Adeno-associated Virus as a Mammalian DNA Vector

Abstract

In the nearly five decades since its accidental discovery, adeno-associated virus (AAV) has emerged as a highly versatile vector system for both research and clinical applications. A broad range of natural serotypes, as well as an increasing number of capsid variants, has combined to produce a repertoire of vectors with different tissue tropisms, immunogenic profiles and transduction efficiencies. The story of AAV is one of continued progress and surprising discoveries in a viral system that, at first glance, is deceptively simple. This apparent simplicity has enabled the advancement of AAV into the clinic, where despite some challenges it has provided hope for patients and a promising new tool for physicians. Although a great deal of work remains to be done, both in studying the basic biology of AAV and in optimizing its clinical application, AAV vectors are currently the safest and most efficient platform for gene transfer in mammalian cells.

FIGURE 1

The adeno-associated virus (AAV) genome is a linear ~4.7-kb single-stranded DNA which is flanked by inverted terminal repeats. The genome contains three promoters that drive transcription of the viruses replication (REP), capsid (CAP) and assembly (AAP) genes. The first two promoters, p5 and p19, drive transcription of the large (78/68 kDa) and small (52/40 kDa) Rep proteins, respectively. In each case an alternate splice at the end of each transcript results in the smaller variant of each protein (Rep68 and Rep40). Transcription from the p40 promoter produces one large mRNA with an intron. A minor splice variant of this message contains the translational initiation codon (AUG) for the largest capsid protein (VP1), while the major splice variant truncates this sequence. The major splice variant contains a nontraditional translation initiation codon (ACG) at the start of VP2, which is often skipped for the downstream AUG of VP3, the smallest and most abundant capsid protein. The p40 transcripts also contain an alternate reading frame that encodes the assembly activating protein (AAP), which is translated from a nontraditional CUG. doi:10.1128/microbiolspec.MDNA3-0052-2014.f1

FIGURE 2

Adeno-associated virus (AAV) genome replication is primed by the 3′ inverted terminal repeat (ITR), and primarily uses the host pol δ (A). The genome is replicated through the 5′ ITR (via strand displacement), yielding a double-stranded (dsDNA) intermediate (B). The newly generated 3′ ITR (produced when copying the 5′ ITR) then primes replication in the opposite direction (C). At the same time, the AAV Rep protein, bindings to the Rep-binding element (RBE) on the original 3′ ITR, and cuts the lower strand at the terminal resolution site (trs), generating a free 3′ end (C), which allows for the replication of the original 3′ ITR (D) and the production of a new single-stranded (ssDNA) genome as well as another dsDNA intermediate (E) which is fed back into the replication cascade. This replication mechanism results in the production of ssDNA genomes of both polarities, which are then packaged into the capsid. In the case of self-complementary (scDNA) vectors, one of the ITRs is mutanted (mITR) to remove the trs, and prevent resolution of the double-stranded intermediate. This resulting replication scheme (detailed in the right column) yields a self-complementary genome that at its center contains the mITR, which is never nicked and which serves as an intermolecular hinge. This hinge acts to form a duplex genome immediately upon uncoating and bypasses the typical need for second-strand synthesis that serves as a bottleneck for ssDNA vectors. doi:10.1128/microbiolspec.MDNA3-0052-2014.f2

FIGURE 3

Adeno-associated virus (AAV) vector production. (A) Tradition AAV vector production begins with transfection of mammalian cells (commonly HEK 293) with three plasmids. The first provides the Cap proteins from the chosen AAV serotype in conjunction with Rep from AAV2. This plasmid lacks inverted terminal repeats (ITRs), ensuring that the Rep/Cap sequences are not packaged into AAV capsids and no replication-competent virus is made. The Genome plasmid contains the chosen transgene sequence flanked by ITRs, which are necessary for packaging and genome replication. In the case of ssDNA vectors, transgene cassettes up to 5 kbp can be packaged with high efficiency, whereas scDNA vectors can accommodate cassettes half that size. The third plasmid provides in trans the adenovirus genes that are necessary for AAV replication. It is common to see this plasmid and the Rep/Cap plasmid combined in a single large construct for simplified production. (B) After 48–72 hr cells are harvested and lysed. Vectors can be purified by either column chromatography or density gradient centrifugation, which can separate AAV from contaminating cellular proteins as well as separating empty capsids from genome-containing particles. It is not uncommon to see at least two purification steps employed for high purity vectors and column-based methods can vary depending on the serotype being purified. (C) Vector quantitation is most often performed by measuring DNase-resistant genomes by real-time PCR. In cases where verification is needed for the separation of full and empty capsids, this genome titering is combined with an ELISA-based assay for capsid protein. doi:10.1128/microbiolspec.MDNA3-0052-2014.f3

FIGURE 4

Adeno-associated virus (AAV) vector infectious pathway. (A) Infection is initiated by capsid binding to cell-surface receptors. In the case of AAV serotype 2 this is a two-step process involving binding to a primary glycan receptor followed by binding to a protein receptor that mediates endocytosis. Classical AAV2 internalization is believed to be a clathrin-mediated, dynamin-dependent process, although other pathways have been implicated. (B) AAV traffics through the endosomal system where it is exposed to a low-pH environment that triggers the externalization of the VP1/2 unique regions and activation of the auto-catalytic capsid protease. Exposure to the low-pH environment is necessary for productive AAV infection. (C) Escape from the endosomal compartment is mediated by the VP1 PLA2 domain, and AAV uses several nuclear localization signaling motifs to traffic to the nucleus and enter, most likely via the nuclear pore complex (NPC). (D) AAV uncoats its genome in the nucleus. In the case of single-stranded DNA vectors, second-strand synthesis must first occur before transcription, creating a bottleneck in the transduction process. Self-complementary (sc) vectors use a single mutant inverted terminal repeat (ITR) in conjunction with both + and − strands to produce a genome that is double-stranded upon uncoating so bypassing second-strand synthesis, albeit at the cost of lower packaging capacity. After a double-stranded genome is formed transcription and translation are driven by the transgene cassette and are not virus-specific. doi:10.1128/microbiolspec.MDNA3-0052-2014.f4