AAV Vectorization of DSB-mediated Gene Editing Technologies

Abstract

Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells

“…Consistent with the observations of numerous other groups, we found AAV to be a particularly effective template for HDR 22, 23, 24, 41. When we attempted to target CAR expression cassette insertion using plasmid DNA or PCR products as the template, the efficiency of gene integration was >10-fold lower than with AAV (data not shown).…”

“…When we attempted to target CAR expression cassette insertion using plasmid DNA or PCR products as the template, the efficiency of gene integration was >10-fold lower than with AAV (data not shown). It is not known whether this is due to the ability of AAV to achieve higher intracellular concentrations or whether the virus interacts with host factors involved in HDR 24, 41. Also consistent with other reports is our observation of a striking increase in gene expression following integration into the genome 25, 26…”

“…HDR with an exogenous DNA sequence has been described previously in T cells using short oligonucleotides paired with CRISPR/Cas9 21 . Other groups have shown that adeno-associated virus (AAV) vectors can be used as a template in conjunction with a site-specific nuclease to achieve high levels of gene insertion via HDR 22, 23, 24. Two groups have also reported the targeted insertion of gene expression cassettes in T cells using MegaTAL nucleases 25 and ZFNs 26 in combination with an AAV6 HDR template.…”

Targeted modulation of E3 ligases using engineered ubiquitin variants

“…Importantly, AAV replicates without incorporating its genome into the host cell chromosomes (in contrast to the lentivirus used above for cellular characterization). AAV is a method of choice in gene therapy and has frequently been used to deliver Cas9 for genome editing in cells and animal models for preclinical research [141]. Similarly, mRNA delivery has been used to introduce genome editing components [142,143].…”

Immunity to CRISPR Cas9 and Cas12a therapeutics

“…These strategies generally build on judicial gRNA and target selection, Cas9 protein engineering, and controlled expression of CRISPR‐Cas9 components, as illustrated in recent reviews . Next is the pursuit of targeted delivery and expression of Cas9 and Cas12a in desired cell types and tissues, which can be achieved through the use of many viral and nonviral means …”