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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]

Presents a study in which an adeno-associated virus vector encoding an antisense RNA was used to transduce stable intracellular resistance to human immunodeficiency virus-1 (HIV-1) in human hemopoietic and non-hemopoietic cell lines. Antisense targets in all HIV-1 transcripts including the TAR sequence; Adeno-associated virus vectors representing a promising form of anti-retroviral gene therapy.

Focuses on efforts to develop a vaccine for chronic myelogenous leukemia (CML). Claim that CML has been responsive to immunotherapy and hematopoietic stem-cell transplantation; Outcome of a clinical trial of a BRC-ABL peptide vaccine for CML; Issues impacting the success of CML vaccines; Impact of using imatinib as a treatment for CML.

I first became aware of Barrie Carter's pioneering work on adeno-associated virus (AAV) nearly four decades ago, when I started my postdoctoral training in the laboratory of Ken Berns, yet another stalwart among what used to be a small group of individuals who were interested in AAV. Barrie also deserves accolades for having recruited Terry Flotte to his laboratory, and Terry has the distinct honor of having performed the very first Phase I clinical trial with a rAAV vector. I joined Barrie's small lab in the summer of 1991, where Roland Owens was studying Rep protein interactions with the inverted terminal repeat (ITR), and Terry Flotte was putting a CFTR transgene into the AAV vector. The first AAV vector to be injected into a human eye was manufactured by Barrie's company, Targeted Genetics Corporation - a gene therapy company that was ahead of its time. [Extracted from the article]

The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34+ cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) vectors are proving to be remarkably successful for in vivo gene delivery. Based upon reports of abundant AAV in the human marrow, we tested CD34+ hematopoietic stem cells for the presence of natural AAV. Here, we report for the first time, the presence of novel AAV variants in healthy CD34+ human peripheral blood stem cells. The majority of healthy peripheral blood stem cell donors were found to harbor AAV in their CD34+ cells. Every AAV isolated from CD34+ cells mapped to AAV Clade F. Gene transfer vectors derived from these novel AAVs efficiently underwent entry and postentry processing in human cord blood stem cells and supported stable gene transfer into long-term, in vivo engrafting human HSCs significantly better than other serotypes. AAVHSC-transduced human CD34+ cells engrafted in vivo and gave rise to differentiated transgene-expressing progeny. Importantly, gene-marked CD34+ stem cells persisted long term in xenograft recipients, indicating transduction of primitive progenitors. Notably, correlation of structure with function permitted identification of potential capsid components important for HSC transduction. Thus, AAVHSCs represent a new class of genetic vectors for the manipulation of HSC genomes. [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]

A recombinant AAV2 (rAAV2) vector encoding antisense RNA to HIV-1 transactivating region (TAR) was evaluated for transduction of human cord blood CD34+CD38- hematopoietic stem cells (HSC) capable of serial engraftment in nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Results revealed long-term multilineage marking in primary and secondary recipients, and significantly, an enrichment of transduced cells in secondary hosts, indicating efficient transduction of multipotential self-renewing HSC. These results were confirmed by the persistence of rAAV marking of clonogenic progenitors in serial analyses of recipient marrow. Upon HIV-1 challenge, the macrophage progeny of transduced CD34+ cells expressed antisense RNA and exhibited sustained and significant inhibition of virus replication as compared with controls in every donor tested, without selective pressure. This study represents a clear in viva demonstration of efficient rAAV2 transduction of human HSC. [ABSTRACT FROM AUTHOR]

Chronic myelogenous leukemia (CML) is characterized by a t(9;22) translocation resulting in expression of BCR-ABL fusion oncoproteins which are unique to the leukemic cells, necessary for oncogenesis, and potentially immunogenic. We have previously shown that human dendritic cells transduced with an adeno-associated virus vector encoding the fusion region of the b3a2 splice variant (p210b3a2) of the BCR-ABL oncoprotein elicit specific T-cell responses in vitro. Two cytotoxic T lymphocyte (CTL) clones generated in this fashion displayed restriction with previously unreported HLA alleles. The first, T1/B9, was CD4+ and restricted by DRB5*0101 (autologous) or DRB1*1101 (allogeneic). The minimum cytotoxic epitope (MCE) binding to DRB5*0101 for this clone was identified as FKQSSKALQ, overlapping the p210b3a2 fusion point (boldface). The MCE of DRB1*1101 for this clone differed from DRB5*0101, but also included the fusion point. The clonality of CTL T1/B9 was verified by analyses of TCRα/β chain usage and DNA sequence analyses. To our knowledge, this is the first description of a single clone recognizing both DRB5*0101 and DRB1*1101. The other CTL clone, T1/33, was CD8+ and recognized HLA-B*3501 or B*3503 complexed with an MCE, RPVASDFEP, derived from the c-abl sequence in proximity to the p210b3a2 fusion point. K562 cells transfected with plasmids encoding HLA-DRA + B5*0101, B*3501, or B*3503 but not controls expressing DRA + DRB1*1501 were lysed by cognate CTL clones, confirming that DRB5*0101 and B*3501/3 could present p210b3a2 joining region epitopes via endogenous processing. The identification of three additional HLA alleles (DRB5*0101, B*3501, and B*3503) presenting the p210b3a2 fusion-region antigen will broaden the application of vaccine strategies for targeting CML cells. The findings of single CTL clones cross-recognizing autologous (DRB5*0101 or B*3501) and allogeneic (DRB1*1101 or B*3503) HLA alleles presenting BCR-ABL fusion-region epitopes implies the potential separation of graft-versus-leukemia from graft-versus-host effects. [ABSTRACT FROM AUTHOR]

An abstract of the article "Comparison of Pseudotyped rAAV Vectors for Transduction of Primary Hematopoietic Stem and Progenitor Cells Assayed In Vitro and In Vivo," by Lijing Li, Sarah Hardy, Leslie Smith-Powell, Sai Srinivas, Stephen J. Forman, K. K. Wong and Saswati Chatterjee is presented.

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]

An abstract of the article "rAAV-2 Transduction of Primitive Human Hematopoietic Stem Cells Capable of Serial Engraftment in Immune Deficient Mice," by Helicia Paz, Leah Santat, Lijing Li, Christie Wong, Stephen J. Forman, K. K. Wong and Saswati Chatterjee is presented.