No abstract

Recombinant adeno-associated virus (rAAV) shows significant promise as a vector for gene transfer in pre-clinical models of human disease, and is currently being evaluated in human clinical trials. As a consequence, increasing attention is being turned to the important tasks of optimizing rAAV titer, purity, and stability. We have observed dramatic variation in divalent cation dependence for thermostability of different rAAV vectors. To further investigate this observation, the thermostability of eight different vector constructs ranging in size from 73 to 107% of wild-type genome size (4.68 kilobases) was determined in the presence and absence of divalent cations. Virions containing smaller genomes (i.e., <85% wild type) were relatively divalent cation independent for thermostability. In contrast, virions containing recombinant genomes close to, or exceeding, wild-type size (i.e., >95% wild type) were dependent on divalent cations for thermostability. Genome sequence also appeared to be a factor in the thermostability of the larger rAAV vectors. These observations are of both practical and theoretical significance. Divalent cations should be included in all buffer solutions used during rAAV purification and storage, and unnecessary heat exposure avoided. These data also demonstrate that different recombinants of a particular virus should not be assumed to possess the same thermostability profile.

Adeno-associated virus (AAV) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in humans, AAV type 2 (AAV2) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an AAV Rep binding element (RBE), a spacer, and a nicking site. Here, we report the DNA sequence of an African green monkey AAV integration site isolated from CV-1 cells. Overall, it has 98% homology to the analogous human site, including identical spacer and nicking sequences. However, the simian RBE is expanded, having five perfect directly repeated GAGC tetramers. We carried out a number of in vitro and in vivo assays to determine the effect of this expanded RBE sequence on the Rep-RBE interaction and AAV targeted integration. Using electromobility shift assays it was demonstrated that AAV4 Rep68 bound the expanded RBE with a sixfold-greater affinity than the human RBE. To determine the basis for the affinity increase, DNase I protection and methylation interference (MI) assays were performed. Comparison of footprints on both the human and simian RBEs revealed nearly identical protection; however, MI analysis suggested greater interaction with the guanine nucleotides of the expanded RBE, thus providing a biochemical basis for the increased binding activity. In vivo, integration targeted to the simian RBE was demonstrated by PCR analysis of latently infected Cos-7 cells. Interestingly, the frequency of site-specific integration was twofold greater in Cos-7 cells than in HeLa cells. Overall, these experiments establish that the simian RBE, identified in CV-1 cells, functions analogously to the human RBE and provide further evidence for a developing model that proposes individual roles for the RBE and the spacer and nicking site elements.To date, eight different but closely related adeno-associated virus (AAV) serotypes (types 1 to 8) which infect primates have been identified (reviewed in references 6 and 16). These viruses are members of the family Parvoviridae, and they have a unique biology that makes them particularly well suited as gene therapy vectors (1,6,16,35,(42)(43)(44). Additionally, AAV (specifically type 2 [AAV2]) has been shown to be the only known mammalian virus having the potential to site-specifically integrate into the human genome, preferentially targeting a site on human chromosome 19q13.3qter (ch19) (6,9,20,21,25,33,34,41) The AAV genome is composed of linear single-stranded DNA ϳ4.7 kb long (42, 49), and while AAV2 is the most thoroughly characterized AAV, studies of other serotypes are under way (10, 12, 38). These include monkey-specific AAV4, which was recently cloned and found to have a high degree of homology to AAV2 (12). The AAV4 genome has two open reading frames, like that of AAV2, and codes for similar Rep proteins (Rep78 [78 kDa], -68, -52, and -40). For example, the AAV4 Rep68 protein has 90% amino acid identity to AAV2 Rep68, with 5% of the divergent amino acids being similar in hydrophobi...

No abstract

No abstract