DELIVERY METHODS AND COMPOSITIONS FOR NUCLEASE-MEDIATED GENOME ENGINEERING IN HEMATOPOIETIC STEM CELLS

• US 20150174169A1
• Filed: 10/16/2014
• Published: 06/25/2015
• Est. Priority Date: 10/17/2013
• Status: Active Grant

• Current Assignee: Fondazione Telethon, Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Therapeutics, Inc.
• Original Assignee: Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Genovese, Pietro, Gregory, Philip D., Holmes, Michael C., Lombardo, Angelo, Naldini, Luigi
• Granted Patent: US 10,117,899 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic cell.

Claims

1. A method for targeted integration into purified hematopoietic stem cells (HSC) and/or progenitor cells (PC), said method comprising the following steps:

(a) delivering a non-integrating lentiviral (IDLV) donor nucleic acid to the HSC and/or PC cells;

(b) culturing the cells obtained by step (a); and

(c) delivering at least one zinc finger nuclease to the cells obtained by step (b) such that the donor nucleic acid is integrated into the genome.

2. The method of claim 1, further comprising treating the HSC and/or PC cells with a compound that preserves stemness of the cells, preferably with an aryl Hydrocarbon Receptor Antagonist such as StemRegenin 1 (SR1) and/or 16.16 dimethyl-prostaglandin E2 (dmPGE2).

3. The method according to claim 1, wherein the at least one zinc finger nuclease is delivered in mRNA form.

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

5. The method according to claim 1, wherein the donor nucleic acid is an exogenous sequence, preferably an exogenous sequence flanked by regions of homology to an endogenous locus, more preferably an exogenous sequence flanked by regions of homology to an endogenous safe harbor locus or a locus downstream the regulatory regions of an endogenous gene.

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

7. 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.

8. The method according to claim 1, wherein the method comprises the following steps:

(a) infecting the HSC and/or PC cells with an IDLV vector comprising an exogenous sequence flanked by regions of homology to an endogenous locus;

(b) culturing the cells of step (a) for 1 to 3 days; and

(c) electroporating mRNA encoding a pair of zinc finger nucleases (ZFNs) specific for the endogenous locus into the cells of step (b) such that the exogenous sequence is integrated into the endogenous locus.

9. The method according to claim 8, 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.

10. The method according to claim 8, wherein the HSC and/or PC cells are selected from the group comprising CD34+ cells, CD34+CD133+ cells, CD34+CD133−

cells and CD34+CD133+CD90+ cells, or combination thereof.

11. A genetically modified HSC or PC or a population of genetically modified HSCs and/or PCs obtained by the method of claim 8.

12. A pharmaceutical composition comprising the genetically modified HSC or PC or population of genetically modified HSCs and/or PCs according to claim 11 and a pharmaceutically acceptable carrier, excipient or diluents.

13. A method of treating a disorder in a subject in need thereof, the method comprising administering the genetically modified HSC or PC of claim 11 to the subject.

14. A method of treating a disorder in a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 12 to the subject.

15. A method of engrafting genetically modified HSC and/or PC cells into a host organism, the method comprising administering the HSC or PC according to claim 11.

16. A method of engrafting genetically modified HSC and/or PC cells into a host organism, the method comprising administering the pharmaceutical composition according to claim 12.