Adeno-associated viruses (AAVs) are single-stranded DNA viruses that are endemic in human populations without known clinical sequelae and are being evaluated as vectors for human gene therapy. To better understand the biology of this virus, we examined a number of nonhuman primate species for the presence of previously uncharacterized AAVs and characterized their structure and distribution. AAV genomes were widely disseminated throughout multiple tissues of a variety of nonhuman primate species. Surprising diversity of sequence, primarily localized to hypervariable regions of the capsid protein, was detected. This diversity of sequence is caused, in part, by homologous recombination of co-infecting parental viruses that modify the serologic reactivity and tropism of the virus. This is an example of rapid molecular evolution of a DNA virus in a way that was formerly thought to be restricted to RNA viruses.A deno-associated viruses (AAVs) belong to the Parvoviridae family, which is characterized as small animal viruses with linear single-stranded DNA genomes that replicate in the presence of helper virus such as adenovirus (1). AAVs are being evaluated as vectors for human gene therapy (2). The initial characterization of this group of viruses was based on serologic crossreactivity by using complement fixation and neutralizing assays (3). Six distinct serotypes of AAV have been described, of which five were initially isolated as contaminants of adenovirus preparations (4-6). Sequence analysis of selected AAV isolates revealed divergence throughout the genome that is most concentrated in hypervariable regions (HVRs) of the capsid proteins (7-10). Epidemiological data indicate that all known serotypes are endemic to primates, although isolation of clinical isolates has been restricted to AAV2 and AAV3 from anal and throat swabs of human infants and AAV5 from a human condylomatous wart (11)(12)(13)(14). No known clinical sequelae have been associated with AAV infection. Vectors based on replication-defective forms of AAV have been evaluated in preclinical and clinical models of gene therapy (2). Detection and Recovery of AAV Sequences. DNA was extracted and analyzed for the presence of AAV DNA by using a PCR strategy to amplify a 255-bp (15) fragment called the ''signature region'' by using conserved oligonucleotides. To directly amplify a 3.1-kb full-length Cap fragment from NHP tissue and blood DNAs, two other highly conserved regions were identified in AAV genomes for use in PCR amplification of large fragments. A primer within a conserved region located in the middle of the Rep gene was selected (AV1ns, 5Ј-GCTGCGTCA ACTGGACCA AT-GAGAAC-3Ј) in combination with the 3Ј primer located in another conserved region downstream of the Cap gene (AV2cas, 5Ј-CGCAGAGACCAAAGTTCAACTGAAACGA-3Ј) for amplification of full-length cap fragments. The PCR products were Topo-cloned (Invitrogen), and sequence analysis was performed by Qiagengenomics (Qiagengenomics, Seattle) with an accuracy of Ն99.9%. A total of 50 capsid clones were i...

Heart disease is the leading cause of morbidity and mortality. Cardiac gene transfer may serve as a novel therapeutic approach. This investigation was undertaken to compare cardiac tropisms of adeno-associated virus (AAV) serotypes 1, 6, 7, 8, and 9. Neonatal mice were injected with 2.5 ϫ 10 11 genome copies (GC) of AAV serotype 1, 6, 7, 8, or 9 expressing LacZ under the control of the constitutive chicken ␤-actin promoter with cytomegalovirus enhancer promoter via intrapericardial injection and monitored for up to 1 year. Adult rats were injected with 5 ϫ 10 11 GC of the AAV vectors via direct cardiac injection and monitored for 1 month. Cardiac distribution of LacZ expression was assessed by X-Gal histochemistry, and ␤-galactosidase activity was quantified in a chemiluminescence assay. Cardiac functional data and biodistribution data were also collected in the rat. AAV9 provided global cardiac gene transfer stable for up to 1 year that was superior to other serotypes. LacZ expression was relatively cardiac specific, and cardiac function was unaffected by gene transfer. AAV9 provides high-level, stable expression in the mouse and rat heart and may provide a simple alternative to the creation of cardiac-specific transgenic mice. AAV9 should be used in rodent cardiac studies and may be the vector of choice for clinical trials of cardiac gene transfer. 1359