Delivery methods and compositions for nuclease-mediated genome engineering in hematopoietic stem cells

• US 10,117,899 B2
• Filed: 10/16/2014
• Issued: 11/06/2018
• 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: Fondazione Telethon, Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Therapeutics, Inc.
• Inventors: Genovese, Pietro, Gregory, Philip D., Holmes, Michael C., Lombardo, Angelo Leone, Naldini, Luigi
• Primary Examiner(s): Wehbe, Anne Marie S
• Application Number:
  US14/516,189
• Publication Number:
  US 20150174169A1
• Time in Patent Office: 1,482 Days
• Field of Search: 4353201, 435325, 435455, 424 9321
• 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), 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.

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

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

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

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

6. 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.

7. 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.

8. 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.

9. 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.

10. 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).

11. 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.