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| Item Type: | Article |
|---|---|
| Title: | Expansion and precise CRISPR-Cas9 gene repair of autologous T memory stem cells from patients with T cell immunodeficiencies |
| Creators Name: | Li, X., Chu, V.T., Kocks, C. and Rajewsky, K. |
| Abstract: | The adoptive transfer of autologous, long-lived, gene-repaired T cells is a promising way to treat inherited T-cell immunodeficiencies. However, adoptive T-cell therapies require a large number of T cells to be manipulated and infused back into the patient. This poses a challenge in primary immunodeficiencies that manifest early in childhood and where only small volumes of blood samples may be available. Our protocol describes the ex vivo expansion of potentially long-lived human T memory stem cells (T(SCM)), starting from a limited number of peripheral blood mononuclear cells (PBMCs). Using the perforin gene as an example, we provide detailed instructions for precise gene repair of human T cells and the expansion of T(SCM). The efficiency of precise gene repair can be increased by suppressing unintended non-homologous end-joining (NHEJ) events. Our protocol yields edited T-cell populations that are ready for phenotyping, genome-wide off-target analysis, and functional characterization. |
| Keywords: | Humans, Memory T Cells, Immunodeficiencies, Gene Therapy, Perforin, CRISPR-Cas9 Systems, Ribonucleoproteins, Adeno-Associated Virus 6 (AAV6), Genome Editing, Gene Editing Efficiency |
| Source: | Bio-protocol |
| ISSN: | 2331-8325 |
| Publisher: | Bio-protocol LLC |
| Volume: | 14 |
| Number: | 20 |
| Page Range: | e5085 |
| Date: | 20 October 2024 |
| Official Publication: | https://doi.org/10.21769/bioprotoc.5085 |
| PubMed: | View item in PubMed |
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