High Efficiency Transduction Of Hematopoietic Stem Cells And Erythroid Lineage-restricted Expression By Capsid-optimized Recombinant Adeno-associated Viral Vectors

BackgroundHemoglobinopathies, such as β-thalassemia and sickle cell disease, are by far the most common monogenic diseases that afflict humans worldwide, with an incidence rate of1:600. Transplantation of patient-derived, genetically-modified autologous transplantation of hematopoietic stem cells (HSCs) is, to date, the most promising alternative to allogeneic transplantation for such kind of diseases, if high-efficiency transduction of HSCs, and erythroid lineage-restricted expression of the human β-globin gene can be achieved. Indeed, in a recent clinical trial, a recombinant lentiviral vector-mediated β-globin gene transfer in an adult patient with severe β-thalassaemia led to transfusion-independence121. However, the observed therapeutic benefit was also accompanied by transcriptional activation of a cellular proto-oncogene, HMGA2, following clonal expansion of myeloid cells. Thus, it is clear that alternatives to lentiviral vectors are needed. The safety as well as clinical efficacy of recombinant vectors based on a non-pathogenic human virus, the adeno-associated virus2(AAV2) has attracted attention in number of Phase Ⅰ-Ⅲ clinical trials. However, controversies abound with reference to the transduction efficiency of AAV2vectors in human HSCs. For example, we and others have reported that AAV2vectors efficiently transduce human CD34+cells at relatively low multiplicities of infection (MOIs), whereas some have reported that successful transduction of human CD34+cells by AAV2vectors requires exceedingly high (>106) MOIs. One group claimed that the alleged transduction of human CD34+cells by AAV2vectors is due to contaminants. The underlying bases of these discrepancies have been reviewed previously.In recent years, a number of additional AAV serotype vectors have become available, but 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.We and others have also documented that site-directed mutagenesis of specific surface-exposed tyrosine (Y) residues on AAV serotype capsids leads to higher transduction efficiency both in vitro and in vivo in various cell types, but how these mutations work on HSCs has not been evaluated, yet. Methods and ResultsIn the present study, we systematically evaluated the transduction efficacy of all available AAV serotype vectors (AAV1through 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.We observed that although there are only six amino acid differences between AAV1and AAV6, AAV1, but not AAV6, transduce mouse HSCs cells well, whereas AAV6, but not AAV1, transduce human HSCs well. None of the10serotypes transduce monkey HSCs well. We also evaluated the transduction efficiency of AAV6vectors containing mutations in surface-exposed tyrosine residues, and observed that tyrosine (Y) to phenylalanine (F) point mutations in residues705, et al., led to a significant increase in transgene expression in human HSCs, both in vitro, and in a mouse xenograft model in vivo.we next generated the wild-type (WT) and tyrosine-mutant AAV6vectors containing the following erythroid cell-specific promoters:β-globin promoter (pp) with the upstream hyper-sensitive site2(HS2) enhancer from the P-globin locus control region (HS2-βp), and the human parvovirus B19promoter at map unit6(B19p6). Transgene expression from the B19p6was significantly higher than that from the HS2-βp, and increased up to30-fold and up to20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34+cells in vitro. Expression from the ubiquitous chicken β-actin promoter (CBAp), on the other hand, remained unchanged. Transgene expression from the B19p6or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6in the WT AAV6capsid, and that from the HS2-βp in the DM-AAV6capsid, transgene expression from the B19p6promoter in the Y705+731F AAV6capsid was significantly higher than that from the HS2-Pp.ConclusionsWe report here that:(ⅰ) AAV1vectors transduce primary murine HSCs most efficiently;(ⅱ) None of the10AAV serotype vectors transduce monkey HSCs well in vitro;(ⅲ) AAV6vectors are the most efficient in transducing primary human HSCs, both in vitro, and in a mouse xenograft model in vivo; and (ⅳ) The transduction efficiency of AAV6vectors is further augmented following site-directed mutagenesis of specific surface-exposed tyrosine residues, both in vitro and in vivo. These studies suggest that the optimized AAV6vectors may prove to be a safe and effective alternative to lentiviral vectors for their potential use in HSC-based gene therapy in humans. These studies suggest that the tyrosine-mutant AAV6serotype 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.We also document that the transgene expression from the erythroid lineage specific promoter B19p6is significantly higher than that from the HS2-βp, and can be further increased following erythroid-differentiation in vitro and in a murine xenograft model in vivo. These data suggest that the scAAV6-B19p6-globin vectors might prove to be useful for the potential gene therapy of human hemoglobinopathies in general, and β-thalassemia and sickle cell disease in particular. These studies suggest that the tyrosine-mutant AAV6serotype 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.