Reducing the Risk of Adeno-Associated Virus (AAV) Vector Mobilization with AAV Type 5 Vectors

Abstract

Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

Insight into the Mechanism of Inhibition of Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex

“…We wanted to determine if a change to the sequence or structural features of the ITR affects MRN inhibition of rAAV. Our group has described ITRs altered in sequence and structure from the serotype 2 ITR that retain the replication and packaging function (43,66). The ITR of serotype 5 AAV shares only ϳ57% nucleotide sequence identity with that of AAV2 and also differs in the spacing of terminal and nicking stem-loop structures and overall length (Fig.…”

Highly Divergent Integration Profile of Adeno-Associated Virus Serotype 5 Revealed by High-Throughput Sequencing

“…AAV-5 integration is also much more localized around Rep binding and nicking sites than the broad peaks generated by AAV-2 (21). These factors, combined with previous observations regarding the relative risks of mobilization (3,26), imply that AAV-5-derived vectors may present a better risk profile for both Rep-mediated and episomal gene therapy applications.…”

“…30% of the population is seropositive for AAV-5 specific antibodies (2,5,6). In addition, it is the most divergent AAV yet discovered (4, 7) and, uniquely, does not cross-complement the replication of other serotypes (3,26,27). AAV-2 is known to integrate at a high frequency in the human genome, with preference for a single locus, AAVS1 (15,18,21).…”

“…Although the DNA sequence motif which binds AAV-5 Rep is similar to other serotypes, the endonuclease cleavage site is completely distinct (3). Underscoring this divergence, AAV-5 Rep protein does not cross-complement the replication of other serotypes (3,26,27). Thus, the primary driver of targeted viral integration differs between AAV-5 and the other known AAVs.…”

Viral vectors for gene delivery to the central nervous system

“…Evidence that vectors may be replicated in the presence of WT AAV and helper-virus infection has been produced from experiments in cell culture and in vivo (Afione et al, 1996; Cheung et al, 1980). To address this, novel ITRs have been developed that are not replicated by Rep proteins of WT AAV and thus, cannot be mobilized (Hewitt et al, 2009; Hewitt and Samulski, 2010). …”