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Adeno-associated virus (AAV)-based vectors have transformed into powerful elements of genetic medicine with proven therapeutic efficacy and a good safety profile. Over the years, efforts to transduce hematopoietic stem cells (HSCs) with AAV2 vectors have, however, been challenging. While there was evidence that AAV2 delivered vector genomes to primitive, quiescent, multipotential, self-renewing, in vivo engrafting HSCs, transgene expression was elusive. In this study, we review the evolution of AAV transduction of HSC, starting with AAV2 vectors leading to the isolation of a family of naturally occurring AAVs from human CD34+ HSC, the AAVHSC. The stem cell-derived AAVHSCs have turned out to have remarkable potentials for genetic therapies well beyond the hematopoietic system. AAVHSCs have tropism for a wide variety of peripheral tissues, including the liver, muscle, and the retina. They cross the blood–brain barrier and transduce cells of the central nervous system. Preclinical gene therapy studies underway using AAVHSC vectors are discussed. We review the notable ability of AAVHSCs to mediate efficient, seamless homologous recombination in the absence of exogenous nuclease activity and discuss the therapeutic implications. We also discuss early results from an AAVHSC-based clinical gene therapy trial that is underway for the treatment of phenylketonuria. Thus, the stem cell-derived AAVHSC, offer a multifaceted platform for in vivo gene therapy and genome editing for the treatment of inherited diseases. [ABSTRACT FROM AUTHOR]

Background aims. Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. Methods. We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. 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, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We 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. Conclusions. 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 increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans. [ABSTRACT FROM AUTHOR]

The search for the ideal vector for stem cell gene therapy continues as recognized problems remain with all existing viral vectors. The recent identification of over 100 new serotypes of AAV significantly expands the repertoire of available gene transfer vectors. Additionally, the capacity to pseudotype rAAV2 genomes in novel capsids facilitates evaluation. We and others have analyzed rAAV2-mediated gene transfer into hematopoietic stem cells (HSC). However, despite efficient gene-marking of primitive HSCs, there are identified limitations to transgene expression especially at early time points. The use of novel capsids has been shown to overcome these limitations in other tissues and expand the host range of AAV. We tested transduction of CD34+ cells from cytokine-primed peripheral blood, CD34+CD38− cells from cord blood (CB) and the CD34+CD38- cell line TF1a with AAV2-dsRed pseudotyped in capsids of AAV1, 2, 5, 7, 8 and 9. Transduced cells were analyzed by flow cytometry 24 and 48 hours post transduction. Results revealed that the highest level of transgene expression was observed with AAV7 in HSCs from all sources while AAV 2 and 9 showed the lowest. The hierarchy of transgene expression in vitro was: AAV7>AAV8≥AAV1>AAV5>AAV2=AAV9 regardless of the source of CD34+cells. These results indicate that the use of certain novel capsids allows circumvention of limitations to expression from AAV2. We next evaluated in vivo engraftment of human HSCs transduced with pseudotyped rAAV in a NOD/SCID xenotransplant model. Transduced CD34+ cells purified from either CB or cytokine primed peripheral blood were transplanted into sublethally irradiated mice. The pseudotyped rAAV2 genome encoded the firefly luciferase gene under the control of the CBA promoter. Serial live imaging of recipients revealed transgene expression from each of the 6 serotypes tested. However the levels and trends for long term expression variedby serotype. At early time points, the highest initial expression was observed with AAV9 followed by AAV7 and 8. AAV2 showed the lowest level of initial expression. However differences emerged over time. AAV7, 9 and 8 capsids emerged as the best serotypes for long-term expression in vivo, followed by AAV2. AAV5 and 1 showed a rapid decline in expression after initial moderately high levels of expression. These differences suggest that the processing of pseudotyped rAAV in vivo varies with the capsid. AAV serotypes also appear to have different transduction properties in vivo as compared with in vitro. The mechanisms underlying these differences are being investigated. The rapid and robust expression seen in vivo with certain serotypes is highly promising and underscores the need for further evaluation of pseudotyped rAAV for stem cell gene transfer.Molecular Therapy (2006) 13, S188–S188; doi: 10.1016/j.ymthe.2006.08.555 [ABSTRACT FROM AUTHOR]