We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

To date, over 1800 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical trials from official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our June 2012 update, we have entries on 1843 trials undertaken in 31 countries. We have analysed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and which genes have been transferred. Details of the analyses presented, and our searchable database are available on The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: http://www.wiley.co.uk/genmed/clinical. We also provide an overview of the progress being made in clinical trials of gene therapy approaches around the world and discuss the prospects for the future.

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Mouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential.

Recombinant adeno-associated viral (rAAV) vectors have shown promise for use in liver-targeted gene delivery, but their effects have not been extensively investigated in the immature liver. Understanding the impact of liver growth on the efficacy of transduction is essential, because many monogenic liver diseases that are amenable to gene therapy will require treatment early in life. Here we show that rAAV2/8 transduces the neonatal mouse liver with high efficiency. With just one doubling in liver weight, however, there is a rapid reduction in vector genome numbers, irrespective of form, and the loss of episomal vector is almost complete by 2 weeks. Stable transgene expression is observed in a small percentage of hepatocytes, often in two- to eight-cell clusters, suggestive of genomic integration. Delivery at serially older ages was associated with progressively improved episome persistence and transgene expression. Vector re-administration was possible following initial neonatal administration, albeit at reduced efficacy because of an anticapsid humoral immune response. We also found that intraperitoneal (i.p.) delivery of rAAV2/8 was highly effective at all ages, and that promoter selection is the critical determinant of the intensity and pattern of transgene expression across the hepatic lobule. We conclude that successful use of rAAV to treat liver disease in early childhood will require optimally efficient vector constructs and probable re-administration.

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34 + progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased >300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.Adeno-associated virus (AAV) is a dependent human parvovirus capable of integration into host DNA (1). Transducing vectors based on AAV are able to transfer nonviral genes into mammalian cells and represent an alternative to the integrating retroviral vectors currently used in many gene therapy protocols. Up to 4.7 kb of foreign DNA can be packaged into stable AAV vector particles as single-stranded DNA molecules of either polarity (2). A wide variety of cell types can be transduced by AAV vectors, including cultured normal human cells (2-6). Although the wild-type provirus often integrates at a specific site on chromosome 19 (7-9), stable transductants usually contain randomly integrated vector sequences (3, 10).The transduction of nondividing cell populations such as hematopoietic stem cells, quiescent lymphocytes, neurons, and normal epithelial cells remains a major goal of gene therapy research. Retroviral vectors based on murine leukemia virus require mitosis for efficient transduction (11-13), limiting their usefulness in many gene therapy protocols. AAV vectors also require cell proliferation for efficient transduction. However, it is S phase of the cell cycle that is important. By using stationary human fibroblast cultures, we found that AAV vectors transduce S-phase cells at >200 times the frequency of non-S-phase cells (3). This preference for S-phase cells was thought to reflect the requirement for host polymerases in the conversion of single-stranded vector genomes to double-stranded transcription templates.Based on these results, we reasoned that the transduction of nondividing cells by AAV vectors might be increased by inducing unscheduled DNA repair synthesis and found that non-S-phase cells were transduced at up to 750-fold higher frequencies after treatment with DNA-damaging agents (14). These agents included y-and UV-irradiation and cisplatinum, all of which reduced the colony-forming ability of treated fibroblasts at the optimal concentrations required for increased transduction. In this report, we have tested otherThe publication costs of this article were defrayed in part by page charge payment....

Vectors based on adeno-associated virus can stably transfer genes by chromosomal integration in recipient cells. In this study we have infected stationary and dividing primary human fibroblast cultures with adeno-associated virus vectors encoding alkaline phosphatase and neomycin phosphotransferase. We find that the transduction frequency of S phase cells is about 200 times that of non-S phase cells. However, neither S phase nor mitosis is essential for transduction. Single-stranded vector genomes survive in stationary cultures and can be recruited for transduction by stimulating these cultures to divide. Stable transductants contain randomly integrated vector sequences. These findings have important implications for the use of adeno-associated virus vectors in gene therapy.Adeno-associated virus (AAV) is a dependent human parvovirus that usually requires the presence of a helper virus such as adenovirus or herpes virus for productive viral infection (1,2). AAV is capable of integration into the host chromosome in the absence ofhelper virus and often integrates in the same location on chromosome 19 (3-5). By expressing essential viral genes in trans, several investigators have developed replication-incompetent, integrating, transducing vectors based on AAV (6-9). These vectors represent an alternative to the retroviral vectors currently used in many gene therapy protocols (10, 11). Retroviral vectors based on murine leukemia virus (MLV) require cell division for efficient transduction (12), and this limits their usefulness in clinical applications. We therefore set out to determine if transduction by vectors based on AAV requires cell division.Little is known about the relationship between the cell cycle and infection by dependent parvoviruses such as AAV. Replication of the related autonomous parvoviruses has been shown to occur in S phase (1,13 (19)(20)(21). Except for stationary cultures, cells were grown in Dulbecco modified Eagle medium (DMEM) with 10% heatinactivated (30 min at 560() fetal bovine serum (FBS) at 370C in 10%(C02. Primary human fibroblasts were obtained from frozen stocks (passage 3 or 4) and maintained in culture for <10 additional passages. Stationary cultures were prepared by changing the medium in confluent cultures to DMEM containing 5% heat-inactivated FBS and 1 AuM dexamethasone and maintaining these cultures for at least 2 weeks while replacing the medium every 3-4 days. Dividing cultures were prepared by treatment with trypsin and plating the cells at a density of either 2.5 x 105 cells per 35-mm well in six-well plates (Falcon) or 4 x 105 cells per 60-mm dish (Corning) the day before infection.Vector Construction and Production. The plasmids pTR, pTRneo, and pTRAAVneo were kindly provided by Sergei Zolotukhin and Nicholas Muzyczka (State University ofNew York, Stony Brook) and were derived from the vector plasmid d13-94 (8). pTR and pTRneo are vector cloning constructs containing no insert or the simian virus 40 early promoter and neomycin phosphotransferase gene (neo), res...