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US-20260124246-A1 - ADHESION MOLECULE INHIBITION FOR STEM CELL THERAPIES

US20260124246A1US 20260124246 A1US20260124246 A1US 20260124246A1US-20260124246-A1

Abstract

An in vitro method of preparing a population of hypoimmune mammalian stem cells includes providing a population of isolated mammalian stem cells, wherein the isolated mammalian stem cells express a cell adhesion molecule; and modifying the expression of the cell adhesion molecule in the population of isolated mammalian stem cells to decrease or knockout expression of the cell adhesion molecule and provide the population of hypoimmune mammalian cells. The population of isolated mammalian stem cells can be pluripotent stem cells, or embryonic stem cells, and can be human or non-human stem cells.

Inventors

  • Matthew Brown

Assignees

  • WISCONSIN ALUMNI RESEARCH FOUNDATION

Dates

Publication Date
20260507
Application Date
20231025

Claims (20)

  1. 1 . An in vitro method of preparing a population of hypoimmune mammalian stem cells, comprising: providing a population of isolated mammalian stem cells, wherein the isolated mammalian stem cells express a cell adhesion molecule; and modifying the expression of the cell adhesion molecule in the population of isolated mammalian stem cells to decrease or knockout expression of the cell adhesion molecule and provide the population of hypoimmune mammalian stem cells.
  2. 2 . The method of claim 1 , wherein the wherein the population of hypoimmune mammalian cells is less immunogenic than the corresponding population of isolated mammalian stem cells.
  3. 3 . The method of claim 1 , wherein the one or more mammalian stem cells are induced pluripotent stem cells, or embryonic stem cells.
  4. 4 . The method of claim 1 , wherein the one or more mammalian stem cells are human stem cells or non-human stem cells.
  5. 5 . The method of claim 1 , wherein the expression of the cell adhesion molecule is modified by gene editing of a gene for the cell adhesion molecule, or by inhibition of expression with an inhibitory RNA.
  6. 6 . The method of claim 1 , wherein the cell adhesion molecule comprises ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, VCAM, MADCAM-1, P-selectin, L-selectin, E-selectin, or a combination thereof.
  7. 7 . The method of claim 1 , wherein the population of isolated mammalian stem cells has reduced or lack expression of an HLA Class I molecule, reduced or lack expression of an HLA Class II molecule, reduced or lack expression of a beta-2 microglobulin, increased expression of CD47, increased expression of PDL1, increased expression of secretin, increased expression CTLA4, or a combination thereof.
  8. 8 . The method of claim 1 , further comprising isolating, expanding, and/or differentiating, or any combination thereof, the population of hypoimmune mammalian stem cells.
  9. 9 . The method of claim 8 , wherein the population of hypoimmune mammalian stem cells differentiated to endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or islet cells.
  10. 10 . The method of claim 9 , wherein the differentiated cell is a cardiomyocyte or a neuron.
  11. 11 . A population of hypoimmune mammalian stem cells produced by the method of claim 1 .
  12. 12 . A population of hypoimmune mammalian stem cells, wherein expression of a cell adhesion molecule is decreased or knocked out by modification by gene editing of a gene for the cell adhesion molecule.
  13. 13 . The population of hypoimmune mammalian stem cells of claim 12 , wherein the cell adhesion molecule comprises ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, VCAM, MADCAM-1, P-selectin, L-selectin, E-selectin, or a combination thereof.
  14. 14 . The population of wherein the population of hypoimmune mammalian stem cells of claim 1 , wherein the hypoimmune mammalian stem cells are differentiated to endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or islet cells.
  15. 14 . A graft comprising the population of differentiated hypoimmune mammalian stem cells of claim 14 .
  16. 15 . A method of treating a mammalian subject, comprising introducing to the mammalian subject in need thereof the graft of claim 14 .
  17. 16 . The method of claim 15 , wherein the mammal is a human, a canine, feline, bovine, equine, swine, ovine, or caprine.
  18. 17 . The method of claim 15 , wherein the mammalian subject is in need of treatment for neurological degeneration, diabetes, vascular disease, myocardial disease, or a combination thereof.
  19. 18 . The method of claim 17 , wherein the mammalian subject is in need of treatment for neurological degeneration, and wherein the graft is administered to the central nervous system, and wherein the mammalian subject has Parkinson's disease, Alzheimers disease, stroke, or ALS.
  20. 19 . The method of claim 17 , wherein the mammalian subject is in need of treatment for diabetes, and wherein the composition is systemically administered.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application 63/380,883 filed on Oct. 25, 2022, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT This invention was made with government support under HD090256 and HL134764 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Oct. 22, 2023, is named “SEQ_LIST--107668_109” and is 62.1 KB (63,629 bytes) in size. The Sequence Listing does not go beyond the disclosure in the application as filed. BACKGROUND One challenge in the cell therapy field is immune-mediated rejection of transplanted donor cells. Technologies are being developed to “hide” these cells from immune cells and/or to make the cells express molecules or factors that prevent immune cells from recognizing these transplanted cells. A careful balance must be struck between preventing immune rejection while not allowing unchecked transplanted cell growth leading to tumor development or to all the cells to become a reservoir for viral infection and uncontrolled replication. In particular, pluripotent stem cell (PSC)-derived cell therapies are promising reparative treatments for a variety of cardiovascular diseases that kill over 655,000 Americans each year. PSC-derived grafts have multiple uniquely attractive attributes, such as near-infinite scalability and a lack of passenger lymphocytes, and they may have diminished susceptibility to the acute and chronic allograft rejection that routinely devastates traditional organ transplantations. Furthermore, PSCs are amenable to CRISPR/Cas9-based gene-editing. Rece Multiple research groups have created hypoimmune “universal cells” (e.g., knock-out [KO] of HLA class I and/or II) have been created which are capable of evading recognition by T cells, donor specific antibodies, and/or NK cell-mediated cytotoxicity in short-term studies. Despite these advances, little is known about the long-term tolerance potential of hypoimmune PSC grafts in patients, including whether such a dramatic intervention as total ablation of HLA I+II increases the risk of deleterious effects (e.g., malignancy, uncontrolled viral replication). Thus, there is a need to develop stem cell (e.g., iPSC or embryonic stem cell) therapies that are functional and curative, and that reduce the risk of rejection by the recipient immune system. This will enable long-term graft function and improvement of patient lives. SUMMARY In an aspect, an in vitro method of preparing a population of hypoimmune mammalian stem cells comprises providing a population of isolated mammalian stem cells, wherein the isolated mammalian stem cells express a cell adhesion molecule; and modifying the expression of the cell adhesion molecule in the population of isolated mammalian stem cells to decrease or knockout expression of the cell adhesion molecule and provide the population of hypoimmune mammalian stem cells. Also included is a population of hypoimmune mammalian stem cells produced by the above-described method. In another aspect, included herein is a population of hypoimmune mammalian stem cells, wherein expression of a cell adhesion molecule is decreased or knocked out by modification by gene editing of a gene for the cell adhesion molecule. The hypoimmune mammalian stem cells can be differentiated to endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or islet cells. Further included is a graft comprising the population of differentiated hypoimmune mammalian stem cells and a method of treating a mammalian subject by introducing the graft. BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A and B show an RNA sequencing analysis of wild-type and B2M KO H1 PSC-AEC cell culture models (CMs) of allorejection inflammation. Expression of adhesion marker ICAM-1 in WT H1 PSC-AECs before and after (1A) 18 h co-culture with allogeneic peripheral blood mononuclear cells, and in B2M KO H1 PSC-AECs after (1B) 48 h stimulation of cells alone with Tumor Necrosis Factor alpha (TNFα). FIGS. 2A and B show transplantation of gene-edited PSC-CVTs into immune-deficient mouse hosts. (2A) Representative flow cytometric AEC phenotype (CD34+CXCR4+ cells sub-gated from CD31+CD144+) is shown. (2B) CMs made from B2M KO H1 PSCs were formed into spheroids and transplanted heterotopically into the receptive kidney capsule transplant site of a NBGSW mouse. After 28 days in vivo, the animal was anesthetized and the graft was examined macroscopically, showing robust contraction and neovascularization with human vessels. FIG. 3 shows direct and indirect pathway of alloreactivity in first-generation hypoimmune PSC-AECs. H1 WT and B2M-KO PSCs were differentiated into AEC t