And there could be more to come, if Novartis sells Sandoz – something that looks on the cards after it recently started a strategic review of the generic business. While this may be because these patients were heavily treated prior to T cell isolation, it does reveal potential limitation of current approach in clinical applications.Novartis’s sale of its stake in Roche, announced today, means the group will soon have $20.7bn burning a hole in its pocket. Notably, in two studies, the authors failed to expand enough edited T cells from a significant fraction of patients (23% 1 and 33%, 5 respectively). Finally, the current protocols for producing patient-derived CRISPR-edited cells are still somewhat inefficient. 5 Given that adverse effects of off-target editing may take several years to manifest, 3 it is important for future studies to evaluate the long-term safety of CRISPR, particularly when the edited cells are long-lived in vivo. Second, while the reported off-target rates were low in all three studies, they were highly dependent on gRNA selection, and multiplex gene editing significantly increased the risk of chromosome translocation. 1, 5 This may be due to the different cell production protocols used in separate studies, but it emphasizes the importance of optimizing and validating each individual CRISPR assay for clinical use. First, the gene-editing efficiency varies significantly across target genes and patients. Nevertheless, these studies also revealed several limitations of existing gene-editing technology. Not only that they provided the eagerly awaited first pieces of evidence supporting the safety and feasibility of CRISPR-based cell therapy in human patients, but also that they established detailed protocols for CRISPR-editing and off-target events tracking in clinical settings which are valuable for designing future trials. These three studies marked an important milestone in the development of CRISPR-based gene therapy. 5 In both studies, the rates of off-target editing were low. While chromosome translocations were detected in the edited cells as would be expected from multiplex editing, no cellular transformation was observed and patients experienced no gene-editing-related severe AEs. The authors reported efficient editing of all target genomic loci and stable engraftment of edited T cells in vivo for up to 9 months. used CRISPR-based multiplex gene editing to ablate the expression of endogenous T cell receptor ( TCR) and PD-1 genes in T cells engineered to express a cancer-specific TCR gene in four patients. The authors found the edited donor cells to persist more than 19 months after transplantation without causing gene-editing-related AEs. transplanted CRISPR-engineered, CCR5-ablated hematopoietic stem/progenitor cells into a patient with HIV infection and acute lymphoblastic leukemia. This finding echoes the conclusions of two other recent clinical studies that also examined the safety and feasibility of CRISPR-based cell therapy. Together, this study showed that using ex vivo CRISPR-edited, PD-1-ablated, patient-derived T cells for cancer treatment is clinically feasible and generally safe. Of note, one patient with the highest PD-1 editing efficiency showed long-term persistence of edited T cells in vivo and stable disease for 76 weeks, suggesting higher editing efficiency may be associated with improved clinical outcomes. The authors did not observe objective response in the treated patients, which may be due to a combination of low editing efficiency, poor T cell expansion, and/or lack of antigen specificity by the edited T cells. No treatment-related dose-limiting toxicities were observed during the 2-year follow-up period, suggesting infusions of PD-1-edited T cells were well-tolerated. Grade 1/2 AEs occurred in 11 of the 12 patients and no grade 3+ AEs were reported. Infused patients were monitored for up to 2 years for treatment-related AEs. Taken together, these results suggested off-target editing by CRISPR was rare. In contrast, no mutations were detected in 2086 predicted off-target sites using the lower coverage (100×) WGS method. Using high coverage (20,000×) NGS, only an average of 0.05% mutation rate was detected in 18 potential off-target sites. To evaluate the impact of off-target editing by CRISPR-Cas9 in edited T cells, the authors performed both targeted NGS and unbiased whole-genome sequencing (WGS) to identify the type and frequency of off-target mutations.
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