# US 10,501,759 B2 · Compounds for improved viral transduction

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,501,759 B2
- Document ID: US10501759B2
- Status: Granted
- Kind: B2
- Application number: 15/997,643
- Filed: 2018-06-04
- Published: 2018-12-20
- Granted: 2019-12-10
- Earliest claimed: 2011-09-30
- Assignees: bluebird bio, Inc.
- Applicants: bluebird bio, Inc.
- Inventors: Garrett Collins Heffner; Abraham Isaac Bassan
- Web page: https://patentgrep.com/patent/US10501759B2

## CPC

- C12N 15/86: Viral vectors
- A61K 38/46: Hydrolases (3)
- A61P 5/38: of the suprarenal hormones
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- A61P 7/06: Antianaemics
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- C12N 15/867: Retroviral vectors
- C12N 2501/02: Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
- C12N 2510/00: Genetically modified cells
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2810/6081: rhabdoviridae, e.g. VSV
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

The present invention provides methods and compositions for improving the efficacy of viral transduction of cells. More particularly, the present invention provides methods and materials useful for safely and reliably improving the efficiency of methods for transducing cells, such as human hematopoietic stem cells (HSC), with viruses and/or viral vectors. The compositions and methods are useful for therapeutic indications amenable to treatment with hematopoietic stem cell gene therapies.

## Claims

### Claim 1

A method for increasing the lentiviral transduction efficiency of CD34+ hematopoietic stem and/or progenitor cells comprising:
contacting the CD34+ hematopoietic stem and/or progenitor cells with a culture medium comprising a lentivirus and prostaglandin E2 (PGE2), 16,16-dimethyl PGE2, or an analogue thereof in vitro,
wherein the lentiviral transduction efficiency is increased in the CD34+ hematopoietic stem and/or progenitor cells contacted with the culture medium compared to the lentiviral transduction efficiency of CD34+ hematopoietic stem and/or progenitor cells contacted with a culture medium comprising the lentivirus in the absence of PGE2, 16,16-dimethyl PGE2, or an analogue thereof.

### Claim 2 (depends on 1)

The method of claim 1, wherein the CD34+ hematopoietic stem and/or progenitor cells are CD34+ hematopoietic stem cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the CD34+ hematopoietic stem and/or progenitor cells are CD34+ hematopoietic progenitor cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the CD34+ hematopoietic stem and/or progenitor cells are CD34+ hematopoietic stem and progenitor cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein a) at least 50% of the CD34+ hematopoietic stem or progenitor cells are transduced; b) at least 75% of the CD34+ hematopoietic stem or progenitor cells are transduced; or c) at least 90% of the CD34+ hematopoietic stem or progenitor cells are transduced.

### Claim 6 (depends on 1)

The method of claim 1, wherein the medium further comprises a histone deacetylase (HDAC) inhibitor.

### Claim 7 (depends on 6)

The method of claim 6, wherein the HDAC inhibitor is selected from the group consisting of: Trichostatin A (TSA), valproic acid (VPA), sodium butyrate, suberoylanilide hydroxamic acid (SAHA), sodium phenylbutyrate, depsipeptide, trapoxin (TPX), cyclic hydroxamic acid-containing peptide 1 (CHAP1), MS-275, LBH589, and PXD-101.

### Claim 8 (depends on 1)

The method of claim 1, wherein the lentivirus is a Human immunodeficiency virus (HIV) virus.

### Claim 9 (depends on 1)

The method of claim 1, wherein the lentivirus is pseudotyped with a vesicular stomatitis virus G-protein (VSV-G) envelope protein.

### Claim 10 (depends on 1)

The method of claim 1, wherein the CD34+ hematopoietic stem and/or progenitor cells are contacted with the culture medium comprising the lentivirus and PGE2, 16,16-dimethyl PGE2, or an analogue thereof for at least twenty-four hours.

### Claim 11 (depends on 1)

The method of claim 1, wherein the lentivirus comprises: a) a left (5′) lentiviral long terminal repeat (LTR) comprising a heterologous promoter; b) an expression control sequence operably linked to a gene of interest; and c) a right (3′) lentiviral SIN LTR.

### Claim 12 (depends on 1)

The method of claim 1, wherein the lentivirus comprises: a) a left (5′) HIV-1 LTR comprising a CMV promoter; b) a Psi packaging sequence (Ψ+); c) an HIV-1 central polypurine tract/DNA flap (cPPT/FLAP); d) a rev response element (RRE); e) a β-globin promoter and a β-globin locus control region (LCR) operably linked to a gene of interest; and f) a right (3′) lentiviral SIN LTR that comprises i) one or more insulator elements, or ii) a rabbit β-globin polyA (rβgpA) sequence.

### Claim 13 (depends on 1)

The method of claim 1, wherein the lentivirus comprises: a) a left (5′) HIV-1 LTR comprising a CMV promoter; b) a Psi (Ψ+) packaging signal; c) a cPPT/FLAP; d) an RRE; e) a myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer-binding site substituted (MND) promoter, operably linked to a polynucleotide encoding a human ATP-binding cassette, sub-family D, member 1 (ABCD1) polypeptide; f) a right (3′) HIV-1 SIN LTR; and g) a rβgpA sequence.

### Claim 14

A method for increasing the lentiviral transduction efficiency of CD34+ hematopoietic stem and/or progenitor cells comprising:
contacting the CD34+ hematopoietic stem and/or progenitor cells with a culture medium comprising a lentivirus and PGE2 in vitro,
wherein the lentiviral transduction efficiency is increased in the CD34+ hematopoietic stem and/or progenitor cells contacted with the culture medium compared to the lentiviral transduction efficiency of CD34+ hematopoietic stem and/or progenitor cells contacted with a culture medium comprising the lentivirus in the absence of PGE2.

### Claim 15 (depends on 14)

The method of claim 14, wherein a) at least 50% of the CD34+ hematopoietic stem or progenitor cells are transduced; b) at least 75% of the CD34+ hematopoietic stem or progenitor cells are transduced; or c) at least 90% of the CD34+ hematopoietic stem or progenitor cells are transduced.

### Claim 16 (depends on 14)

The method of claim 14, wherein the lentivirus is an HIV virus.

### Claim 17 (depends on 14)

The method of claim 14, wherein the lentivirus is pseudotyped with a VSV-G envelope protein.

### Claim 18 (depends on 14)

The method of claim 14, wherein the CD34+ hematopoietic stem and/or progenitor cells are contacted with the culture medium comprising the lentivirus and PGE2 for at least twenty-four hours.

### Claim 19 (depends on 14)

The method of claim 14, wherein the lentivirus comprises: a) a left (5′) lentiviral LTR comprising a heterologous promoter; b) an expression control sequence operably linked to a gene of interest; and c) a right (3′) lentiviral SIN LTR.

### Claim 20 (depends on 14)

The method of claim 14, wherein the lentivirus comprises: a) a left (5′) HIV-1 LTR comprising a CMV promoter; b) a Psi packaging sequence (Ψ+); c) an HIV-1 cPPT/FLAP; d) an RRE; e) a β-globin promoter and a β-globin LCR operably linked to a gene of interest; and f) a right (3′) lentiviral SIN LTR that comprises i) one or more insulator elements, or ii) a rβgpA sequence.

### Claim 21 (depends on 14)

The method of claim 14, wherein the lentivirus comprises: a) a left (5′) HIV-1 LTR comprising a CMV promoter; b) a Psi (Ψ+) packaging signal; c) a cPPT/FLAP; d) an RRE; e) an MND promoter, operably linked to a polynucleotide encoding a human ABCD1 polypeptide; f) a right (3′) HIV-1 SIN LTR; and g) a rβgpA sequence.

### Claim 22

A method for increasing the lentiviral transduction efficiency of CD34+ hematopoietic stem and/or progenitor cells comprising:
contacting the CD34+ hematopoietic stem and/or progenitor cells with a culture medium comprising a lentivirus and 16,16-dimethyl PGE2 in vitro,
wherein the lentiviral transduction efficiency is increased in the CD34+ hematopoietic stem and/or progenitor cells contacted with the culture medium compared to the lentiviral transduction efficiency of CD34+ hematopoietic stem and/or progenitor cells contacted with a culture medium comprising the lentivirus in the absence of 16,16-dimethyl PGE2.

### Claim 23 (depends on 22)

The method of claim 22, wherein a) at least 50% of the CD34+ hematopoietic stem or progenitor cells are transduced; b) at least 75% of the CD34+ hematopoietic stem or progenitor cells are transduced; or c) at least 90% of the CD34+ hematopoietic stem or progenitor cells are transduced.

### Claim 24 (depends on 22)

The method of claim 22, wherein the lentivirus is an HIV-1 virus.

### Claim 25 (depends on 22)

The method of claim 22, wherein the lentivirus is pseudotyped with a VSV-G envelope protein.

### Claim 26 (depends on 22)

The method of claim 22, wherein the CD34+ hematopoietic stem and/or progenitor cells are contacted with the culture medium comprising the lentivirus and 16,16-dimethyl PGE2 for at least twenty-four hours.

### Claim 27 (depends on 22)

The method of claim 22, wherein the lentivirus comprises: a) a left (5′) lentiviral LTR comprising a heterologous promoter; b) an expression control sequence operably linked to a gene of interest; and c) a right (3′) lentiviral SIN LTR.

### Claim 28 (depends on 22)

The method of claim 22, wherein the lentivirus comprises: a) a left (5′) HIV-1 LTR comprising a CMV promoter; b) a Psi packaging sequence (Ψ+); c) an HIV-1 cPPT/FLAP; d) an RRE; e) a β-globin promoter and a β-globin LCR operably linked to a gene of interest; and f) a right (3′) lentiviral SIN LTR that comprises i) one or more insulator elements, or ii) a rβgpA sequence.

### Claim 29 (depends on 22)

The method of claim 22, wherein the lentivirus comprises: a) a left (5′) HIV-1 LTR comprising a CMV promoter; b) a Psi (Ψ+) packaging signal; c) a cPPT/FLAP; d) an RRE; e) an MND promoter, operably linked to a polynucleotide encoding a human ABCD1 polypeptide; f) a right (3′) HIV-1 SIN LTR; and g) a rβgpA sequence.