350. Toward Genosafe Endonuclease-Boosted Gene Targeting Using Breakthrough CRISPR/Cas9 for Next Generation Stem Cell Gene Therapy Culminating in Efficient Ex Vivo/In Vivo Gene Repair/Genomic Editing
Authors: Roger Bertolotti
Journal: Molecular Therapy (2015)

Bacterial CRISPR-associated protein Cas9 acts as an in vitro programmable site-specific endonuclease directed to its DNA target by a 100-nt single-guide RNA (sgRNA) and drives a gene targeting breakthrough in human cells based on efficiency and easy cost-efficient sgRNA customization. Such a breakthrough substitutes simple digital RNA-DNA base-pairing for complex analogic protein-DNA binding of zinc-finger/TALE nucleases and is a key upgrade for the endonuclease-boosted gene targeting arm of our proposed 2006 Universal Stem Cell Gene Therapy Platform. However, Cas9 clinical translation is still pending on the final improvement of 1) DNA target specificity and 2) on-target genosafety. DNA TARGET SPECIFICITY of Cas9 relies on a 20-bp sequence complementary to the 5'-end of sgRNA and on adjacent 5'-NGG-3' PAM recognized by Cas9 itself, thereby involving a DNA sequence longer than the 18-bp specificity reference for a human genome. However, Cas9 can accomodate up to 5 mismatches, mainly on the 5'-end of sgRNA, resulting in off-target genotoxic double-strand breaks (DSBs). Cas9 is thus subjected to strategic improvements including 1) truncation of 2/3 nt at the 5'-end of sgRNA and 2) use of single (nickase) and double (inactive dCas9) Cas9 mutants for DSB genesis by paired nickases and by dimers of FokI-dCas9 fusion protein. Another promising approch relies on the delivery of pre-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes using either nucleofection or ex vivo/in vivo cationic-lipid transfection. ON-TARGET GENOSAFETY is a major clinical concern. Gene targeting is boosted by site-specific endonucleases because it relies on homologous recombination, a DSB repair pathway mediating DNA exchanges between chromosomal and transfecting/transducing DNA, thereby providing the means to modify at will chromosomal DNA sequences (genomic editing) culminating in gene repair or targeted transgene integration. However, the aforedescribed homology-directed repair (HDR) pathway is challenged by non-homologous end-joining (NHEJ) DSB-repair leading to insertion/deletion mutations (indels): NHEJ prevails in the absence of homologous donor DNA (eg: off-target DSBs, targeted gene inactivation) and hampers on-target HDR while generating genotoxic indels. Therefore, under off-target-free condition, the on-target HDR/NHEJ ratio has to be increased to a clinical-trial genosafety level. We are thus resuming our former approach based on recombinase-DNA nucleoprotein filaments for the transfer of donor DNA in its active presynaptic state. Our approach is thus discussed in terms of ex vivo nucleofection and in vivo cationic-lipid transfection of nucleoprotein complexes comprising Cas9-sgRNA RNPs and donor-DNA presynaptic-filaments for efficient genosafe gene repair/genomic editing of Next Generation Stem Cell Gene Therapy focussed on hazard-free patient-specific iPS cells and expandable tissue-specific converted stem cells.