Controlling CRISPR with small molecule regulation for somatic cell genome editing.

Biomedical research has been revolutionized by the introduction of many CRISPR-Cas systems that induce programmable edits to nearly any gene in the human genome. Nuclease-based CRISPR-Cas editors can produce on-target genomic changes but can also generate unwanted genotoxicity and adverse events, in part by cleaving non-targeted sites in the genome. Additional translational challenges for in vivo somatic cell editing include limited packaging capacity of viral vectors and host immune responses. Altogether, these challenges motivate recent efforts to control the expression and activity of different Cas systems in vivo. Current strategies utilize small molecules, light, magnetism, and temperature to conditionally control Cas systems through various activation, inhibition, or degradation mechanisms. This review focuses on small molecules that can be incorporated as regulatory switches to control Cas genome editors. Additional development of CRISPR-Cas-based therapeutic approaches with small molecule regulation have high potential to increase editing efficiency with less adverse effects for somatic cell genome editing strategies in vivo.

Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells.

Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.

Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain. METHODS: Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG™ xenograft neuroblastoma model. RESULTS: In comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model. CONCLUSIONS: This study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.