# US 10,117,899 B2 · Delivery methods and compositions for nuclease-mediated genome engineering in hematopoietic stem cells

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- Number: US 10,117,899 B2
- Document ID: US10117899B2
- Status: Granted
- Kind: B2
- Application number: 14/516,189
- Filed: 2014-10-16
- Published: 2015-06-25
- Granted: 2018-11-06
- Earliest claimed: 2013-10-17
- Assignees: Sangamo Therapeutics, Inc.; Ospedale San Raffaele S.R.L.; Fondazione Telethon
- Applicants: Sangamo BioSciences, Inc.; Ospedale San Raffaele srl; Fondazione Telethon
- Inventors: Pietro Genovese; Philip D. Gregory; Michael C. Holmes; Angelo Leone Lombardo; Luigi Naldini
- Web page: https://patentgrep.com/patent/US10117899B2

## CPC

- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- C12N 2510/00: Genetically modified cells
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- 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)
- 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 disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic cell.

## Claims

### Claim 1

A method for targeted integration into purified hematopoietic stem cells (HSC) and/or progenitor cells (PC), the method comprising the following steps:
(a) stimulating the cells;
(b) delivering a non-integrating lentiviral (IDLV) donor nucleic acid to the cells one day after stimulation;
(c) culturing the cells obtained by step (b) for one day; and
(d) delivering at least one zinc finger nuclease to the cells obtained by step (c) such that the donor nucleic acid is integrated into the genome, wherein the at least one zinc finger nuclease is delivered in mRNA form.

### Claim 2 (depends on 1)

The method of claim 1, further comprising treating the HSC and/or PC cells with an aryl Hydrocarbon Receptor Antagonist.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the method comprises delivering at least one zinc finger nuclease by electroporation.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the donor nucleic acid is an exogenous sequence.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the nuclease comprises a pair of ZFNs.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the HSC and/or PC cells are selected from the group comprising CD34+ cells, CD34+CD133+ cells, CD34+CD133− cells, CD34+CD133+CD90+ cells, or a combination thereof.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the IDLV vector comprises an exogenous sequence flanked by regions of homology to an endogenous locus and the mRNA is delivered by electroporation.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the exogenous sequence is integrated into an endogenous safe harbor locus or downstream of the regulatory regions of an endogenous gene, such that expression of the exogenous sequence is driven by the endogenous regulatory regions.

### Claim 9 (depends on 7)

The method according to claim 7, wherein the HSC and/or PC cells are selected from the group comprising CD34+ cells, CD34+CD133+ cells, CD 34+CD133− cells and CD34+CD133+CD90+ cells, or combination thereof.

### Claim 10 (depends on 1)

The method of claim 1, further comprising the steps of:
(e) culturing the cells of step (d), and
(f) isolating the cultured cells of step (e).

### Claim 11 (depends on 10)

The method of claim 10, further comprising formulating the isolated cultured cells of step (f) into a pharmaceutical composition and administering the pharmaceutical composition to a subject.