ARF
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AAV Dirk Grimm DKFZ, Heidelberg, Germany [email protected]
Definition Adeno-associated viruses (AAV) are small DNA-containing viruses that belong to the family of Parvoviridae. So far six ! serotypes of adeno-associated viruses (AAV-1 to AAV-6) have been isolated from humans and primates. According to current knowledge, none of these serotypes are pathogenic in humans. AAV type 2 (AAV-2) has been studied for over 30 years and is thus the best characterized AAV serotype. All AAV serotypes are currently being developed and evaluated as gene transfer ! vectors for the human ! gene therapy of various inherited or acquired diseases, including different types of cancer.
Characteristics As with all other members of the ! Parvovirus family, AAV are non-enveloped, icosahedral capsids of about 18-24 nm in diameter. These
capsids carry linear, single-stranded DNA genomes of approximately 4.6-4.8 kilobases. The genomes of all known AAV serotypes have been cloned and sequenced. With the exception of AAV-5, which is distinct from the other serotypes at both the nucleotide and amino acid level, the AAV genomes display a high level of sequence homology. Consequently, the genomic structure is also very similar among the various AAV serotypes. As an example, the organization of the 4681 nucleotide AAV-2 genome is described (Fig. 1). The AAV-2 genome consists of two large open reading frames (orf), one at the left end encoding the nonstructural proteins (replication, ! rep orf), and one at the right end encoding the structural proteins (capsid, cap orf). In addition, a single intron sequence is found in the middle of the genome, where the rep and cap orfs overlap. The AAV-2 rep gene actually encodes four closely related proteins (Rep proteins) with partially overlapping amino acid sequences. On the basis of their molecular weights, these proteins were designated Rep78, Rep68, Rep52 and Rep40. Unspliced and spliced transcripts originating from a promoter located at map unit 5 (p5) are translated into the two large Rep proteins, Rep78 and Rep68. Rep52 and Rep40 are expressed from identically spliced mRNAs that initiate from a second promoter, p19. The third AAV-2 promoter, p40, controls transcription of the cap gene. Translation of differentially spliced cap mRNAs results in expression of the three proteins that form the AAV-2 capsid, VP1, VP2 and VP3. The two ends of the viral genome are constituted by short (AAV-2: 145 nucleotides) inverted terminal repeats (! ITR). These contain palindromic sequences which are able to fold into T-shaped stem loop structures. The ITRs
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AAV
are necessary and sufficient for replication and encapsidation of the viral genome during a productive infection of cells. Moreover, they are important for integration and rescue of the AAV DNA into or from the infected cell's genome, respectively. Cellular Characteristics (Viral Life Cycles) The AAV serotypes belong to the Parvovirus genus Dependovirus. This classification indicates their requirement for a second helpervirus to undergo a productive infection
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