One-step generation of modular CAR-T cells with AAV–Cpf1

Abstract

Enhancing production efficiency, stability, effector function, and other desired features is of prime interest for chimeric antigen receptor engineered T cells (CAR-Ts). Here, we developed a new system for efficient generation of CAR-T with significantly enhanced features by streamlined genome engineering. Leveraging tracrRNA-independent CRISPR/Cpf1 systems with adeno-associated virus (AAV), building a stable CAR-T with homology-directed repair (HDR) knockin and checkpoint knockout (KIKO CAR-T) was achieved at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knockin of two different CARs in the same T cell. Compared to Cas9-based methods, the AAV-Cpf1 system generates double knockin CAR-Ts more efficiently. Dual-targeting CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to Cas9 CAR-Ts in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T cell immune engineering with simplicity and precision.

A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

“…Cas12a can be programmed to induce DNA doublestrand breaks (DSBs) at specific genomic loci in vivo [14,15,17,18,20,25,26]. To test if CeCas12a and BfCas12a could be harnessed for gene editing in mammalian cells, we optimized the codons of two genes and attached a Cterminal and an N-terminal nuclear localization signals (NLS) for optimal expression and nuclear targeting in human cells.…”

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

“…Applying CRISPR‐Cas9 under ex vivo conditions may be technically less challenging but equally valuable for disease therapy. For instance, ex vivo genome editing in hematopoietic stem cells (HSCs) could potentially provide a cure for patients carrying sickle cell disease (SCD) and β‐thalassaemia, and targeted insertions via CRISPR strategies may provide a valuable alternative to T‐cell engineering for cancer immunotherapy . A proof‐of‐concept study by Dewitt et al has reported an efficient correction of the E6V mutation at sickle alleles in patients' HSCs, through CRISPR‐HDR‐based replacement with the assistance of oligonucleotide donors.…”

“…A proof‐of‐concept study by Dewitt et al has reported an efficient correction of the E6V mutation at sickle alleles in patients' HSCs, through CRISPR‐HDR‐based replacement with the assistance of oligonucleotide donors. Using AAV‐delivered Cpf1, Dai et al built stable CAR‐T cells with immune‐checkpoint knockout (KIKO CAR‐T cells) via the HDR‐based strategy in one step.…”

“…For instance, ex vivo genome editing in hematopoietic stem cells (HSCs) could potentially provide a cure for patients carrying sickle cell disease (SCD) and β-thalassaemia, 85 and targeted insertions via CRISPR strategies may provide a valuable alternative to T-cell engineering for cancer immunotherapy. 86 which identified TOP2A and CDK6 as new genes responsible for the drug resistance to purine analogue 6-thioguanine. 93 Subsequent screenings also discovered other genes critical to cancer development and treatment, [94][95][96] such as BCR and ABL, which could be potentially regarded as new therapeutic targets in leukaemia and colorectal carcinoma.…”

Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of genetic payloads for rapid cell engineering

“…The Sadelain group inserted a CAR into the TRAC locus by electroporating T cells with Cas9/gRNA RNPs and delivering the CAR transgene using rAAV6 (Eyquem et al 2017). The Chen group also recently generated CAR-T cells by electroporating Cpf1 mRNA while delivering gRNA and donor DNA sequences via AAV, a method which enabled genetic disruption of a second location (Dai et al 2019). The Marson lab similarly delivered Cas9/gRNA RNP into T cells and used high-output PCR products as donor templates to correct a pathological IL2RA defect and replace endogenous TCR genes with a cancer antigen-specific transgenic TCR (Roth et al 2018).…”