Challenges of CRISPR-Based Gene Editing in Primary T Cells

Abstract

Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.

Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining

“…Precise editing by HDR has a broad application in genome and cell engineering by CRISPR genome editing but is often limited by the low efficiency of HDR ( 1 , 3 , 52 ). Many approaches have been taken to improve HDR-mediated genome editing ( 3 , 9 ).…”

Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes

“…To test whether editing could be improved, we performed two or three sequential PERC treatments and ultimately reached 35% editing efficiency. These findings establish PERC as a straightforward means vectors 2,3 , but the prevailing delivery strategies for ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing of human lymphocytes rely on electroporation, which is a substantial source of cytotoxicity, cost and manufacturing burden in cell therapy pipelines 4 . Peptide-enabled delivery to cells is an attractive alternative involving a low-cost reagent that is simply mixed with genome-editing cargo before application to cells in culture.…”

“…Engineered immune cells have entered clinical trials to treat cancers, infections, genetic diseases and autoimmune disorders 1 , but manufacturing barriers in cell therapy pipelines persist, hindering cost-effective development of these products. Precise T-cell engineering offers advantages over pseudo-random integration with lentiviral or gammaretroviral (gRV) vectors 2 , 3 , but the prevailing delivery strategies for ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing of human lymphocytes rely on electroporation, which is a substantial source of cytotoxicity, cost and manufacturing burden in cell therapy pipelines 4 . Peptide-enabled delivery to cells is an attractive alternative involving a low-cost reagent that is simply mixed with genome-editing cargo before application to cells in culture.…”

Molecular and therapeutic effect of CRISPR in treating cancer

“…According to the research on inhibitory receptors and immune checkpoint such as PD1, TGF beta and CTLA-4 important hallmarks of those in cancer treatment were found out. Inhibitory receptors (iRs) are important in function of adaptive immune cells and in T cell exhaustion which is a common event in cancer [ 38 , 39 ] by blocking checkpoint molecules re-establishing the potency of T cells is one important part in the cancer treatment process via immune-checkpoint therapy [ 40 , 41 ]. CRISPR is a good safeguarding tool to derange immune checkpoint by protecting from checkpoint inhibition via knock out immune checkpoint molecules in CAR T cells.…”