Cloning of an Avian Adeno-Associated Virus (AAAV) and Generation of Recombinant AAAV Particles

Abstract

Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The entire genome of AAAV displays 56 to 65% identity at the nucleotide level with the other known AAVs. The AAAV genome has inverted terminal repeats of 142 nucleotides, with the first 122 forming the characteristic T-shaped palindromic structure. The putative Rep-binding element consists of a tandem (GAGY) 4 repeat, and the putative terminal resolution site (trs), CCGGT/CG, contains a single nucleotide substitution relative to the AAV 2 trs. The Rep open reading frame of AAAV displays 50 to 54% identity at the amino acid level with the other AAVs, with most of the diversity clustered at the carboxyl and amino termini. Comparison of the capsid proteins of AAAV and the primate dependoviruses indicate that divergent regions are localized to surface-exposed loops. Despite these sequence differences, we were able to produce recombinant AAAV particles carrying a lacZ reporter gene by cotransfection in 293T cells and were able to examine transduction efficiency in both chicken primary cells and several cell lines. Our findings indicate that AAAV is the most divergent AAV described to date but maintains all the characteristics unique to the genera of dependovirus.Adeno-associated viruses (AAVs) are small, nonpathogenic parvoviruses that require coinfection with a helper virus, such as adenovirus or herpesvirus, for productive infection (2). To date, eight AAV isolates (AAV types 1 to 8 [AAV1 to -8]) have been characterized and sequenced (2,4,19,20,25,32,51,56), with AAV2 having been the most extensively studied.AAV virions are approximately 20 to 25 nm in diameter and are composed of a mixture of assembled proteins (VPs) that encapsidate a linear ϳ4.7-kb single-stranded DNA (ssDNA) of plus or minus polarity (7, 43). The genome of AAVs is flanked by inverted terminal repeats (ITRs), which in the case of AAV 2 are 145 nucleotides in length. The ITR is organized as three interrupted palindromes that can fold in an energetically favored T-shaped hairpin structure, which can exist in two orientations, termed flip and flop (42). The ITRs serve as origin of replication and contain cis-acting elements required for rescue, integration, excision from cloning vectors, and packaging (41,42,49,58).The genetic map of the AAVs has been derived primarily from studies of AAV2 but is conserved in all serotypes (26,27,29,36,42,45,46,58,60,64). Two major open reading frames (rep and cap ORFs) and three transcriptional active promoters (P 5 , P 19 , and P 40 ) have been identified in the genome of AAV2. The P 5 and P 19 promoters encode the nonstructural rep...

Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype

“…In fact, nearly all of the AAV vector DNAs pseudotyped to date were based on the AAV-2 prototype, which seems paradoxical considering that ITRs from at least eight different AAVs were cloned or sequenced thus far (4,8,9,55,59,68). Perhaps this was mostly due to practical reasons, in particular, the limited availability of genotyped vector plasmids which are as flexible and convenient as the many existing AAV-2 variants.…”

“…With few exceptions (see below), these sites are largely conserved among all eight AAV isolates that have been fully cloned to date, namely, AAV-1 through -6, as well as avian (AAAV) and bovine (BAAV) AAVs. Likewise maintained is the capability of all single-stranded ITRs to assume a secondary hairpin structure despite variations in total length from 142 (AAAV) to 167 (AAV-5) nucleotides and DNA homologies as low as 55% (4,24,59) (AAV-5 and the rest).…”

AAV Hybrid Serotypes: Improved Vectors for Gene Delivery

“…AAV10 and AAV11 were isolated from cynomolgus monkey tissue using a similar PCR strategy, and demonstrated the ability to infect both human and monkey cells [Mori, S et al, 2004]. In addition to human and primates, AAV has also been isolated from different species including cow [Schmidt, M et al, 2004], chicken [Bossis. I and Chiorini, JA, 2003], sheep [Clarke, JK et al, 1979], snake [Farkas, SL et al, 2004], lizard [Jacobson, ER et al, 1996], and goat [Olson, EJ et al, 2004].…”

A role for adeno-associated viral vectors in gene therapy

“…AAV genomes have not been isolated only from primates, but also from other species such as horse (Dutta, 1975), lizard (Jacobson et al, 1996), chicken (Bossis and Chiorini, 2003), cow (Schmidt et al, 2004), snake (Farkas et al, 2004) and goat (Olson et al, 2004;Arbetman et al, 2005). Among these, AAV isolates from bovine, avian and caprine species have been developed into vectors for gene transfer studies (Schmidt et al, 2004;Bossis and Chiorini, 2003;Arbetman et al, 2005).…”

“…Among these, AAV isolates from bovine, avian and caprine species have been developed into vectors for gene transfer studies (Schmidt et al, 2004;Bossis and Chiorini, 2003;Arbetman et al, 2005).…”