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US-20260124241-A1 - MODIFIED HEMATOPOIETIC STEM CELLS AND PROGENIES THEREOF

US20260124241A1US 20260124241 A1US20260124241 A1US 20260124241A1US-20260124241-A1

Abstract

The present disclosure in various aspects and embodiments relates to cell compositions (and methods for making or using the same) comprising hematopoietic stem cells (HSCs) and/or hematopoietic stem progenitor cells (HSPCs), where the HSCs and/or HSPCs have one or more endogenous genes modified in their expression, to thereby avoid or reduce targeting of these cells by targeted therapies. In an aspect, the disclosure provides such HSCs and/or HSPCs from gene-edited human induced pluripotent stem cells (iPSCs).

Inventors

  • Dhvanit SHAH

Assignees

  • GARUDA THERAPEUTICS, INC.

Dates

Publication Date
20260507
Application Date
20231005

Claims (20)

  1. 1 . A method for preparing a population of hematopoietic stem cells (HSCs) and/or hematopoietic stem progenitor cells (HSPCs), the method comprising: preparing a human induced pluripotent stem cell (iPSC) population; modifying one or more endogenous genes in the iPSC population that are tumor-associated antigens, thereby disrupting the expression of the tumor associated antigen; inducing differentiation of the iPSC population to a CD34+ population; and inducing endothelial-to-hematopoietic transition (EHT) of the CD34+ population for at least 2 days and no more than 12 days, to prepare a population comprising HSCs and/or HSPCs having reduced expression of the tumor-associated antigen.
  2. 2 . The method of claim 1 , wherein the tumor associated antigen is expressed or overexpressed in one or more of cytogenetically normal acute myeloid leukemia (CN-AML), acute myeloid leukemia (AML), acute lymphoid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphoid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.
  3. 3 . The method of claim 2 , wherein the endogenous gene is expressed in immune cells.
  4. 4 - 9 . (canceled)
  5. 10 . The method of claim 1 , wherein at least one of the endogenous genes is selected from CD33, CD19, CD7, CD123, and CD371.
  6. 11 - 25 . (canceled)
  7. 26 . The method of claim 1 , wherein the iPSC population is derived from lymphocytes, cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or human primary tissues.
  8. 27 . (canceled)
  9. 28 . The method of claim 26 , wherein the iPSCs are homozygous for one or more HLA Class I and/or Class II genes.
  10. 29 . The method of claim 28 , wherein the iPSCs are homozygous for HLA-DRB1.
  11. 30 . (canceled)
  12. 31 . The method of claim 28 , wherein the iPSCs are gene-edited to delete one or more HLA Class I genes, delete one or more Class II genes, and/or delete one or more genes governing HLA or MHC expression or presentation capacity.
  13. 32 - 34 . (canceled)
  14. 35 . The method of claim 31 , wherein the iPSCs are: HLA-A neg , homozygous for both HLA-B and HLA-C, HLA-DPB1 neg , HLA-DQB1 neg , and homozygous for HLA-DRB1.
  15. 36 . The method of claim 1 , wherein the disruption of the one or more endogenous genes is generated by introducing (a) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease, and (b) a nucleic acid molecule encoding a guide RNA (gRNA) directing the mutation or deletion of nucleotide sequences of the endogenous gene by the Cas9 endonuclease.
  16. 37 - 39 . (canceled)
  17. 40 . The method of claim 36 , wherein the endothelial-to-hematopoietic transition (EHT) generates an HSC population comprising one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem progenitor cells.
  18. 41 - 45 . (canceled)
  19. 46 . The method of claim 1 , wherein the induction of endothelial-to-hematopoietic transition (EHT) comprises Piezol activation; wherein the Piezol activation is by contacting the iPSCs or cells derived from the iPSCs, with one or more Piezol agonists.
  20. 47 - 51 . (canceled)

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/413,550 filed Oct. 5, 2022, the contents of which are hereby incorporated by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on Sep. 26, 2023, is named GRU-0010PC_Sequence_Listing.xml and is 30,045 bytes in size. BACKGROUND An ideal antigen for targeted therapy is a disease-specific antigen, for example, a cancer-specific antigen. A cancer-specific antigen is an antigen that is exclusively expressed in malignant cells, thereby providing a unique target that will lead to maximal disease elimination with minimal off-target toxicity. However, such disease antigens are rare, as most antigens that are expressed by diseased or malignant cells are also found in normal tissues. For example, targeting the myeloid marker CD33 (e.g., using CAR-T cells) in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. To circumvent the loss or destruction of the normal cells, hematopoietic stem cells (HSCs) that have been genetically modified to disrupt expression of the targeted antigen provide a promising therapeutic approach to replenish normal cells that are lost or damaged due to the destruction of normal cells. However, methods for making clinically relevant numbers of genetically-modified HSCs, and/or cell lineages therefrom, and having clinically advantageous phenotypes, remains a significant hurdle. There is a need to generate reliable, off-the-shelf, scalable HSCs (and progenies thereof) with one or more genetic modifications of cancer-associated antigens to treat diseases including but not limited to hematological cancers. In various aspects and embodiments, the invention meets these objectives. SUMMARY OF THE DISCLOSURE The present disclosure in various aspects and embodiments relates to cell compositions (and methods for making or using the same) comprising hematopoietic stem cells (HSCs) where the HSCs have one or more endogenous genes modified in their expression, to thereby avoid or reduce targeting of these cells by targeted therapies. In an aspect, the disclosure provides such HSCs from gene-edited human induced pluripotent stem cells (iPSCs). In one aspect, the present disclosure provides a method for preparing a population of HSCs and/or hematopoietic stem progenitor cells (HSPCs) that are useful for replenishing hematopoietic cells in subjects undergoing a therapy that targets for destruction one or more hematopoietic lineages. The method comprises preparing a human iPSC population and modifying one or more endogenous genes in the iPSC population. Generally, the one or more endogenous genes comprise one or more cancer-associated antigens, and thus the present disclosure involves disrupting the expression of the cancer associated antigen(s) in the iPSC-derived HSCs or HSPCs. The method further comprises differentiating the iPSC population to a CD34+; population (e.g., recovered from dissociated embryoid bodies) and inducing endothelial-to-hematopoietic transition (ELT) of the CD34+ population to prepare a population comprising HSCs and/or HSPCs having reduced expression of the cancer-associated antigen. When these HSCs and/or HSPCs (or a cell population derived therefrom) are administered in connection with a cancer therapy that targets the cancer-associated antigen, targeting of the HSCs and/or HSPCs or their progeny can be reduced or avoided entirely. In embodiments, the one or more endogenous genes are selected from CD33, CD19, CD7, CD123, and CD371, among others. The HSCs and/or HSPCs can be derived from gene-edited iPSCs. In various embodiments, the iPSCs are prepared by reprogramming somatic cells, such as (without limitation) CD34+ cells isolated from peripheral blood. In embodiments, the iPSCs can be further gene edited to delete one or more of HLA-A, HLA-B, and HLA-C, and to delete one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, the iPSCs retain expression of at least one HLA Class I (e.g., of HLA-A, HLA-B, HLA-C, and ILA-E) and at least one HLA Class II complex. In certain embodiments, iPSCs are homozygous for at least one retained Class I and Class II loci. In some embodiments, the iPSCs are gene edited to be HLA-Aneg, homozygous for both HLA-B and HLA-C, and HLA-DPB1neg and HLA-DQB1neg. In some embodiments, the iPSCs are further homozygous for HLA-DRB1. In some embodiments, the process of producing a population comprising HSCs and/or HSPCs can comprise generating CD34+-enriched cells from the differentiated pluripotent stem cells (e.g., EBs) and inducing endothelial-to-hematopoietic differentiation. HSCs comprising relatively high frequency of LT-HSCs can be generated from the cell populations using various stimuli or factors, i