Application of CRISPR-Based Gene Editing in Primary T Cells
Immune protection relies on a functional adaptive immune system. Its essential parts are T cells characterized by the expression of a unique clone-specific cell-surface protein complex, the T-cell receptor (TCR). Immune protection is enhanced by TCR recognition of peptide antigens derived from intracellularly processed proteins and presented in the context of major histocompatibility complex molecules on the surface of antigen-presenting cells. Another promising approach for treating immunocompromised patients that lack functional T-cell responses has been the adoptive transfer of T cells. Transferred T cells provide protective immunity against harmful complications caused by infectious diseases or malignant tumors.
Although those cells proved to be clinically effective, at least against certain tumor types, their widespread use was limited with the number of cells available for adoptive transfer. The first two FDA-approved CAR-T products were produced by lentiviral (Kymriah) or gamma-retroviral (Yescarta) transduction. ZFNs are recombinant proteins composed of a bacterial restriction enzyme (Fok1) that serves as a nuclease and a DNA-binding zinc finger domain (ZFN).
They induce double-strand breaks (DSBs) at specific sites into the genome, thus inducing genome editing. TALENs contain the bacterial cleavage domain Fok1 to cause DSBs at specific cellular repair mechanisms. The CRISPR/Cas system originated from a defense mechanism found initially in bacteria and archaea to counteract infections with viruses known as bacteriophages. The system consists of the Cas protein with endonuclease activity and the guide RNA (gRNA) that guides this protein to the correct cleavage site in the genome. By providing the chemically synthesized gRNAs, the Cas protein can be programmed to target a specific site in the genome. This allows for the induction of precise DSB and, eventually, gene editing in eukaryotic cells.
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