Combining Engineered Nucleases with Adeno-associated Viral Vectors for Therapeutic Gene Editing

Abstract

With the recent advent of several generations of targeted DNA nucleases, most recently CRISPR/Cas9, genome editing has become broadly accessible across the biomedical community. Importantly, the capacity of these nucleases to modify specific genomic loci associated with human disease could render new classes of genetic disease, including autosomal dominant or even idiopathic disease, accessible to gene therapy. In parallel, the emergence of adeno-associated virus (AAV) as a clinically important vector raises the possibility of integrating these two technologies towards the development of gene editing therapies. Though clear challenges exist, numerous proof-of-concept studies in preclinical models offer exciting promise for the future of gene therapy.

Keywords: AAV; CRISPR/Cas9; Gene editing; Gene therapy; Zinc-finger nuclease.

Figure 1. Cellular mechanisms of DNA repair following DNA double-strand break

When a double-strand DNA break occurs, one of two cellular mechanisms repairs the damage. (a) In non-homologous end joining (NHEJ), polymerases and nucleases clean up the damaged ends by adding or deleting small numbers of nucleotides until they can be rejoined by ligases. The final ligated product contains small insertions or deletions (indels) at the damage site, often resulting in a frameshift. (b) In homology-directed repair (HDR), the 3’ overhang strand at a site of DNA damage can displace a strand in a separate DNA duplex with homology to that strand (a donor template). Polymerases extend the damaged end according to the homologous template DNA duplex, and the strand either returns to its original complementary strand, annealing to the other original damaged end; or the donor template strand and the previously damaged strand can undergo complete crossover and recombination. Either option results in a repaired strand.