherein the sense and antisense regions of the target gene specific insert each comprise a length of 19-30 nucleotides.

### Claim 3 (depends on 2)

The retroviral vector of claim 2, wherein the sense and antisense regions of the target gene specific insert each comprise a length of 19-25 nucleotides.

### Claim 4 (depends on 3)

The retroviral vector of claim 3, wherein the sense and antisense regions of the target gene specific insert each comprise a length of 19-23 nucleotides.

### Claim 5

A modified Lentivirus vector for carrying double stranded RNA into a cell in order to modify the expression of a target gene having a sense strand and an antisense strand, wherein:
(a) the endogenous CMV promoter of the Lentivirus has been removed, said modified Lentivirus vector comprising:
(i) a REV element that binds to a REV response element (RRE) is inserted;
(ii) a U6 promoter sequence of
(SEQ ID NO:7)
ttcccatgattccttcatatttgcatatacgatacaaggctgttagagag
ataattagaattaatttgactgtaaacacaaagatattagtacaaaatac
gtgacgtagaaagtaataatttcttgggtagtttgcagtttttaaaatta
tgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcga
tttcttgcctttatatatcttgtggaaaggacgaaacaccg; and
(b) a polylinker region comprising a nucleotide sequence of: gatcc gctgggactcctttgcatg ttcaagaga catgcaaaggagtcccagc ttttt ggaa a (SEQ ID NO:4);
wherein said double stranded RNA comprises a sense portion that is complementary to a portion of the antisense strand of the target gene, and an antisense portion that is complementary to the sense portion so that the sense portion and antisense portion anneal, and the double stranded RNA folds back upon itself.

### Claim 6 (depends on 5)

The modified Lentivirus vector of claim 5, further comprising a reporter gene.

### Claim 7 (depends on 5)

The modified Lentivirus vector of claim 5, wherein said reporter gene is selected from the group consisting of Blasti and hrGFP.

### Claim 8 (depends on 7)

The modified Lentivirus vector of claim 7, wherein said vector is pLenti-U6-Blasti, which comprises the nucleotide sequence of SEQ ID NO:8.

### Claim 9

A modified lentivirus pLenti-U6-Blasti, comprising the nucleotide sequence of SEQ ID NO:8.

### Claim 10 (depends on 9)

A cell transformed or transfected with the modified lentivirus of claim 9.