Abstract
Recombinant AAV vectors are some of the most attractive vehicles for therapeutic gene delivery available today, for many reasons. One is that AAV capsids are highly amenable to genetic engineering, permitting the creation and identification of vectors with novel properties including target cell specificities. One potent strategy is to insert a randomized peptide library into an exposed AAV2 capsid region, followed by iterative selection on desired cells to enrich single peptides binding to cell-surface receptors and hence mediating AAV transduction. However, several problems hamper the use of AAV2 as scaffold for peptide display: (i) neutralizing anti-AAV2 antibodies that are prevalent in the human population may interfere with transduction of AAV2 peptide mutants, and (ii) if not fully ablated, the AAV2 tropism for its genuine receptors will dampen retargeting efficiencies. Here, we aimed to overcome both problems by developing 11 other AAV serotypes as templates for peptide display, AAV1, 3-9, rh.10, po.1 and 12. We first engineered their capsid genes to contain unique restriction sites and then exploited these for insertion of 18 different oligonucleotides encoding peptides which were pre-selected in AAV2 on various cells. The resulting >200 AAV variants were subsequently produced as YFP-expressing vectors and screened in over 80 human or murine cells, including primary hepatocytes or keratinocytes, as well as T-cells and stem/iPS cells. Strikingly, many capsid-peptide combinations vastly outperformed the AAV2 counterparts, especially those based on AAV serotypes 1, 7-9 and rh.10, and those carrying 7mer peptides with an NxxRxxx motif (x = any amino acid). In addition, we found that disruption of residue R585 in AAV2 boosted the potency of several peptide display mutants by up to two orders of magnitude, whereas the same mutation had no effect in the chimeric capsid AAV-DJ, despite a common region surrounding R585. Another puzzling notion was that some serotypes were inactivated by peptide display, tempting us to model all 12 capsids after insertion of a prototype peptide. This revealed that in contrast to our prediction from linear sequence alignments, the exact position of the displayed peptide differed by up to four residues between the 12 capsids. Indeed, once adjusted accordingly, AAV3 and AAV6 were also markedly enhanced upon peptide insertion. Collectively, our data imply an enormous potential of alternative AAV serotypes as scaffolds for peptide display and provide a set of vital guidelines for their further development as gene therapy vectors. To aid in this process, we also present a protocol for AAV spotting and drying in 96/384-well plates which permits their long-term storage and shipping, and thus facilitates their evaluation in other labs.
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