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Genetically recombinant hematopoietic stem cells for treating tumors and preparation method of genetically recombinant hematopoietic stem cells

A technology of hematopoietic stem cells and genetic recombination, which is applied in the therapeutic field, can solve the problems of short effective time, inflammatory response cytokines, and disease recurrence, and achieve the effects of improving efficiency, increasing concentration, and reducing myeloid differentiation

Inactive Publication Date: 2016-12-14
侯昌禾 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, Car-T therapy still has the following problems: 1. Car-T has an excellent killing effect on non-solid tumors. When the tumor load in the patient is large, it will cause a large number of tumor cell necrosis, causing severe inflammatory reactions and cell Factor storm, and even patient death, there is no sure way to avoid or eliminate this adverse reaction
2. Car-T is a terminally differentiated mature T cell with limited proliferation ability and lifespan. It must be constructed and expanded in vitro once and then infused back into the body. It cannot proliferate for a long time in the body, and the effective time is short, so the short-term effect Significant and long-term disease recurrence is still possible

Method used

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  • Genetically recombinant hematopoietic stem cells for treating tumors and preparation method of genetically recombinant hematopoietic stem cells
  • Genetically recombinant hematopoietic stem cells for treating tumors and preparation method of genetically recombinant hematopoietic stem cells

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Experimental program
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Effect test

Embodiment 1

[0034] Patients with hCD19-positive acute lymphoblastic leukemia or chronic lymphocytic leukemia were subcutaneously injected with G-SCF 10ug / kg for 5 days to mobilize hematopoietic stem cells. Or not mobilize hematopoietic cells.

[0035] Use special equipment for collecting peripheral blood mononuclear cells, and circulate adsorption to obtain mononuclear cells.

[0036] Mononuclear cells were screened with magnetic beads labeled with anti-hCD34. After elution, hCD34+ mononuclear cells are obtained, which are HSCs.

[0037] Select a specific scFv, such as anti-human CD19 monoclonal antibody, and truncate the gene encoding its Fab end as scFv. The gene encoding the CD8 transmembrane domain was selected. The gene encoding CD137 intracellular domain (4-1BB) was selected. The gene encoding the intracellular domain of CD3 was selected. Conventional genetic engineering technology sequentially connects the above coding genes to form an anti-hCD19 Car fusion gene (for the struc...

Embodiment 2

[0042] For patients with HER2 positive breast cancer, routine bone marrow puncture is performed to extract bone marrow fluid, which is centrifuged with lymphocyte separation medium to obtain mononuclear cells in the middle layer.

[0043] Mononuclear cells were screened with magnetic beads labeled with anti-hCD34. After elution, hCD34+ mononuclear cells are obtained, which are HSCs.

[0044] Select a specific scFv, such as anti-human HER2 monoclonal antibody, and truncate the gene encoding its Fab end as scFv. The gene encoding the CD8 transmembrane domain was selected. The gene encoding CD137 intracellular domain (4-1BB) was selected. The gene encoding the intracellular domain of CD3 was selected. Conventional genetic engineering technology sequentially connects the above-mentioned coding genes to form an anti-hHER2 Car fusion gene (for the structure, see figure 2 ).

[0045] Select the upstream promoter part of CD8b1 (200bp upstream of the transcription start point), s...

Embodiment 3

[0049] For patients with HER2 positive breast cancer, routine bone marrow puncture is performed to extract bone marrow fluid, which is centrifuged with lymphocyte separation medium to obtain mononuclear cells in the middle layer.

[0050] Mononuclear cells were screened with magnetic beads labeled with anti-hCD34. After elution, hCD34+ mononuclear cells are obtained, which are HSCs.

[0051] Select a specific scFv, such as anti-human HER2 monoclonal antibody, and truncate the gene encoding its Fab end as scFv. The gene encoding the CD8 transmembrane domain was selected. The gene encoding CD137 intracellular domain (4-1BB) was selected. The gene encoding the intracellular domain of CD3 was selected. Conventional genetic engineering technology sequentially connects the above-mentioned coding genes to form an anti-hHER2 Car fusion gene (for the structure, see figure 2 ).

[0052] The CD8a promoter and CD8-TG-g enhancer were selected and spliced ​​with the Car fusion gene in...

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Abstract

The invention discloses genetically recombinant hematopoietic stem cells for treating tumors, chronic infection or autoimmune diseases and the like and a preparation method of the genetically recombinant hematopoietic stem cells. The recombinant hematopoietic stem cells are formed by inserting Car genes and CD3+CD8+CD4-T cell specific promoters and enhancers into genome of hematopoietic stem cells of a patient. According to the invention, actually a segment of DNA sequence containing Car is input into the hematopoietic stem cells of the patient, and specific expression of Car in a differentiation stage of CD8+CD4-T cells is controlled through a gene recombination technology. Meanwhile, the hematopoietic stem cells are efficiently and directionally differentiated into the CD8+CD4-T cells due to the gene recombination technology, myeloid differentiation, B cell differentiation and CD8+CD4-T cell differentiation can be reduced, the concentration of CAR-T in peripheral blood is increased, and the efficiency is improved.

Description

technical field [0001] The present invention relates to the technical field of treatment of tumors, chronic infections or autoimmune diseases, etc., and specifically relates to a genetically recombined hematopoietic stem cell and a preparation method thereof. Background technique [0002] Car-T is a new treatment method. Simply put, it is to artificially construct a CAR molecule targeting the specific antigen of the target cell, insert the DNA sequence encoding the molecule into the recombinant T cell, and return the recombinant T cell to the patient. kill target cells. The constructed CAR molecule expressed on the surface of recombinant T cells has an extracellular domain of scFv (single-chain variable fragment), and an intracellular domain that includes the CD3 intracellular domain. The former can directly interact with target cell surface markers independent of MHC I antigen presentation. (for example, for ALL and CLL, acute and chronic lymphocytic leukemia, the surface ...

Claims

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Application Information

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IPC IPC(8): C12N5/10A61P35/00A61P31/00A61P37/02
CPCC07K16/28C07K14/7051C07K14/70521C07K14/70575C07K14/70578C07K2317/622C07K2319/33C07K2319/74
Inventor 侯昌禾任沁沁
Owner 侯昌禾
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