Gene Editing: Paving the Way for Accelerated Clinical Development of Adoptive Cell Therapies
Precision genome engineering requires technologies that allow efficient and reproducible delivery of DNA, mRNA and Ribonucleoprotein (RNP)-based reagents into a range of primary and stem cells. In addition, clinical gene editing requires a transfection platform that is inert and does not affect genome stability and cell viability. The CRISPR-Cas9 system of genome editing has the potential to treat a variety of genetic diseases. However, unintended off-target double-stranded breaks introduced by Cas9 cleavage could have unforeseen clinical complications. Here we share data characterizing genome integrity in clinically relevant human hematopoietic stem and progenitor cells (HSPCs) following transfection of CRISPR-Cas9 using MaxCyte® electroporation technology. There was no evidence of significant structural or nucleotide sequence variations within the genomes of clonal isolates from human HSPCs following electroporation and/or Cas9 treatment.
Primary human HSPCs were electroporated using two Cas9 RNPs that were engineered to target two separate loci, CXCR4 and AAVS1. Gene-edited HSPCs and controls were then subject to single cell cloning in 96 well plates. Whole genome sequencing (WGS) and bioinformatic analysis were performed as an unbiased method of assessing genomic integrity and characterization of Cas9 activity.