Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle

Abstract

Reengineering the receptor footprints of adeno-associated virus (AAV) isolates may yield variants with improved properties for clinical applications. We generated a panel of synthetic AAV2 vectors by replacing a hexapeptide sequence in a previously identified heparan sulfate receptor footprint with corresponding residues from other AAV strains. This approach yielded several chimeric capsids displaying systemic tropism after intravenous administration in mice. Of particular interest, an AAV2/AAV8 chimera designated AAV2i8 displayed an altered antigenic profile, readily traversed the blood vasculature, and selectively transduced cardiac and whole-body skeletal muscle tissues with high efficiency. Unlike other AAV serotypes, which are preferentially sequestered in the liver, AAV2i8 showed markedly reduced hepatic tropism. These features of AAV2i8 suggest that it is well suited to translational studies in gene therapy of musculoskeletal disorders.

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

“…The second approach is to use error-prone PCR to generate a library of AAV variants and select for NAb escape mutants in the presence of NAb in vitro (31,41,56). The third approach is to randomly modify AAV capsids and to escape NAbs (2,36,37) and yield novel capsids with preferred tropism. The fourth approach is to use other types of AAV which have shown no or low NAb cross-reactivity in mice (27,30,38,60,65).…”

“…These findings present a concern to the gene therapy community as to how we can avoid antagonistic NAb activity during systemic delivery of AAV vectors. To overcome this concern, several approaches have been exploited in our lab and by other groups, including (i) the utilization of polymers to cover the AAV capsid and block NAb recognition (11,32,33), (ii) the development of NAb escape mutants by error-prone PCR in vitro (31,41,56), (iii) the application of other types of AAV vectors (27,30,38,60,65), and (iv) the generation of chimeric types via AAV shuffling (2,36,37). In this study, we have systematically explored the possibility of using different types and AAV mutants as alternative vectors for intramuscular gene delivery in mice preimmunized against different AAV types.…”

Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

“…All structural models were visualized using PyMOL, with residues forming the galactose binding site (AAV9 VP1 numbering Asp-271, Asn-272, Tyr-446, Asn-470, Ala-472, Val-473, and Trp-503) (13) and heparan sulfate binding site (AAV2 VP1 numbering Arg-487, Lys-527, Lys-532, Arg-585, and Arg-588) (10 -12, 18) highlighted in orange and purple, respectively. The i8 motif (AAV8 VP1 numbering 588-QQNTAP-593) was colored in deep blue (19). In VP3 trimer models, different monomers were colored in pale green, light blue, and light pink.…”

“…Amino acid residues involved in Gal recognition and other flanking residues from AAV9 were remarkably well tolerated on different AAV serotype capsids because the packaging efficiencies of these AAV chimeras are comparable with parental strains. Multiple AAV chimeras on the basis of AAV serotypes 1, 2, 6, and 8 and the previously engineered AAV2i8 mutant (19) were obtained (at titers ranging from 5 ϫ 10 11 to 5 ϫ 10 12 viral genome copies/ml, data not shown). We first carried out a detailed characterization of the dual glycanbinding AAV chimera, dubbed AAV2G9 (where "g" stands for the Gal footprint and the numbers identify the recipient and donor capsid serotypes, respectively).…”

“…The resulting chimera, AAV2i8G9, was thus constructed on the basis of an AAV2i8 chimera (19), thereby enabling studies solely focused on the Gal motif in the AAV2 capsid context. The original AAV2i8 chimera has been described by our laboratory earlier (19). As shown in Fig.…”