Non-viral engineering of CAR-T cells using an enhanced version of a CRISPR/Cas system

CAR-T cells present a revolutionary therapeutic option for various malignant diseases based on their ability to specifically recognize the selected tumor surface markers in MHC independent manner, triggering cell activation and cytokine production that results in killing cancerous cells expressing surface markers recognized by the chimeric antigen receptor (CAR). The high success in CD19 malignancies therapy led also to the FDA approval of this therapeutic approach. Usually CAR-T cell production relies on autologous donors, of which cells are then ex vivo virally transduced to stabile express CAR constructs for long-term therapeutic benefits. The main drawback of viral mediated CAR-T cells production is random integration process that can result in an insertional mutagenesis, when viral integration can impair the functionality of the normal genetic landscape. This can lead to possible oncogenic changes and abnormalities, also the final T cell product can be less homogenous with lesser desired phenotype predictability. To overcome that, site-specific targeted insertion is preferred, which could be accomplished by CRISPR/Cas system mediated genome integration of transgenes, expressing CAR constructs by the delivered homology directed repair (HDR) DNA template. Nevertheless, the efficiency of HDR is still quite low, therefore improvements of CRISPR orchestrated CAR-T cell generation should to be introduced. In this project we propose to employ our developed enhanced version of CRISPR/Cas system that showed increased efficiency in NHEJ and HDR mediated cell repair mechanism, leading to higher percentage of genomic modification events to non-virally produced CAR-T cells. The enhanced version of CRISPR/Cas system is based on tethering of Cas9 protein to the exonuclease via a coiled-coil (CC)-forming heterodimeric peptide pairs, termed CCExo (CRISPR-Coiled-coil--Exonuclease), where one peptide is linked to the Cas9 protein and the other heterodimeric peptide is genetically fused to the exonuclease. Formation of a CC heterodimer results in bringing the exonuclease into close proximity of Cas9 mediated DNA genome breaks in order to form complementary single strandedDNA ends. The same event occurs on the DNA template, coding for precisely positioned insertion of a CAR construct at specific genomic site with high efficiency. Additional approach of CCExo system mediated increase in genome editing will be the use of streptavidin-binding aptamere within gRNA and the presence of biotinylated HDR template. By non-viral delivery (electroporation or lipid nanoparticle delivery) we aim to produce high yield of CAR-T cells with precisely defined phenotype with favorable immunotherapeutic properties. By implementing non-viral production of CAR-T cells by the developed CCExo system genome targeted insertion, we aim to prepare final cell therapeutic product with no shortcomings that may occur in viral production. By incorporation of streptavidin-biotin mechanism we also plan to increase DNA template trafficking into cell nucleus, with the desire to further increase the yield of precisely integrated CAR-T cells, whose activity we will test in mouse cancer models. This proposed project thoroughly compare viral and non-viral preparation of CAR-T cell characteristics, thus highlighting the importance of non-viral CRISPR mediated generation of CAR-T cells. This technique of high yield site-directed genomic modification could be implemented not only on CAR-T cell production but also on other gene therapy aspects, where a therapeutic transgene is incorporated into the genome. We are convinced that site-specific genome integration will become the new standard for therapeutic cell production.