Methods and compositions for gene editing in hematopoietic stem cells

Patent metadata

Field	Value
Publication number	US-10548922-B2
Application number	US-201815970605-A
Country	US
Kind code	B2
Filing date	May 3, 2018
Priority date	Nov 4, 2015
Publication date	Feb 4, 2020
Grant date	Feb 4, 2020

Abstract

The present invention relates to compositions and methods of generating modified hematopoietic stem or progenitor cells. One aspect of the invention includes a modified hematopoietic stem or progenitor cell comprising a nucleic acid capable of decreasing expression of an endogenous gene or a portion thereof, wherein the endogenous gene encodes a polypeptide comprising an antigen domain targeted by a chimeric antigen receptor (CAR). Another aspect of the invention includes a method for generating a modified hematopoietic stem or progenitor cell. Also included are methods and pharmaceutical compositions comprising the modified cell for adoptive therapy and treating a condition, such as an autoimmune disease or cancer. In certain embodiments, the invention includes a modified hematopoietic stem or progenitor cell comprising a nucleic acid capable of decreasing expression of CD33. In certain embodiments, the invention includes compositions and methods for treating cancer comprising administering CD33 CAR-T cells and modified hematopoietic stem or progenitor cells that are resistant to CD33-targeted therapy.

First claim

What is claimed is:

1. A method for generating a population of modified human hematopoietic stem and progenitor cells (HSPCs) that are resistant to a CD33 CAR-T therapy, comprising:
introducing into the HSPCs a CRISPR system comprising a guide nucleic acid, wherein the CRISPR system produces an insertion and/or deletion in an endogenous CD33 coding sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 4, and wherein the insertion and/or deletion is capable of downregulating gene expression of CD33,
thereby generating the population of modified human HSPCs.

2. The method of claim 1 , wherein the CRISPR system further comprises a homology-directed repair (HDR) template comprising a single-stranded oligonucleotide donor (ssODN) sequence.

3. The method of claim 2 , wherein the ssODN sequence comprises (i) the nucleotide sequence set forth in SEQ ID NO: 4 with an extra adenine inserted or (ii) the nucleotide sequence set forth in SEQ ID NO: 5.

4. The method of claim 1 , wherein the HSPCs are human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

5. The method of claim 1 , wherein the HSPCs are CD34+ HSPCs.

6. A method for generating a population of modified human hematopoietic stem and progenitor cells (HSPCs) that are resistant to a CD33 CAR-T therapy, comprising introducing into the HSPCs a CRISPR system comprising:
a guide nucleic acid; and
a single-stranded oligonucleotide donor (ssODN) sequence, wherein the ssODN sequence comprises the nucleotide sequence set forth in SEQ ID NO:5,
wherein the CRISPR system produces an insertion and/or deletion in an endogenous CD33 coding sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 4, and wherein the insertion and/or deletion is capable of downregulating gene expression of CD33,
thereby generating the population of modified human HSPCs.

7. The HSPCs of claim 6 , wherein the HSPCs are human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

8. The HSPCs of claim 6 , wherein the HSPCs are CD34+ HSPCs.

9. A population of modified human hematopoietic stem and progenitor cells (HSPCs) that are resistant to a CD33 CAR-T therapy, wherein the HSPCs comprise an insertion and/or deletion in an endogenous CD33 coding sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 4, wherein the insertion and/or deletion is mediated by a CRISPR system comprising a guide nucleic acid, and wherein the insertion and/or deletion is capable of downregulating gene expression of CD33.

10. The HSPCs of claim 9 , wherein the CRISPR system further comprises a homology-directed repair (HDR) template comprising a single-stranded oligonucleotide donor (ssODN) sequence.

11. The HSPCs of claim 10 , wherein the ssODN sequence comprises (i) the nucleotide sequence set forth in SEQ ID NO:4 with an extra adenine inserted or (ii) the nucleotide sequence set forth in SEQ ID NO:5.

12. The HSPCs of claim 9 , wherein the HSPCs are autologous human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

13. The HSPCs of claim 9 , wherein the HSPCs are CD34+ HSPCs.

14. A method of treating acute myeloid leukemia (AML) in a subject in need thereof, the method comprising:
administering to the subject a CD33-targeted therapy comprising a CD33 CAR-T cell; and
administering to the subject a population of modified human hematopoietic stem and progenitor cells (HSPCs) that are resistant to the CD33-targeted therapy,
wherein the HSPCs comprise an insertion and/or deletion in an endogenous CD33 coding sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 4 that is introduced by a CRISPR system comprising a guide nucleic acid, and wherein the insertion and/or deletion is capable of downregulating gene expression of CD33,
thereby treating AML in the subject.

15. The method of claim 14 , wherein the CRISPR system further comprises a homology-directed repair (HDR) template.

16. The method of claim 15 , wherein the HDR template comprises a single-stranded oligonucleotide donor (ssODN) sequence.

17. The method of claim 16 , wherein the ssODN sequence comprises (i) the nucleotide sequence set forth in SEQ ID NO:4 with an extra adenine inserted or (ii) the nucleic acid sequence set forth in SEQ ID NO:5.

18. The method of claim 14 , wherein the HSPCs are administered to the subject prior to the CD33-targeted therapy.

19. The method of claim 14 , wherein the HSPCs are autologous to the subject and obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

20. The method of claim 14 , wherein the HSPCs are CD34+ HSPCs.

Assignees

• Univ Pennsylvania

Inventors

• Gill Saar
• Kim Miriam

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• A61P35/02

  specific for leukemia · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• A61K45/05

  Immunological preparations stimulating the reticulo-endothelial system, e.g. against cancer · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title