Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo

Abstract

Background Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention owing to their safety and efficacy in number of Phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a handful of additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. Methods Here, we evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey, and human HSCs, respectively. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. Results We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduce mouse HSCs cells well, whereas AAV6, but not AAV1, transduce human HSCs well. None of the 10 serotypes transduce cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues, and observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705, and 731, led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. Discussion These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs, and that it is possible to further increase the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.

Gene Transfer Properties and Structural Modeling of Human Stem Cell-derived AAV

“…The use of AAV capsids with site-directed mutagenesis of surface exposed tyrosine residues led to a substantial increase in the level of transduction of in vivo engrafting stem cells. 24,26,27 Similarly, the use of pseudotyped recombinant AAV using capsids of other serotypes was also found to result in higher transduction efficiencies, 26 suggesting that significant improvement in gene transfer efficiency could be addressed through capsid-based strategies.…”

“…20,21 We and others have previously reported AAV transduction of human CD34 + cells, a population enriched for hematopoietic stem cells (HSCs). [22][23][24][25][26][27][28][29][30] Although transduction of in vivo engrafting CD34 + stem cells was noted, the efficiencies were modest. The use of AAV capsids with site-directed mutagenesis of surface exposed tyrosine residues led to a substantial increase in the level of transduction of in vivo engrafting stem cells.…”

Development of novel AAV serotype 6 based vectors with selective tropism for human cancer cells

“…Considering the similarity of capsid structures between AAV2 and AAV6 serotypes (44,45), we hypothesized and experimentally proved that mutations of these important conserved residues could lead to the improvement of transduction efficiency of AAV6 vectors in several different cell types (23,27,46,47).…”

“…These studies resulted in the development of novel AAV vectors containing one or more mutations on the capsid that transduce various cell types and tissues more efficiently compared with wild-type (WT) AAV vectors (20,(22)(23)(24)(25)(26)(27).…”

Engineering the AAV capsid to optimize vector–host-interactions

“…Substitution of Y to F resulted in improved AAV2 transduction efficiencies in permissive and nonpermissive cells both in cell culture and in vivo. Success of the strategy was preserved for other serotype capsids [22] and for alternative targets of phosphorylation [23 ], arguing for proteasomal degradation as a general barrier limiting efficiency of AAV vectors.…”