# US 7,745,179 B2 · Lentiviral vectors featuring liver specific transcriptional enhancer and methods of using same

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- Number: US 7,745,179 B2
- Document ID: US7745179B2
- Status: Granted
- Kind: B2
- Application number: 11/626,192
- Filed: 2007-01-23
- Published: 2007-08-02
- Granted: 2010-06-29
- Earliest claimed: 2001-05-14
- Assignees: Gbp IP, LLC
- Inventors: James G. McArthur; Dale J. Talbot; Andrew D. Simmons; Ryan McGuinness; Michael Kelly; Lisa V. Tsui; Thomas Dull
- Web page: https://patentgrep.com/patent/US7745179B2

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C07K 14/755: Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)}
- C12N 9/644: {Coagulation factor IXa (3.4.21.22)}
- C12N 15/86: Viral vectors
- C12Y 304/21022: Coagulation factor IXa (3.4.21.22)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2740/10043: viral genome or elements thereof as genetic vector
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2740/16052: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N 2830/008: cell type or tissue specific enhancer/promoter combination
- C12N 2830/15: chimeric enhancer/promoter combination
- 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)
- C12Y: ENZYMES
- 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)

## Abstract

Recombinant lentiviruses and transfer vectors for transgene delivery as well as methods for gene therapy using such vectors are disclosed. The invention provides a third generation lentiviral packaging system and a set of vectors for producing recombinant lentiviruses, as well as novel tissue specific enhancer and promoter elements useful for optimizing liver specific transgene delivery. The transgene is preferably a blood clotting factor such as human factor IX (hFIX) or human factor VIII (hFVIII) and can be used for treatment of hemophilia.

## Claims

### Claim 1

A lentiviral producer cell comprising:
(a) a gag gene, or a pol gene, or gag and pol genes;
(b) an env gene; and
(c) a lentiviral transfer vector that comprises a transgene operably linked to an expression control sequence, wherein the expression control sequence comprises the nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

### Claim 2 (depends on 1)

The producer cell of claim 1, wherein the expression control sequence comprises the nucleotide sequence of SEQ ID NO: 10.

### Claim 3 (depends on 1)

The producer cell of claim 1, wherein the expression control sequence consists of the nucleotide sequence of SEQ ID NO: 10.

### Claim 4 (depends on 1)

The producer cell of claim 1, wherein the expression control sequence further comprises a mouse transthyretin (mTTR) promoter.

### Claim 5 (depends on 1)

The producer cell of claim 1, wherein the expression control sequence further comprises an α-1-microglobulin/bikunin enhancer and a human factor VIII endogenous promoter (L-F8).

### Claim 6 (depends on 1)

The lentiviral producer cell of claim 1, wherein the elements of (a), (b) and (c) are stably integrated into the genome of the lentiviral producer cell.

### Claim 7 (depends on 1)

The lentiviral producer cell of claim 1, wherein the lentivirus is a human immunodeficiency virus (HIV).

### Claim 8 (depends on 7)

The lentiviral producer cell of claim 7, wherein the HIV is HIV-1.

### Claim 9 (depends on 1)

The lentiviral producer cell of claim 1, further comprising a rev gene.

### Claim 10 (depends on 1)

The lentiviral producer cell of claim 1, which lacks functional genes selected from tat, vif, vpr, vpu, vpx and nef, or a combination thereof.

### Claim 11 (depends on 1)

The lentiviral producer cell of claim 1, wherein the expression control sequence further comprises a liver specific promoter.

### Claim 12 (depends on 11)

The lentiviral producer cell of claim 11, wherein the liver specific promoter comprises a mouse transthyretin (mTTR) promoter.

### Claim 13 (depends on 1)

The lentiviral producer cell of claim 1, wherein the expression control sequence further comprises an α-1-microglobulin/bikunin enhancer and a human factor VIII endogenous promoter (L-F8).

### Claim 14

A set of lentiviral vectors comprising:
(a) a first packaging vector comprising a gag gene, or a pol gene, or gag and pol genes operably linked to an expression control sequence;
(b) a second packaging vector comprising a heterologous env gene operably linked to an expression control sequence; and
(c) a lentiviral transfer vector that comprises a transgene operably linked to an expression control sequence;
wherein the expression control sequence comprises the nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

### Claim 15 (depends on 14)

The set of claim 14, wherein the expression control sequence comprises the nucleotide sequence of SEQ ID NO: 10.

### Claim 16 (depends on 14)

The set of claim 14, wherein the expression control sequence consists of the nucleotide sequence of SEQ ID NO: 10.

### Claim 17 (depends on 14)

The set of claim 14, wherein the expression control sequence further comprises a mouse transthyretin (mTTR) promoter.

### Claim 18 (depends on 14)

The set of claim 14, wherein the expression control sequence further comprises an α-1-microglobulin/bikunin enhancer and a human factor VIII endogenous promoter (L-F8).

### Claim 19 (depends on 14)

The set of claim 14, wherein the transgene encodes a blood clotting factor.

### Claim 20 (depends on 14)

The set of claim 14, wherein the transgene encodes human factor IX (hFIX).

### Claim 21 (depends on 14)

The set of claim 14, wherein the transgene encodes human factor VIII (hFVIII).

### Claim 22 (depends on 14)

The set of claim 14, wherein the lentivirus is a human immunodeficiency virus (HIV).

### Claim 23 (depends on 22)

The set of claim 22, wherein the HIV is HIV-1.

### Claim 24 (depends on 14)

The set of claim 14, further comprising a fourth vector that comprises a rev gene.

### Claim 25 (depends on 14)

The set of claim 14, which lacks functional genes selected from tat, vif, vpr, vpu, vpx and net or a combination thereof.

### Claim 26 (depends on 14)

A method of producing a lentiviral producer cell comprising transforming a cell with the set of vectors of claim 14.

### Claim 27 (depends on 1)

A method of producing a recombinant lentivirus comprising culturing a producer cell of claim 1 in a medium and recovering recombinant lentivirus from the medium.

### Claim 28 (depends on 1)

A method of delivering a transgene to a cell comprising contacting the cell with a recombinant lentivirus produced by the lentiviral producer cell of claim 1 under conditions permitting transformation of the cell.