Peptide Ligands Incorporated into the Threefold Spike Capsid Domain to Re-Direct Gene Transduction of AAV8 and AAV9 In Vivo

Abstract

Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes.

Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant ‐7m8

“…The other vectors did not benefit from 7m8 insertion. These findings are consistent with previous studies that demonstrate that peptide functionality is largely determined by the capsid scaffold, thus preventing a direct transfer of lead sequences from AAV2 onto other capsids and rather requiring a firsthand selection for each new serotype (Grimm et al, 2008;Michelfelder et al, 2011;Varadi et al, 2012;Ying et al, 2010).…”

“…We used previously reported potential capsid regions amenable for the insertion of the 7m8 peptide that may re-direct the natural viral tropism ( Fig. 2B-E Michelfelder et al, 2011). We hypothesized that the 7m8 peptide may improve transduction on its own or in conjunction with its surrounding amino acids.…”

“…All rights reserved 15 has been identified as its co-receptor and AAV8 binding to lamR is mediated by the amino acids 491 to 547 and 593 to 623 (Akache et al, 2006). Moreover, peptide insertions on AAV8 capsid at position 590 allow modification of its tropism (Michelfelder et al, 2011;Raupp et al, 2012) but amino acids 588 to 592 are not necessary for AAV8 binding and uptake. This region is thus involved in but not necessary for interaction with cellular receptors (Raupp et al, 2012).…”

Engineering the AAV capsid to optimize vector–host-interactions

“…Nevertheless, using rational design approaches, the N 0 -terminus of VP2 and the tips of the protrusions at the 3-fold symmetry axis were identified as the as of yet most promising positions [29,36]. These sites were initially identified and explored for cell entry targeting in the context of AAV2, but homologous sites showed promise also in the context of other serotypes such as AAV8 or AAV9 [37,38]. In line with results obtained for non-genetic targeting approaches, insertion of receptor-binding ligands conferred AAV targeting vectors with the ability to transduce cell types permissive for the parental serotypes with improved efficiency and -even more important -opened up the avenue for modification of non-permissive cells types ( [6,29,39] and Table 1).…”

“…Functionality of peptides selected by AAV2 peptide display across serotypes was proven for endothelial cell targeting, one of the main target cell types in AAV-based cell entry targeting approaches [37], and for the ESGLSQS peptide selected by in vivo AAV2 peptide display in a xenograft tumor model [38]. Variations of the initial strategy were more recently explored.…”