Genome Engineering Using Adeno-associated Virus: Basic and Clinical Research Applications

Abstract

In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.

Figure 1

Adeno-associated virus (AAV) structure and genome organization. (a) Surface representation of the AAV2 capsid structure. The residues important for heparin binding, Arg 484, Arg 487, Lys 532, Arg 585, and Arg 588, are colored blue (PDB ID: 1LP3). (b) Structure of the wild-type AAV genome. Rep78 and Rep68 are expressed from the p5 promoter, and Rep52 and Rep40 are expressed from the p19 promoter. VP1, 2, 3, and the assembly-activating protein (AAP) are translated from the p40 transcript encoded by the cap gene. Solid black boxes indicate the inverted terminal repeats (ITRs).

Figure 2

AAV integration into the human genome. Wild-type AAV vectors encoding the rep gene can facilitate AAV integration into a region of human chromosome 19 termed AAVS1, denoted by a blue circle. Wild-type and recombinant AAV vectors can also integrate into random chromosomal sites via nonhomologous end joining (denoted by blue circles). When the AAV vector genome is modified to contain a genomic sequence homologous to a specific chromosomal site, homologous recombination (HR) between the AAV vector and target site can occur (denoted by blue DNA fragment). Co-delivering a targeted nuclease within the same or a separate AAV particle can further enhance the frequency of HR.

Figure 3

Overview of AAV-mediated gene targeting. AAV vectors containing DNA sequences homologous to a specific chromosomal site can be recombined with the matching genomic locus. By modifying the DNA sequence between the homology arms, targeted modifications (i.e., transgenes, single-base substitutions) can be introduced into the host genome. Dashes denote homologous regions of DNA. Black boxes indicate inverted terminal repeats (ITRs).