Excitement over recent breakthroughs in autologous cellular therapies has been tempered by the expense of manufacturing with viral gene delivery and concerns over random integration and the safety of viral vectors. MaxCyte offers a non-viral alternative that allows for site-specific delivery of transgenes through homology-directed repair (HDR) and circumvents complex and expensive manufacturing procedures. Although CRISPR-Cas9 technology has led to rapid advances in genetic engineering, the efficiency of knockin (KI) and absolute yield of live KI cells remain challenging, particularly for clinical and therapeutic applications.
In 2020, researchers in the Marson lab demonstrated that an HDR template with Cas9 target sequences (CTS) shuttle more efficiently to the cell nucleus, allowing RNPs to bind and facilitate KI. Even with improved KI, the absolute yield of engineered cells remained limited due to double-stranded DNA (dsDNA) toxicity1. Now, Shy, et al have developed an innovative single-stranded construct with two short regions of double-stranded CTS at the sequence ends. This hybrid oligonucleotide allows Cas9 to bind more efficiently while overcoming the problem of dsDNA cytotoxicity. A recent BioRxiv report demonstrates the integration efficiency of these single stranded Cas9 target sequence (ssCTS), in a range of construct sizes, genetic loci, and cell types2.
MaxCyte electroporation technology enabled the delivery of long ssCTS HDR templates into difficult-to-transfect primary cells. This approach resulted in highly efficient integration (46-62%) of a B cell maturation antigen (BCMA)-CAR sequence into the TRAC locus of T cells. MaxCyte electroporation is clinically validated and scalable facilitating increased production of engineered cells for patient treatment. Finally, the delivery of BCMA-CAR sequences had minimal cell toxicity when CAR T cells were manufactured at clinical scale (>1.5 x 109 CAR+ cells) enabling the commercial production of quality, clinically active, genetically engineered cells for cancer treatment.