Identification and Characterization of Novel Adeno-Associated Virus Isolates in ATCC Virus Stocks

Abstract

Adeno-associated viruses (AAVs) depend on a helper virus for efficient replication. To identify novel AAV isolates, we screened a diverse set of virus isolates for the presence of AAV DNA. AAVs found in 10 simian adenovirus isolates showed greater than 96% homology to AAV1 and AAV6 but had distinct biological properties. Two representatives of this group, AAV(VR-195) and AAV(VR-355), were studied in more detail. While the novel AAVs had high sequence homologies and required sialic acid for cell binding and transduction, differences were observed in lectin competition, resulting in distinct tropisms in human cancer cell lines.Adeno-associated virus (AAV) is a member of the Parvoviridae, a virus family characterized by a single-stranded linear DNA genome enclosed by a capsid with icosahedral symmetry. A hallmark of the AAV life cycle is its dependency on a helper virus for efficient productive replication. Adenovirus was originally identified as the AAV helper virus, but other viruses, such as herpesvirus and cytomegalovirus (7), can provide helper functions for AAV replication. Virus stocks have therefore served as a rich source for the discovery of AAV isolates; AAV1, AAV2, AAV3, AAV4, AAV6, and bovine AAV were all found as contaminants of adenovirus preparations (1-3, 8, 10-12, 15).In this article, we describe the analysis of virus stocks from the American Type Culture Collection (ATCC) for the presence of AAV DNA. Our goals were to identify ATCC virus isolates that contain AAV contaminations and, if novel, to characterize the isolates.Virus stocks supplied by the ATCC were analyzed for the presence of AAV DNA by a PCR-based assay, as described previously (5). AAV DNA was detected in 13 of 137 samples analyzed. AAV contaminations were frequently detected in adenovirus isolates (26%) but not in herpesvirus, retrovirus, coronavirus, orthomyxovirus, poxvirus, or reovirus stocks. The entire coding regions for the AAV Rep and Cap open reading frames were PCR amplified and subcloned, and several clones from each isolate were sequenced and analyzed. Adenovirusfree stocks of the novel recombinant AAVs were produced by standard cotransfection protocols (14).Ten of the AAVs detected in simian adenovirus stocks displayed at least 96% homology on the DNA level and 98% identity in the capsid amino acid sequence either to AAV1, to AAV6, or to each other. To determine whether these minor sequence changes could affect the biological activity of the isolates, 2 of the 10 isolates, AAV , and AAV(VR-355), isolated from ATCC respectively (GenBank accession numbers DQ180604 and DQ180605), were studied in greater detail. The VP1 capsid proteins of AAV and AAV(VR-355) differ by 7 and 6 amino acids, respectively, from that of AAV6. The locations of the divergent amino acids within the capsid were identified by superimposing the AAV(VR-195) and AAV(VR-355) VP1 sequences onto a pseudoatomic structure for AAV6 (Fig. 1). Divergent amino acids were found to cluster on the capsid surface around the threefold axis of symmetry, an area ...

Differential internalization and nuclear uncoating of self-complementary adeno-associated virus pseudotype vectors as determinants of cardiac cell transduction

“…[1][2][3][4][5][6][7][8][9][10][11][12] Multiple AAV serotypes have been isolated in recent years which show grossly different tissue and organ tropisms. [14][15][16][17][18][19][20] This may be due to different cellular receptor affinities of the respective AAV virions, but for only few of the currently known AAV types the cellular receptors are currently known. 14,22,23,35 The choice of AAV serotype and the optimal design of the AAV vector for a given therapeutic purpose also remain challenging because the molecular mechanisms beyond the receptor level, which determine their different potential to support transgene expression in various cell types, are incompletely understood.…”

“…[14][15][16][17][18][19][20] This may be due to different cellular receptor affinities of the respective AAV virions, but for only few of the currently known AAV types the cellular receptors are currently known. 14,22,23,35 The choice of AAV serotype and the optimal design of the AAV vector for a given therapeutic purpose also remain challenging because the molecular mechanisms beyond the receptor level, which determine their different potential to support transgene expression in various cell types, are incompletely understood. One well-documented factor limiting AAV-based transgene expression is the second-strand synthesis after infection with monomeric AAV vector.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12] Recent reports have shown that pseudotyping of AAV vectors as well as artificial capsid modifications 13 significantly alter their transgene targeting in vivo. Different AAV pseudotypes [14][15][16][17][18][19][20][21] have different receptorbinding sites in their capsids, providing a possible molecular basis to explain their differential tissue targeting. For only few AAV subtypes, however, the cellular receptor complements mediating cellular attachment and internalization are known so far [21][22][23] and therefore, the extent by which AAV targeting is determined at the receptor level could not yet be fully elucidated.…”

A role for adeno-associated viral vectors in gene therapy

“…Up to date, several AAV serotypes and over 100 AAV new isolates have been acquired from adenovirus stocks or from human/nonhuman primate tissues (Atchison et al, 1965;Hoggan et al, 1966;Parks et al, 1967;Bantel-Schaal and zur Hausen, 1984;Rutledge et al, 1998;Gao et al, 2002;Gao et al, 2004;Mori et al, 2004;Schmidt et al, 2006). The over 100 new AAV isolates are called AAV variants since their serology is not currently available.…”

“…In the past 4 years, several novel AAV serotypes, including AAV7, 8 and 9, and over 100 AAV variants have been found in human or nonhuman primate tissues (Gao et al, 2002;Gao et al, 2004;Mori et al, 2004;Schmidt et al, 2006;Gao et al, 2005). Different from AAV1 to 6, the new AAV serotypes and variants were not isolated as live virus forms; instead, they were isolated as DNA sequences using a specific PCR-based strategy (Gao et al, 2004;Gao et al, 2005).…”