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Expression of the transduced human  -globin gene in bone marrow cells from recipient mice 6 months posttransplantation. Equivalent amounts of total RNA isolated from bone marrow samples obtained from control (lane 1) and transplanted (lanes 2–9) mice were used to amplify DNA products specific for the human  -globin gene (A) and the mouse  -actin gene (B) transcripts, respectively, in the presence ( ϩ ) or absence ( Ϫ ) of reverse transcriptase and analyzed on Southern blots using the indicated probes as described under Materials and Methods. The size of the RT-PCR product from the human  -globin gene is 291 bp and that from the mouse  -actin gene is 354 bp.
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Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. Here, we report successful AAV-mediated stable transduction and high-efficiency, long-term, erythroid lineage-restricted expression of a human β-globin gene in primary murine hematopoietic stem cells in vivo....
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... cells in recipient mice. Total RNA isolated from bone marrow cells obtained 6 months posttransplantation was subjected to RT-PCR analyses. Primers for PCR, following reverse transcription, were designed in such a way as to eliminate amplification of the mouse endogenous  -globin mRNA as well as to distinguish amplification products from the transduced viral sequences. The RT reactions were performed using primers specific for the mouse  -actin gene as well to ensure the quality and structural integrity of RNA preparations as described under Materials and Methods. The results of expression analysis by RT- PCR are shown in Fig. 4. As can be seen, the human  -globin transcripts were present only in bone marrow cells from recipient animals transplanted with the AAV-  -globin-transduced donor cells (Fig. 4A). The quality of RNA from cells from all animals was confirmed to be good using the mouse  -actin gene transcript-specific primer pair (Fig. 4B). No RT-PCR product was generated in the absence of RT in either case. Semiquantitative RT-PCR assays were performed to determine the level of expression of the transduced human  -globin gene relative to that of the endogenous mouse  -globin gene. These results are shown in Fig. 5. The highest level of expression was determined to be up to 35% compared with that of the endogenous B6.c- kit W 41/41  -globin gene. It was also of interest to substantiate whether multilineage reconstitution and erythroid lineage-restricted expression of the transduced human  -globin gene occurred in the transplanted mice. This was carried out as follows. Granulocytic, lymphocytic, and erythroid cell populations obtained from peripheral blood from three recipient mice 10 months posttransplantation were subjected to PCR and RT-PCR analyses, respectively. These results are shown in Fig. 6. As can be seen in Fig. 6A, both granulocytic and lymphocytic, but not erythroid, cell populations contained the transduced human  -globin gene sequences, suggesting that multilineage reconstitution had indeed occurred. However, the transgene expression could be detected only in the erythroid cell population in each of the three transplanted animals, and no RT-PCR product was generated in the absence of RT (Fig. 6B), documenting erythroid lineage-restricted transgene expression. Similar results were obtained in hematopoietic progenitor cell assays performed with bone marrow cells obtained from a transplanted animal in which a human  -globin gene-specific RT-PCR product could be amplified in BFU-E colonies, but not in CFU-GM colonies (Fig. 6B). We also wished to determine whether the transduced human  -globin gene sequences were stably integrated into the mouse chromosomal DNA. To this end, total genomic DNA samples isolated from bone marrow cells from two transplanted animals 14 months posttransplantation were digested with restriction enzymes that do not cleave within the recombinant AAV genome ( Cla I), cleave once ( Bam HI), and cleave twice ( Eco RI) and analyzed on Southern blots using a human  -globin DNA-specific probe. A DNA sample from bone marrow cells obtained from an untransplanted mouse was used as a negative control. These results are shown in Fig. 7. The restriction pattern obtained is consistent with the conclusion that the recombinant AAV proviral sequences were stably integrated into the mouse chromosomal DNA. Human hemoglobinopathies, such as  -thalassemia and sickle-cell disease, are among the likely candidates for their potential treatment provided that the pluripotent hematopoietic stem cell (HSC) can be stably transduced and that long-term, regulated expression of a functional  -globin gene in the erythroid progenitor cell can be achieved. Indeed, attempts have been made to success- fully transduce the HSC with retroviral vectors, but the structural instability of the LCR elements, low levels of transgene expression, and transcriptional silencing of the transgene have yielded less than optimal results (29 –37). However, the use of lentiviral vectors, containing larger segments of the LCR elements, has recently been shown to lead to the production of potentially therapeutic levels (13–24%) of normal  -globin (38). Of note, the use of shorter segments of the LCR elements failed to lead to sustained expression of the transgene over a 24-week pe- riod in homozygous  -thalassemic mice in vivo. In contrast, even with the use of shorter segments of the LCR elements in a recombinant AAV vector used in the present studies, we were able to document high levels (up to 35%) of erythroid lineage-restricted expression of a normal human  -globin gene in progenitor cells derived from HSC obtained from homozygous  -thalassemic mice for up to 40 weeks. Two key differences between our current studies and those reported by May et al. (38) should also be emphasized. First, in our studies, the donor mice were not treated with 5-fluorouracil and second, the target cells were transduced without the exposure to cy- tokines and without in vitro culture for 18 h. Furthermore, in our studies, the recipient animals were not lethally irradiated, and as a consequence, the level of engraftment of donor cells was well below 50% as determined by hemoglobin gel electrophoresis (data not shown). We, nonetheless, observed relatively higher levels as well as significantly longer term expression of the transduced  -globin gene in our studies. It should be acknowledged, however, that RT-PCR analyses are not indicative of globin protein production, and this was not attempted in our studies. In addition to demonstrating erythroid cell lineage- restricted expression, we were able to document the presence of the transduced human  -globin gene in multiple lineages of hematopoietic progenitor cells in recipient animals, suggesting that transduction of a pluripotent hematopoietic stem cell had occurred. This was further corroborated by documenting that the recombinant proviral genome sequences were stably integrated into the mouse chromosomal DNA. Interestingly, nearly identical restriction patterns of the integrated proviral genomes were obtained with two different animals, suggesting the following two possibilities. First, integration occurred in a site-specific manner, and second, similarly transduced HSC reconstituted the bone marrow in the two animals. In view of the observed loss of site specificity of integration by recombinant AAV vectors (39, 40), we favor the second possibility since HSCs from several donors were transduced in a mixed population. It is clear, however, that further studies with a larger number of animals would be needed to define these issues at the molecular level. As pointed out above, due to the limited colony size of our homozygous  -thalassemic mice, and the lack of analyses of transgene expression at the protein level, it is difficult to ascertain whether the level of expression of the transduced human  -globin gene is in the therapeutic range (41, 42). Additional studies with homozygous  -thalassemic mice as transplant recipients will be needed to determine whether AAV-mediated  -globin gene transfer will lead to improvements in peripheral blood hemat- ocrit, red blood cell (RBC) volume, RBC hemoglobin content, RBC morphology, reticulocyte counts, and, ultimately, the average life span of transplanted ...
Citations
... Concerning β-type hemoglobinopathies, to both in vitro and in vivo studies, more often are used specialized recombinant AAV vectors (rAAVs) or selfcomplementary (scAAVs) in order to overcome some major limitations in comparison to the typical ones, such as the limited cargo capacity and the slow onset of gene expression [142,143]. Notably, successful transductions in both murine and human HSCs using recombinant rAAVs vectors have shown great potentials and especially in the case of the AAV2 and AAV6 serotypes [144][145][146]. Recently, Yang and co-workers (2020) suggested that AAV6, under certain circumstances, can be used for gene therapy in β-type hemoglobinopathies [147]. ...
For decades, various strategies have been proposed to solve the enigma of hemoglobinopathies, especially severe cases. However, most of them seem to be lagging in terms of effectiveness and safety. So far, the most prevalent and promising treatment options for patients with β-types hemoglobinopathies, among others, predominantly include drug treatment and gene therapy. Despite the significant improvements of such interventions to the patient’s quality of life, a variable response has been demonstrated among different groups of patients and populations. This is essentially due to the complexity of the disease and other genetic factors. In recent years, a more in-depth understanding of the molecular basis of the β-type hemoglobinopathies has led to significant upgrades to the current technologies, as well as the addition of new ones attempting to elucidate these barriers. Therefore, the purpose of this article is to shed light on pharmacogenomics, gene addition, and genome editing technologies, and consequently, their potential use as direct and indirect genome-based interventions, in different strategies, referring to drug and gene therapy. Furthermore, all the latest progress, updates, and scientific achievements for patients with β-type hemoglobinopathies will be described in detail.
... 68 Although AAV2 vectors performed better than retroviral vectors, therapeutic levels of human β-globin gene expression were not achieved in normal and β-thalassaemic mice. 69 TDT patients. 73 By removing the insulator domains and replacing the 5 0 long-terminal repeat with a CMV promoter this vector was found to have a 3-4 times more transduction efficiency in CD34 + and also 3-4 times higher titre than the HPV569 vector. ...
Beta‐thalassaemia is one of the most significant haemoglobinopathies worldwide resulting in the synthesis of little or no β‐globin chains. Without treatment, β‐thalassaemia major is lethal within the first decade of life due to the complex pathophysiology, which leads to wide clinical manifestations. Current clinical management for these patients depends on repeated transfusions followed by iron‐chelating therapy. Several novel approaches to correct the resulting α/β‐globin chain imbalance, treat ineffective erythropoiesis and improve iron overload are currently being developed. Up to now, the only curative treatment for β‐thalassemia is haematopoietic stem‐cell transplantation, but this is a risky and costly procedure. Gene therapy, gene editing and base editing are emerging as a powerful approach to treat this disease. In β‐thalassaemia, gene therapy involves the insertion of a vector containing the normal β‐globin or γ‐globin gene into haematopoietic stem cells to permanently produce normal red blood cells. Gene editing and base editing involves the use of zinc finger nucleases, transcription activator‐like nucleases and clustered regularly interspaced short palindromic repeats/Cas9 to either correct the causative mutation or else insert a single nucleotide variant that will increase foetal haemoglobin. In this review, we will examine the current management strategies used to treat β‐thalassaemia and focus on the novel therapies targeting ineffective erythropoiesis, improving iron overload and correction of the globin chain imbalance.
... L'entrée dans la cellule se fait par endocytose après de nombreux contacts entre le virus humain, mais cela reste encore à explorer (Tan et al. 2001;Daya and Berns 2008). Une étude in vivo chez la souris a montré que 10% des AAV recombinants injectés ont été intégrés dans le génome cellulaire de cellules hépatiques dont 3,5% à proximité d'oncogènes (Rossi and Salvetti 2016). ...
La surdité et les troubles vestibulaires sont des pathologies fréquentes, source de handicap et d’altération de la qualité de vie. Actuellement, il n’existe pas de traitement curatif pour ces pathologies. La thérapie génique utilisant les AAVr semble une alternative prometteuse notamment dans le traitement des surdités et troubles vestibulaires d’origine génétique. Cependant, de nombreux défis restent à relever avant d’envisager une application chez l’Homme. Dans ce travail, nous avons cherché à identifier les étapes clés à franchir pour une application clinique de la thérapie génique pour 2 surdités génétiques humaines, le syndrome USH1G et la surdité DFNB9, à l'aide des modèles murins correspondants, d'études chez le primate non-humain et d'un modèle d’explant d’organes vestibulaires humains. Nous avons pu montrer que la fenêtre thérapeutique était un facteur majeur à prendre en compte dans un objectif translationnel. Le stade de maturation de l’oreille interne influe grandement sur l’efficacité de la thérapie, d’autant plus lorsque la pathologie implique des anomalies de développement comme dans le syndrome USH1. Pourtant, nous avons pu apporter la preuve d’une extension de la fenêtre thérapeutique chez la souris Ush1g-/-, et montrer que la thérapie génique permettait une restauration à un niveau proche de la normale de la fonction vestibulaire et dans une moindre mesure de la fonction auditive après injection à un stade mature. Dans la surdité DFNB9 pour laquelle il n’existe pas d’anomalie de développement, nous avons pu montrer que la thérapie génique permettait une restauration complète de l’audition, et posé les fondements d’une future thérapie chez l’Homme.
... We have had a long-term interest in the pursuit of the potential gene therapy of thalassemia and sickle cell disease with adeno-associated virus (AAV) vectors 16 . In these previous studies, while AAV2 vectors performed better (transgene expression ranging from 3-7%) than retroviral vectors, therapeutic levels of human -globin gene expression could not be achieved in normal and -thalassemic mice [16][17] since AAV2 vectors do not efficiently transduce mouse hematopoietic stem cells (HSCs). ...
... We have had a long-term interest in the pursuit of the potential gene therapy of thalassemia and sickle cell disease with adeno-associated virus (AAV) vectors 16 . In these previous studies, while AAV2 vectors performed better (transgene expression ranging from 3-7%) than retroviral vectors, therapeutic levels of human -globin gene expression could not be achieved in normal and -thalassemic mice [16][17] since AAV2 vectors do not efficiently transduce mouse hematopoietic stem cells (HSCs). In our subsequent studies, we identified AAV1 and AAV7 serotype vectors to be significantly more efficient than AAV2 vectors in transducing normal mouse HSCs, but these serotype vectors failed to transduce HSCs from sickle cell disease mice [18][19][20] . ...
We have reported that of the 10 most commonly used adeno-associated virus (AAV) serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs) in vitro as well as in vivo. More recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for human serum albumin (HSA) for ex vivo expansion of HSCs. Since HSA has been shown to increase the transduction efficiency of AAV serotype vectors, we evaluated whether PVA could also enhance the transduction efficiency of AAV6 vectors in primary human HSCs. We report here that up to 12-fold enhancement in the transduction efficiency of AAV6 vectors can be achieved in primary human HSCs with PVA. We also demonstrate that the improvement in the transduction efficiency is due to PVA-mediated improved entry and intracellular trafficking of AAV6 vectors in human hematopoietic cells in vitro as well as in murine hepatocytes in vivo. Taken together, our studies suggest that the use of PVA is an attractive strategy to further improve the efficacy of AAV6 vectors. This has important implications in the optimal use of these vectors in the potential gene therapy and genome editing for human hemoglobinopathies such as β-thalassemia and sickle cell disease.
... In retrospect, the use of AAV2 (the only available serotype vector available at the time) was not ideal since AAV2 transduces murine hematopoietic stem/ progenitor cells (HSPCs) poorly. Thus, despite our sustained efforts, [8][9][10][11] the use of a murine model to demonstrate the feasibility of AAV2 vectors for the potential gene therapy of hemoglobinopathies did not succeed. It wasn't until 2013 when we identified AAV6 as the most efficient serotype for transducing human HSPCs, 12,13 and it is my fervent hope that the optimized AAV6-B19-b-globin vector will prove to be a useful alternative for the potential gene therapy of human hemoglobinopathies in the not-too-distant future, based on recent success with AAV6 vectors and human HSPCs. ...
The mentor. I have been blessed to have George Stamatoyannopoulos as a mentor for the past 18 years. My encounter with Dr. Stam had an outsized impact on the path my career and my life took after I met him in 1998, early after obtaining my Greek board certification in hematology. I have had the considerable privilege of being supervised by him during my first steps in gene therapy and closely interacting with his personal authenticity, passionate commitment to research, and selfless work ethic. At that time, I had also the privilege to witness some of his legendary verbal fights over scientific matters with Thalia, his loveliest and toughest competitor ever, that were generating a unique learning atmosphere in the lab. Upon my return to Greece, George has been instrumental in helping me to set up a gene and cell therapy program at the G. Papanicolaou Hospital in Thessaloniki, and since then, a long-standing collaboration between the two institutions has been established, translated in two clinical trials and studies on the optimization of mobilization and graft sources for thalassemia gene therapy. Our mentor–mentee interaction continues up to now, and I often seek his advice on critical scientific questions or dilemmas. I am and I will be eternally grateful for his mentorship!
The father. George has been a father figure in my life, offering guidance the times I felt lost and overwhelmed and teaching me lessons that impacted my life. In times of tragedy, his wise words and paradigms retrieved from Greek legends and history have softened the soul pain and created meaning.
... In HSCs, which must divide to give rise to progenitor cells, the proviral genome would be lost if not stably integrated into host cell chromosomal DNA. In our previous studies, we documented stable integration of the proviral genome into chromosomal DNA in pluripotent HSC transduced with ssAAV1 and AAV2 vectors in a small number of animals[Tan et al., 2001;Zhong et al., 2006a]. Therefore, further studies with a larger number of animals are needed to define this issue at the molecular level. ...
... Recombinant vectors based on a non-pathogenic human virus, the adeno-associated virus 2 (AAV2) have been developed and shown to be safe and effective in a number of recent clinical trials [2], [3]. Others and we have generated recombinant AAV2-globin vectors [4]–[8], but the transduction efficiency of these vectors in primary human HSCs has not been evaluated. ...
... The development of lentiviral vectors by a number of investigators has indeed achieved these objectives [38]–[45], but their long-term safety still remains an open question [1]. We and others have described the development of the first generation of recombinant AAV2 vectors for the potential gene therapy of b-thalassemia and sickle cell disease [4]–[8], but in retrospect, it has become clear that the use of the WT AAV2 capsid, and the single-stranded nature of the vector genome, were major obstacles to achieving therapeutic levels of the human b-globin gene [6], [8], [46]–[48]. In addition, the use of murine models of these diseases was not predictive of the potential efficacy of a number of alternative serotypes of AAV vectors [27], [28]. ...
... The development of lentiviral vectors by a number of investigators has indeed achieved these objectives [38]–[45], but their long-term safety still remains an open question [1]. We and others have described the development of the first generation of recombinant AAV2 vectors for the potential gene therapy of b-thalassemia and sickle cell disease [4]–[8], but in retrospect, it has become clear that the use of the WT AAV2 capsid, and the single-stranded nature of the vector genome, were major obstacles to achieving therapeutic levels of the human b-globin gene [6], [8], [46]–[48]. In addition, the use of murine models of these diseases was not predictive of the potential efficacy of a number of alternative serotypes of AAV vectors [27], [28]. ...
We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34(+) cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34(+) cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.
... In subsequent studies, using a ssAAV vector containing the human β-globin gene under the control of its own promoter with an upstream mini-LCR cassette consisting of HS2, HS3 and HS4 enhancer elements (Fig. 12.2B), and enriched HSCs from homozygous β-thalassemic mice, long-term, erythroid lineage-restricted expression of a human β-globin gene could be achieved. 66 Although the expression level of the transduced human β-globin gene reached up to 35% of the endogenous murine β-globin gene, expression analyses were based on RT-PCR assays at the RNA level, that are not indicative of globin protein production. ...
Human hemoglobinopathies, such as β-thalassemia and sickle cell disease (SCD), are the most common human genetic diseases worldwide, and are an attractive target for potential gene therapy. This potential could be realized if a functional β-globin gene could be safely and efficiently introduced into hematopoietic stem cells (HSCs), and lineage-restricted expression of the β-globin protein exceeding 15% could be achieved in erythroid progenitor cells. The use of first generation retroviral vectors remained limited due to the inability to achieve therapeutic levels of β-globin expression. Although therapeutic levels of β-globin expression could be achieved by second-generation retroviral vectors, their use in gene therapy trials for immuno-deficiency resulted in insertional mutagenesis leading to leukemia in several children, which raised serious concerns. The use of lentiviral vectors expressing β-globin has led to phenotypic correction of β-thalassemia and SCD in mouse models, but long-term safety issues with lentiviral vectors remain to be evaluated. This chapter will provide a brief review of obstacles that were encountered, and achievements that have occurred over the past two decades, including the development of an alternative vector system based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV), for the potential gene therapy of β-thalassemia and sickle cell disease.
... Recombinant AAV2 vectors have also been reported to transduce a wide variety of cells and tissues in vitro and in vivo (Flotte et al., 1993; Muzyczka, 1992; Snyder et al., 1997; Xiao, Li, and Samulski, 1996). Several fundamental steps in the life cycle of AAV2 vectors, such as viral binding and entry (Kashiwakura et al., 2005; Qing et al., 1999; Summerford, Bartlett, and Samulski, 1999; Summerford and Samulski, 1998), intracellular trafficking (Douar et al., 2001; Hansen et al., 2000; Sanlioglu et al., 2000; Zhao et al., 2006), uncoating (Thomas et al., 2004; Zhong et al., 2004c), second-strand DNA synthesis and transgene expression (Ferrari et al., 1996; Fisher et al., 1996; Qing et al., 1997; Zhong et al., 2004a; Zhong et al., 2004b; Zhong et al., 2004d; Zhong et al., 2008b), and viral genome integration into host cell chromosome (McCarty, Young, and Samulski, 2004; Tan et al., 2001;), have been studied extensively. ...
We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by approximately 68% and approximately 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.
... In HSCs, which must divide to give rise to progenitor cells, the proviral genome would be lost if not stably integrated into host cell chromosomal DNA. In our previous studies, we documented stable integration of the proviral genome into chromosomal DNA in pluripotent HSC transduced with ssAAV1 and AAV2 vectors in a small number of animals [Tan et al., 2001;Zhong et al., 2006a]. Therefore, further studies with a larger number of animals are needed to define this issue at the molecular level. ...
Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system.
