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Allograft tolerance without the need for systemic immune suppression

A technology of allograft and graft, applied in the field of transplantation, can solve problems such as systemic immune suppression

Pending Publication Date: 2020-03-06
SINAI HEALTH SYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In another approach, ES cells were engineered to express PD-L1 and CTLA4-Ig, which improved survival in allogeneic hosts (Rong et al., Cell Stem Cell. 14:121-30 (2014)) , but with the serious limitation that CTLA4-Ig can lead to systemic immune suppression

Method used

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  • Allograft tolerance without the need for systemic immune suppression
  • Allograft tolerance without the need for systemic immune suppression
  • Allograft tolerance without the need for systemic immune suppression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0407] Example 1: Materials and methods

[0408] Construction of vectors expressing target genes essential for allogeneic tolerance

[0409] The plasmid containing the cDNA sequence of the gene involved in allogeneic tolerance was obtained as follows:

[0410] PD-L1: Mount Sinai Hospital, clone #V102001

[0411] FasL: Mount Sinai Hospital, #75719

[0412] Cd47: Mount Sinai Hospital, #V75535

[0413] Cd200: GE Dharmacon, ID#17470

[0414] H2-M3: Mount Sinai Hospital, clone #8188

[0415] Ccl21: Mount Sinai Hospital, clone #V77120

[0416] Mfge8: Mount Sinai Hospital, clone #V72614

[0417] Spi6: Mount Sinai Hospital, clone #V8907

[0418] The Gateway cloning system (Thermo Fisher) was used to generate expression vectors containing these target genes (GOI) or luciferase. Cd47, Ccl21, Mfge8 and Spi6 cDNA were obtained in the form of containing cDNA flanking attB sites. For H2-M3, Cd200, FasL and PD-L1, design primers to amplify the cDNA sequence and add attB sites ( Figure 15 (a)). After PCR...

Embodiment 2

[0433] Example 2: Generation of masked cells

[0434] The transgenes encoding the genes in Table 1 were cloned into an expression vector, and sequence verification was performed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing, all using standard methods known in the art.

[0435] A group of constructs (group 1) containing transgenic Cd47, Cd200, FasL and H2-M3 were transfected into mouse embryonic stem cells derived from the inventor's C57BL / 6 mouse ES line (C2). The existence of the transgene was verified by PCR, and the expression of the expressed protein was recorded by immunohistochemistry ( Figure 1A -D). The second group of constructs (group 2) containing the transgenes Ccl21, Mfge8, TGF-β and Spi6 was transfected into FVB / N-derived ES cells (ES line C2).

[0436] A similar method was used to generate masked B16F10 melanoma cells except that the medium used DMEM medium containing 10% fetal bovine serum (FBS).

Embodiment 3

[0437] Example 3: Screening method for inhibiting T cell activation

[0438] A modified in vitro mixed lymphocyte response (MLR) assay is used to screen transgenic combinations to produce the most effective inhibition of T cell activation. Cell lines transfected with the masked transgene from Example 1 were used. The donor OT-1 splenocytes were labeled with carboxyfluorescein succinimidyl ester CFSE, and 60,000 cells were added to each well of a 96-well plate. Mix ES or melanoma cells with egg cell expressing cells 10:1. Add 10,000 of these to each well of spleen cells. IL-2 was added as a general activator, and T cell proliferation was measured by flow cytometry after 3 days ( Figure 2A-2E ). The cells were initially gated to include only CD8+ cells, and all conditions were set in quadruplicate.

[0439] The negative control (spleen cells only) produced a baseline 6.12% proliferation rate ( Figure 2A ). Wild-type B16 melanoma (+10% egg cell expression) cells resulted in a s...

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Abstract

A cell genetically modified to comprise at least one mechanism for providing a local immunosuppression at a transplant site when transplanted in an allogeneic host, and methods for making and using the same are provided. The cell comprises a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting, and whose function is one or more of: to mitigate antigen presenting cell activation and function; to mitigate graft attacking leukocyte activity or cytolytic function; to mitigate macrophage cytolytic function and phagocytosis of allograft cells; to induce apoptosis in graft attacking leukocytes; to mitigate local inflammatory proteins; and to protect against leukocyte-mediated apoptosis. The transgenes comprise two or more of the followinggenes: PD-L 1, HLAG or H2-M3, Cd47, Cd200, FASLG or Fasl, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6.

Description

[0001] Invention field [0002] The present disclosure generally relates to the field of transplantation. The present disclosure further relates to methods for generating local immune suppression in the environment of transplanted cells. [0003] Background of the invention [0004] The emergence of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells has a paradigm shift in regenerative medicine and translational medicine. These cells can renew themselves indefinitely in a pluripotent state, while retaining the ability to differentiate into any cell type in the human body. Such properties enable researchers to better understand the causes of human development and developmental disorders. They also provide modern medicine with powerful new tools for diseases that are difficult or impossible to treat with conventional medicine, such as spinal cord injury, diabetes, blindness, multiple sclerosis, and cancer, to name a few. As the understanding of how to control s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735A61K35/12A61K35/545A61K38/17A61K39/395A61K48/00A61K45/06A61L27/38A61P37/06C07K14/47C12N15/11C12N15/12
CPCA61K35/12A61K35/545A61P37/06A61K38/1709A61K38/1774A61K38/1841C12N5/0606C12N2510/00A61L27/38A61P1/16A61P27/02A61P7/04C07K14/70503A61K45/06A61P19/02A61P3/00A61P9/10C07K14/70532A61K9/0021A61P25/16A61P3/10C07K14/70575A61P25/28C07K14/47C12N15/09C07K14/521A61K2039/505
Inventor A.纳吉J.哈丁K.纳吉
Owner SINAI HEALTH SYST
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