REGULATION OF GENE EXPRESSION USING ENGINEERED NUCLEASES

• US 20200131490A1
• Filed: 01/09/2020
• Published: 04/30/2020
• Est. Priority Date: 08/24/2016
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Miller, Jeffrey C., Rebar, Edward J.
• Granted Patent: US 11,845,965 B2
• Time in Patent Office: Days
• 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 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• 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. An isolated population of cells comprising genetically modified stem cells in which the BCL11A locus of the cells is specifically modified by a zinc finger nuclease (ZFN) comprising left and right ZFNs, the left ZFN comprising the amino acid sequence as shown in SEQ ID NO:

29 and the right ZFN comprising the amino acid sequence as shown in SEQ ID NO;

31 and further wherein the genetically modified stem cells exhibits increased expression of gamma (γ

) and/or beta (β

) globin as compared to cells without the genomic modification.

2. The isolated population of cells according to claim 1, wherein the left ZFN is encoded a polynucleotide according to SEQ ID NO:

28 and the right ZFN is encoded by a polynucleotide according to SEQ ID NO;

30.

3. The isolated population of cells according to claim 1 comprising CD34+ hematopoietic stem cells.

4. The isolated population of cells according to claim 1, wherein the cells are human cells.

5. The isolated population of cells according to claim 1, wherein the genomic modification in the BC11A locus comprises an insertion and/or a deletion.

6. An isolated population of genetically modified cells propagated, descended or differentiated from the genetically modified stem cells according to claim 1.

7. The isolated population of cells according to claim 1, wherein the ratio of γ

globin to β

globin is increased as compared to cells without the genetic modification.

8. The isolated population of cells according to claim 1, wherein the ratio of γ

globin to alpha (α

) globin is increased as compared to cells without the genetic modification.

9. The isolated population of cells according to claim 1, wherein γ

globin mRNA and/or protein levels are increased as compared to cells without the genetic modification.

10. The isolated population of cells according to claim 9, wherein γ

globin mRNA levels are increased at least 1.5 to 2-fold.

11. The isolated population of cells according to claim 9, wherein γ

globin protein levels are increased at least 3- to 4-fold.

12. A pharmaceutical composition comprising the cells according to claim 1.

13. A method of increasing globin production in a subject, the method comprising:

administering the pharmaceutical composition according to claim 12 to the subject.

14. The method of claim 13, wherein the subject is a human patient and the cells are human stem cells and/or human precursor cells.

15. The method of claim 14, wherein the cells are infused into the patient and the cells engraft, differentiate and mature in the subject.

16. The method of claim 13, wherein the subject has a hemoglobinopathy.

17. The method of claim 13, wherein the hemoglobinopathy is a beta-thalassemia or sickle cell disease.

18. A kit comprising the pharmaceutical composition according to claim 12.