1066. A Novel Class of Miniature Stabilized Double-Stranded AAV (msdsAAV) Vectors for the In Vivo Expression of Short Hairpin RNAs
Authors: Dirk Grimm, Joyce S. Lee, Konrad L. Streetz, Theresa A. Storm, Mark A. Kay
Journal: Molecular Therapy (2006)

Double-stranded adeno-associated viral (dsAAV) vectors are currently emerging as a powerful tool for the in vitro and in vivo expression of short hairpin RNA (shRNA), to induce RNAi-mediated gene silencing. In these vectors, the shRNA expression cassette is typically flanked by a full-length AAV-2 ITR (inverted terminal repeat) and a mutant form devoid of the terminal resolution site (trs), blocking the AAV DNA replication cycle and creating the ds phenotype. However, two inherent drawbacks have prevented the clinical evaluation of dsAAVs thus far: 1) the near sequence identity of the mutant and intact ITRs promotes their frequent recombination, resulting in repair of the mutation or in partial ITR deletion, and thus loss of the ds phenotype; and 2) the usually small size of shRNA expression cassettes, and the presumed lower DNA packaging limit of AAV capsids, require the additional and undesirable encapsidation of stuffer DNA. Here, we present an improved dsAAV vector design which overcomes both limitations and thus represents a better candidate for clinical evaluation. The major advance was replacement of the mutant AAV-2 ITR with a trs-deleted AAV-4 repeat, sharing <80% nucleotide homology with AAV-2. The increase in sequence diversity virtually eliminated the risks of gene conversion between the two ITRs, thus stabilizing the ds phenotype. In fact, in over 60 stabilized ds (sds) AAV preparations made thus far, we found no evidence for ITR deletion or repair. In addition to higher stability over regular dsAAV, sdsAAV particle yields were also increased and identical to those obtained with traditional single- stranded AAVs. Importantly, a unique property of sdsAAVs is their ability to accomodate inserts with sizes of as little as 0.2, and up to 2.4, kilobases, without effects on vector DNA stability, replication or packaging. Notably, we observed that regardless of vector DNA size, the AAV capsid preferentially packages a single copy of the viral genome. This corroborates the current model of AAV DNA encapsidation, in which a single pore in the capsid takes up the viral genome, before a conformational change prevents further DNA packaging. For in vivo evaluation, we next generated a miniature sds (msds) construct with a 0.6 kb insert, comprising our most robust U6 promoter-driven shRNA directed against human alpha-1- antitrypsin (hAAT). The vector was pseudotyped with AAV-8 (to maximize liver transduction) and peripherally infused into hAAT- transgenic mice. As compared to a full-length sdsAAV (including 1.7 kb stuffer DNA), we found no difference in long-term vector copy numbers, shRNA expression levels, or hAAT knockdown efficiencies (>80% on protein level). Notably, both double-stranded vectors (sds/msds) were significantly more potent than an identical dose of a conventional single-stranded AAV-8 expressing the same shRNA. In conclusion, msdsAAVs represent a novel class of powerful vectors for persistent and robust in vivo shRNA expression, which should be particularly interesting for clinical development due to the uniqueabsence of stuffer DNA sequences.