Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction

Abstract

Recombinant adeno-associated virus has emerged as one of the most promising gene therapy delivery vectors. Development of these vectors took advantage of key features of the wild-type adeno-associated virus (AAV), enabled by basic studies of the underlying biology and requirements for transcription, replication, and packaging of the viral genome. Each step in generating and utilizing viral vectors involves numerous molecular interactions that together determine the efficiency of vector production and gene delivery. Once delivered into the cell, interactions with host proteins will determine the fate of the viral genome, and these will impact the intended goal of gene delivery. Here, we provide an overview of known interactions of the AAV genome with viral and cellular proteins involved in its amplification, packaging, and expression. Further appreciation of how the AAV genome interacts with host factors will enhance how this simple virus can be harnessed for an array of vector purposes that benefit human health.

Keywords: DNA replication; adeno-associated virus; gene therapy; vector genome; vector production; vector transduction.

Figure 1.

The AAV genome and its products. (A) Diagram of the wtAAV genome, illustrating the layout of the rep (orange) and cap (green) genes, viral promoters (p5, p19, p40), polyadenylation signal (pA, gray), and ITR sequences. All elements are drawn to scale. (B) Magnified view of the ITR (boxed region in A), detailing the complementary A/A′ (blue), B/B′ (green), and C/C′ (red) sequences. The D-sequence (brown) is contiguous with the ssDNA genome body. The terminal resolution site (TRS) consists of the cleavage site (black arrow) and the flanking bold bases. (C) The nine known protein products of the AAV genome. Transcripts (gray lines) are aligned with their respective promoters in (A). Boxes labeled with protein names constitute ORFs translated from the rep (orange) and cap (green) genes. +1 indicates these protein products are translated from a +1 nucleotide shifted ORF within the cap gene. The Rep enzymatic domains relevant to interactions with the AAV genome are indicated above. (D) Schematic of an rAAV vector genome, illustrating the maximum cassette size that can be packaged of 5 kb (drawn to scale with A). The CMV promoter (pink) is shown as an example, with the blue bar representing available space for the gene of interest. CMV, cytomegalovirus; ITR, inverted terminal repeat; ORF, open reading frames; pA, polyadenylation signal; rAAV, recombinant adeno-associated virus; RBE, Rep-binding element; ssDNA, single-stranded DNA; TRS, terminal resolution site; wt, wild-type.

Figure 2.

The cellular path of AAV genome fates and interactions. Schematic representation of (A) the wtAAV infection pathway and (B) the decoupling of this pathway in rAAV vector production and transduction. The AAV gene products are denoted in colors corresponding to Fig. 1C. Major steps of the pathway are enumerated in (A) as follows: (1) Entry. (2) Trafficking. (3) Uncoating. (4) Second strand synthesis. (5) Transcriptional activation. (6) Genome replication. (7) Packaging. Diagrams are not to scale and are not exhaustive of the processes involved. For example, transgene expression occurs from monomeric vector genomes in addition to concatemers. (C) Summary of host and helper viral proteins that interact with the AAV genome, arranged by the processes and phases with which they have been identified. Green text indicates factors involved in positive regulation, and red text denotes negative regulators of viral replication or vector transduction. The production phase of this panel employs similar factors as in replication, and thus they were omitted from the production diagram for simplicity; there are no known factors that interact with the AAV genome exclusively in the vector production context. *Factors that interact with the AAV genome in an AdV-dependent manner; ^†factors that bind the AAV genome as part of a complex with the Rep proteins. AdV, adenovirus.