Regulation of gene expression using engineered nucleases

• US 10,563,184 B2
• Filed: 02/21/2019
• Issued: 02/18/2020
• 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.
• Primary Examiner(s): Hill, Kevin K
• Application Number:
  US16/281,751
• Publication Number:
  US 20190177709A1
• Time in Patent Office: 362 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 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.

2. One or more isolated polynucleotides encoding left and right ZFNs according to claim 1.

3. The polynucleotide of claim 2, wherein the polynucleotide is mRNA.

4. The polynucleotide of claim 3, comprising SEQ ID NO:

28 or SEQ ID NO;

30.

5. An isolated cell comprising one or more polynucleotides of claim 2, wherein the zinc finger nuclease is expressed in the cell.

6. The cell of claim 5, wherein the cell is a stem cell or precursor cell.

7. The cell of claim 6, wherein the cell is a human cell.

8. The cell of claim 5, wherein the genome of the cell is modified by the zinc finger nuclease.

9. The cell of claim 8, wherein the genomic modification is selected from the group consisting of insertions, deletions and combinations thereof.

10. The cell of claim 8, wherein the genomic modification comprises an indel in a BCL11A locus.

11. An isolated population of genetically modified cells produced from the cell of claim 10, wherein less than 0.15% genetic modifications are outside the BCL11A locus.

12. An isolated partially or fully differentiated cell descended from the isolated population of genetically modified cells of claim 11.

13. The isolated population of genetically modified cells of claim 11, wherein the cells exhibits increased expression of gamma and/or beta globin as compared to cells without the genomic modification.

14. A pharmaceutical composition comprising the cell of claim 13.

15. A method of modifying an endogenous BCL11A enhancer sequence in a cell, the method comprising administering one or more polynucleotides of claim 2 to the cell such that the endogenous BCL11A enhancer sequence is modified.

16. The method of claim 15, further comprising introducing an exogenous sequence into the cell such that the exogenous sequence is inserted into the endogenous BCL11A enhancer sequence.

17. The method of claim 15, wherein the modification comprises a deletion.

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

administering the cells of claim 13 to the subject.

19. The method of claim 18, wherein the subject is a human and the cells are human stem cells or human precursor cells.

20. The method of claim 19, wherein the cells are infused into the patient and the cells engraft, differentiate and mature in the subject.

21. The method of claim 18, wherein the subject has a hemoglobinopathy.

22. The method of claim 21, wherein the hemoglobinopathy is a beta-thalassemia or sickle cell disease.

23. A method of producing a genetically modified cell comprising a genomic modification within an endogenous BCL11A enhancer sequence, the method comprising the steps of:

a) contacting a cell with the one or more polynucleotides according to claim 2;

b) subjecting the cell to conditions conducive to expressing the fusion protein from the polynucleotide; and

c) modifying the endogenous BCL11A enhancer sequence with the expressed fusion protein sufficient to produce the genetically modified cell.

24. The method of claim 23, further comprising stimulating the cell with at least one cytokine.

25. A kit comprising one or more polynucleotides of claim 2.

26. A kit comprising one or more cells of claim 5.