Non-immunogenic engineered tissue and methods of producing and using the same

A non-immunogenic, engineered technology, applied in biochemical equipment and methods, tissue culture, bone/connective tissue cells, etc., can solve problems such as transplant rejection

Pending Publication Date: 2021-06-11
L 杰罗姆罗
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the above-mentioned methods, while producing, for example, functional cardiac tissue or functional skeletal tissue, still face the problem of transplant rejection if the pluripotent stem cells used are derived from an allogeneic donor and / or are not histocompatible

Method used

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  • Non-immunogenic engineered tissue and methods of producing and using the same
  • Non-immunogenic engineered tissue and methods of producing and using the same
  • Non-immunogenic engineered tissue and methods of producing and using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Production of MHC I molecules and more stem cells containing immunomodulatory proteins in their surface

[0093] β2-microglobulin (B2M) knockout

[0094] A multi-capable cell line 50039 is obtained from NINDS human cells and databases. The cell line can also be obtained from Lonza, Baggbaderane et al. (Baggbaderane et al. (2015), STEM CELL Reprots, 5: 647-6659). Baggbaderane et al. Also disclose standard conditions for maintaining the cell line. Unlike this, the pluripotent stem cells are in STEMMACS TM Maintenance in the IPS-BREW medium. The extracellular matrix is ​​provided using CTG layer adhesion protein-521 (Biolamina) or GelTrex (Thermo Scientific).

[0095] In order to destroy all the copies of the B2M gene, in accordance with the manufacturer's instructions, Integrated Dnatechnologies is used. CRISPER / CAS technology. Depending on the sequence of Crispr-Cas9 crrna, Alt-R Cas9 nuclease can specifically introduce double-strand breakage, which may result...

Embodiment 2

[0111] Example 2: Producing biological engineering myocardial tissue with modified PSC

[0112]The scheme described in WO 2015 / 040142 and Tiburcy et al. (Tiburcy, (2017), Circulation, 135: 1832-1847), and WO 2015 / 025030, starting from PSC, engineered myocardium begins. The plurality of stem cells, particularly clones, 18, 20, and 34, in Example 1, can be used in this embodiment. The scheme includes the step of induced induced embryo layers, cardiac differentiation, and heart maturation as described in WO 2015 / 040142, which is then directed to form a tissue in type I collagen hydrogel as described in WO 2015 / 025030.

[0113] In the first step, the PSC must differentiate into cardiomyocytes. This step can be carried out in accordance with, for example, Tiburcy et al. (Tiburcy et al., (2017), Circulation, 135: 1832-1847, and the first method disclosed in WO 2015 / 040142. Embodiment 1 Multi-capable cells (PSC) can be 5 × 10 on the masstel of 1:30 4 To 1 × 10 5 Cell / cm 2 Plate on a p...

Embodiment 3

[0119] Example 3: A modified PSC produces biological engineered myocardial tissue

[0120] The HIPSCs (see Example 1) obtained herein are differentiated as cardiomyocytes (CM) based on the protocols described in Example 2 described above (Tiburcy et al., 2017, WO 2015 / 025030). HLA-E ki HIPSC derived CMS showed the expression of muscle subinoproteins; α-ciliacin and myocardial kerceculin T (CTNT), together with> 90% cuisurine protein + cell high purity ( Figure 10 A).

[0121] Flow cytometry analysis showed wild type (WT) CMS expression B2M and HLA I molecules; HLA-B and C, but did not express HLA-E under basic conditions. IFN-γ treatment induces B2M and HLA-B, C molecules, and HLA-E is slightly lower (~ 60%). As a negative control, if expected, B2M KO CMS displays no HLA expression. HLA-E ki cloned (dimer and trimer) expressed B2M and HLA-E after IFN-γ, and did not show any other HLA I expression. CMS from HLA-E trimer HIPSC is slightly high in initial analysis compared to the co...

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Abstract

The invention provides a method of producing a non-immunogenic (bio)engineered tissue from pluripotent stem cells or pluripotent stem cell derivatives, the respective cells being deficient of MHC class I molecules and comprising an immunomodulatory protein on their surface, wherein the method comprises inducing the differentiation of the pluripotent stem cells into a cell type that is essential for the function of the engineered tissue under conditions that also allow the formation of the engineered tissue,thereby rendering the engineered tissue to be non-immunogenic to a recipient of the engineered tissue. The present invention further relates to an engineered tissue, a pharmaceutical composition comprising the engineered tissue, medical treatments using the engineered tissue and uses of the engineered tissue.

Description

[0001] Cross-reference [0002] The present application claims priority equity of European Patent Application No. 199183294.0, filed on July 13, 2019, which is incorporated herein by reference. Technical field [0003] The present invention provides a method of producing non-immunogenic engineering from a pluripotent stem cell that lacks MHC I molecules and contains immunomodulatory proteins thereof, wherein the method comprises organizing an engineering tissue. In the presence of at least one cell type necessary for the function, the engineering tissue is formed under conditions allowed to form the engineering tissue, wherein the at least one cell type has been passed through the engineering. Under the conditions of the tissue, induce multivariable stem cells to differentiates to at least one cell type necessary for the function of the engineered tissue, thereby causing the engineering tissue to the engineered tissue. of. The invention also relates to an engineered tissue, a phar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2510/00C12N2500/90
Inventor L·杰罗姆罗
Owner L 杰罗姆罗
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