Pluripotent stem cells expressing shRNA/shRNA-miR targeting CTLA-4 or derivatives thereof

A CTLA-4, pluripotent stem cell technology, applied in DNA/RNA fragments, cells modified by introducing foreign genetic material, recombinant DNA technology, etc., can solve tumorigenicity and virus infection, loss of antigen presentation ability, Insufficient immune compatibility, etc., to relieve immune suppression, restore T cell activity, and remove tumor cells

Pending Publication Date: 2022-04-15
FUTURE HOMO SAPIENS INST OF REGENERATIVE MEDICINE CO LTD (FHSR) +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these schemes either have incomplete immune compatibility and still cause allogeneic immune rejection through other channels; or completely eliminate the allogeneic immune rejection response, but make the cells of the donor-derived graft lose the ability to present antigens at the same time. ability, which poses a great risk to recipients for diseases such as tumorigenicity and viral infection

Method used

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  • Pluripotent stem cells expressing shRNA/shRNA-miR targeting CTLA-4 or derivatives thereof
  • Pluripotent stem cells expressing shRNA/shRNA-miR targeting CTLA-4 or derivatives thereof
  • Pluripotent stem cells expressing shRNA/shRNA-miR targeting CTLA-4 or derivatives thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0299] Example 1 Detection method for expressing pluripotent stem cells comprising CTLA-4 repressor molecule exosomes or derivatives thereof

[0300] Knock the protocols of each experimental group in Table 6 and Table 7 into the genomic safety site AAVS1 of iPSCs, MSCs, NSCs, and EBs cells, culture in a 37°C, 0.5% CO2 incubator, collect the culture supernatant, and use the exosome extraction kit (BestBio, lot#BB-3901) extract exosomes in the culture supernatant after culturing pluripotent stem cells expressing exosome-encapsulated CTLA-4 repressor molecules and their derivatives. The culture supernatant was centrifuged at 3000g for 15min at 4°C. After collecting the supernatant, centrifuge at 10,000g for 20min at 4°C. After collecting the supernatant, add extract A at a ratio of 4:1, invert upside down for 1min, and overnight at 4°C. Centrifuge at 10,000 g for 60 min at 4°C to collect the exosome pellet.

[0301] The effect of blocking CTLA-4 repressor molecules expressed by ...

Embodiment 2

[0306] Example 2 Anti-tumor effect of pluripotent stem cells expressing CTLA-4 repressor molecules or derivatives thereof

[0307] Knock the protocols of each experimental group in Table 6-Table 7 into the genomic safety site AAVS1 of iPSCs, MSCs, NSCs, and EBs cells to obtain cells expressing CTLA-4 repressor molecules, using 51 Cr release test was used to test its antitumor effect.

[0308] The N (control) group refers to T cells not treated with the culture supernatant of pluripotent stem cells expressing CTLA-4 repressor molecules.

[0309] Table 9 Effects of CTLA-4 repressor molecules expressed by each experimental group on T cells killing tumor cells

[0310]

[0311]

[0312] Through the above experiments, it can be proved that the exosome-encapsulated CTLA-4 expressed by the pluripotent stem cells prepared in the present invention or their derivatives can effectively inhibit CTLA-4 and play an anti-tumor effect.

Embodiment 3

[0313] Example 3 CTLA-4 repressor molecules are applied in the treatment of various tumors

[0314] We selected B2M and CIITA gene knockout protocol groups (Aa3, Ab3, Ac3, Ad3) in immune-compatible cells (hPSCs, MSCs, NSCs, EBs) for testing.

[0315] In the humanized NSG mouse tumor model, we injected each group of experimental cells to observe its therapeutic effect on RCC kidney cancer, MC colon cancer and HCC liver cancer. In order to avoid immune compatibility problems, the immune cells, hPSCs and hPSCs-derived derivatives we use are all derived from the same person.

[0316] The N (control) group refers to the NSG mouse tumor model that was not injected with the experimental cells. Independent samples T-test (*p<0.01).

[0317] Table 10 Anti-tumor effect of pluripotent stem cells expressing CTLA-4 repressor molecules or derivatives thereof

[0318]

[0319] Through the above experiments, it can be proved that the exosome-encapsulated CTLA-4 expressed by the pluripot...

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Abstract

The invention discloses a pluripotent stem cell for expressing an exosome containing a CTLA-4 repression molecule or a derivative of the pluripotent stem cell. An expression sequence of a CTLA-4 repression molecule and an exosome processing synthesis gene are introduced into the genome of the pluripotent stem cell or the derivative thereof, and the exosome processing synthesis gene can express an exosome containing the CTLA-4 repression molecule. After the CTLA-4 repression molecules expressed by the pluripotent stem cells or the derivatives of the pluripotent stem cells are wrapped by the exosome, the exosome carries the CTLA-4 repression molecules to be combined with target cells and release the CTLA-4 repression molecules contained in the target cells, so that a CTLA-4 pathway is blocked, inhibition is relieved, an immune system is activated, T cell activity is recovered, and tumor cells can be effectively removed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a pluripotent stem cell or a derivative thereof expressing shRNA / shRNA-miR targeting CTLA-4. Background technique [0002] Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), also known as CD152, is a transmembrane protein encoded by the CTLA-4 gene, expressed on activated T cells, and on the surface of T cells The co-stimulatory molecule receptor (CD28) has a high degree of homology and shares B7 molecular ligands (B7-1 / CD80, B7-2 / CD86), and CTLA-4 can negatively down-regulate T cells after binding to B7 molecules It is involved in the negative regulation of the immune response and is therefore an immune co-inhibitory molecule. The mechanism, one is to compete with CD28 to bind B7 or recruit phosphatase to the intracellular domain of CTLA-4 to degrade the signal of T cell receptor (TCR) and CD28, and the other is to reduce the expression of CD80 / CD86 i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113A61K35/28A61K31/7105A61K48/00A61P35/00
Inventor 王淋立陈月花杨建国莫健
Owner FUTURE HOMO SAPIENS INST OF REGENERATIVE MEDICINE CO LTD (FHSR)
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