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Modified pluripotent cells

a technology of pluripotent cells and modified cells, applied in the field of regenerative cell therapy, can solve the problems that rh(d) and rh(c) antigens confer significant risk of hemolytic disease of fetus and newborns

Pending Publication Date: 2022-07-07
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]In a specific embodiment, the invention provides hypoimmune pluripotent ABO blood type O Rhesus Factor negative (HIPO−) cells that evade rejection by the host allogeneic immune system and avoid blood antigen type rejection. In some embodiments, the HIPO− cells are engineered to reduce or eliminate HLA-I and HLA-II expression, increase expression of an endogenous protein that reduces the susceptibility of the pluripotent cell to macrophage phagocytosis, and comprise a universal blood group O Rh− (“O−”) blood type. The universal blood type may be achieved by eliminating ABO blood group A and B antigens and Rh factor expression, or by starting with an O− cell line. These novel HIPO− cells evade host immune rejection because they have an impaired antigen presentation capacity, protection from innate immune clearance, and lack blood group rejection.
[0026]The invention also provides one or more cells that are derived or differentiated from the induced ABO blood group O− Rh factor (−) pluripotent (e.g., PSCO−, iPSCO, ESCO−) cells described herein. These cells comprise an ABO blood group type O (O) and a Rhesus Factor (Rh) blood type negative (−), rendering them less susceptible to rejection when transplanted into a subject when compared with cells that are ABO blood group or Rh factor mismatched to the subject.

Problems solved by technology

Antibodies to Rh antigens can be involved in hemolytic transfusion reactions and antibodies to the Rh(D) and Rh(c) antigens confer significant risk of hemolytic disease of the fetus and newborn.

Method used

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  • Modified pluripotent cells
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Examples

Experimental program
Comparison scheme
Effect test

example 1

n of Human iPSCs

[0224]The Human Episomal iPSC Line was derived from CD34+ cord blood (Cat. No. A33124, Thermo Fisher Scientific) using a three-plasmid, seven-factor (SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen) EBNA-based episomal system from ThermoFisher. This iPSC line is considered to have a zero footprint as there was no integration into the genome from the reprogramming event. It has been shown to be free of all reprogramming genes. The iPSCs have a normal XX karyotype and endogenous expression of pluripotent markers like OCT4, SOX2, NANOG (as shown by RT-PCR) OCT4, SSEA4, TRA-1-60 and TRA-1-81 (as shown by ICC). In directed differentiation and teratoma analyses, these hiPSCs retained their differentiation potential for the ectodermal, endodermal, and mesodermal lineages. In addition, vascular, endothelial, and cardiac lineages were derived with robust efficiencies.

[0225]Several gene-delivery vehicles for iPSC generation were successfully used, in...

example 2

n of Human HIP Cells

[0229]Hypoimmune pluripotent (HIP) cells were generated as disclosed in WO2018 / 132783 and U.S. Prov. App. Nos. 62 / 698,941, 62 / 698,965, 62 / 698,973, 62 / 698,978, 62 / 698,981, and 62 / 698,984, each of which are incorporated by reference herein in their entirety. Human Cas9 iPSC underwent 2 gene-editing steps. In the first step, CRISPR technology was performed by a combined targeting of the coding sequence of human beta-2-microglobuline (B2M) gene with the CRISPR sequence 5′-CGTGAGTAAACCTGAATCTT-3′ and the coding sequence of human CIITA gene with the CRISPR sequence 5′-GATATTGGCATAAGCCTCCC-3′. Linearized CRISPR sequence with T7 promoter was used to synthesize gRNA as per the kit's instructions (MEGAshortscript T7 Transcription Kit, Thermo Fisher). The collected in-vitro transcription (IVT) gRNA was then purified via the MEGAclear Transcription Clean-Up Kit. For IVT gRNA delivery, singularized cells were electroporated with 300 ng IVT gRNA using a Neon electroporation sy...

example 3

Rejection in Rhesus Macaque Monkeys and Pigs

[0231]10 million hypoimmunogenic B2M− / − CIITA− / − rhesus CD47 tg iPSC derived endothelial cells (expressing luciferase) were injected subcutaneously into rhesus macaque monkeys and cells were longitudinally followed using bioluminescence imaging; each animal was injected intravenously with 100 mg / ml D-luciferin (Perkin Elmer, San Jose, Calif.) via a peripheral vessel for in vivo imaging using a Xenogen IVIS® 200 Series imaging system (Caliper Life Sciences, Alameda, Calif., Cat. No. 122799). Our pre-transplant screening assays and validation studies predicted that the cells would not be rejected, resulting in stable BLI signals. The BLI-signals decreased, however, on day 6 and were not detected by day 16. In addition, “bumps” were observed on the injection sites (data not shown). Blood was drawn from the same monkeys and T cell, cytotoxic T cell, NK cell (FIG. 1A), B cell (DSA; donor specific antibodies), or macrophages activation (FIG. 1B)...

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Abstract

The invention discloses for the first time pluripotent cells, including induced pluripotent stem cells, embryonic stem cells, and hypo-immune pluripotent cells that are ABO blood type O Rhesus Factor negative and evade rejection resulting from blood type antigen mismatch. The invention further provides universally acceptable “off-the-shelf” pluripotent cells and derivatives thereof for generating or regenerating specific tissues and organs.

Description

I. CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 846,399, filed May 10, 2019 and U.S. Provisional Application No. 62 / 855,499, filed May 31, 2019, each of which are incorporated herein by reference in their entirety.II. FIELD OF THE INVENTION[0002]The invention relates to regenerative cell therapy. In some embodiments, the regenerative cell therapy comprises transplanting cell lines into patients in need thereof. In some embodiments, the cell lines comprise O Rh− hypoimmunogenic pluripotent cells. In some embodiments, the regenerative cell therapy reduces the propensity for the cell transplant recipient's immune system to reject allogeneic material. In some embodiments, the regenerative cell therapy is used in the treatment of injured organs and tissue. In some embodiments, the regenerative cell therapy is used for rehabilitating damaged tissues after myocardial infarction.III. BACKGROUND ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/0735C12N15/113C12N5/074
CPCC12N5/0606A61K35/545C12N5/0696C12N15/1138C12N9/22C12N2310/20C12N2510/00A61K35/34C12N5/0657C12N5/069C12N2506/45C12N2506/11A61K2035/124C12N2800/80C12N15/113
Inventor SCHREPFER, SONJADEUSE, TOBIAS
Owner RGT UNIV OF CALIFORNIA
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