ALBERT

Description: Retroviral insertion site analysis was used to track the contribution of retrovirally transduced primitive progenitors to hematopoiesis after autologous transplantation in the rhesus macaque model. CD34-enriched mobilized peripheral blood cells were transduced with retroviral marking vectors containing the neo gene and were reinfused after total body irradiation. High-level gene transfer efficiency allowed insertion site analysis of individual myeloid and erythroid colony-forming units (CFU) and of highly purified B- and T-lymphoid populations in 2 animals. At multiple time points up to 1 year after transplantation, retroviral insertion sites were identified by performing inverse polymerase chain reaction and sequencing vector-containing CFU or more than 99% pure T- and B-cell populations. Forty-eight unique insertion sequences were detected in the first animal and also in the second animal, and multiple clones contributed to hematopoiesis at 2 or more time points. Multipotential clones contributing to myeloid and lymphoid lineages were identified. These results support the concept that hematopoiesis in large animals is polyclonal and that individual multipotential stem or progenitor cells can contribute to hematopoiesis for prolonged periods. Gene transfer to long-lived, multipotent clones is shown and is encouraging for human gene therapy applications.

Description: To determine whether the multidrug resistance gene MDR1could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that theMDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

Description: Until recently, the risk of insertional mutagenesis using retroviral vectors for gene therapy has been estimated to be low. Owing to reports of proto-oncogenes activation in mice and humans, this estimation is being re-evaluated in regards to the nature of susceptible loci and the number of genetic hits needed for oncogenesis. Separating the impact of overexpressing a growth-altering transgene from the insertional events themselves is particularly important to address in long-term repopulating hematopoietic stem cells. We here report a high frequency of proviral insertions at the MDS1-EVI1 locus in the engrafted gene-modified hematopoiesis of non-human primates. We have recovered vector-genome junction sequences from mature granulocytes and mononuclear cells of 22 rhesus macaques that were transplanted 6 months to 6 years previously with autologous mobilized CD34+ peripheral blood stem cells transduced with an amphotropic Moloney murine leukemia virus-derived vector containing a neomycin-resistance marker gene. Using a modified LAM-PCR method, we have retrieved and analyzed 702 independent integration sites that mapped to a unique genomic location. While several transcription units harbor two or three proviral insertions, we have identified, in 9 animals, an unexpected 13 unique integration events within the two first introns of the MDS1 gene. MDS1 is adjacent to EVI1, a well-known retrovirally-activated zinc finger transcription factor in a number of murine leukemogenesis studies. We used insertion-specific primers to confirm that the fusion sequences between the MDS1 locus and the 5′-LTR of the vector were detectable in four of the animals for which we had the longest follow-up (4 to 6 years). The fusion sequence was present both in purified granulocytes and lymphocytes of one of the animal, and only in granulocytes in the other three animals. In order to determine if the cells carrying proviral insertions at the MDS1-EVI1 locus have a selective growth advantage, we performed quantitative PCR experiments with neomycin and MDS1/5′-LTR-specific probes. The two animals analyzed to date do not have any evidence of clonal expansion of the MDS1-targeted granulocyte population, and the number of retrovirally-transduced circulating cells remains stable 5 and 6 years after transplantation. As retroviral insertion upstream of a proto-oncogene may lead to its activation, we investigated MDS1-EVI1 expression by RT-PCR in neo+ CFU that harbor a proviral insertion at the MDS1 locus. The 8 colonies screened from two animals do not express any of the various MDS1-EVI1 transcripts, suggesting that the transcriptional regulation of the locus has not been altered. Our study suggest that the MDS1-EVI1 locus is particularly susceptible to retroviral integration but the competing hypothesis that proviral insertion within this region favors engraftment and long-term contributions to hematopoiesis will be difficult to eliminate, since specific insertions may have favored engraftment of such clones. It is however important to mention that the long-term follow-up of these primates has revealed completely normal hematopoiesis and lack of any progression towards neoplasia. Systematic analysis of proviral insegration sites in this appropriate pre-clinical model is essential as it provides decisive information for risk assessment in the development of integrating vectors, as well as offering further insights into the mechanisms of retroviral insertion.

Description: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking. One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites. In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive. By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector. We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

Description: Abstract 3759 For genetic blood diseases, such as primary immunodeficiencies, gene therapy targeted to hematopoietic stem cells (HSCs) is a feasible and now proven effective therapeutic option for patients who lack a histocompatible HSC. However, the risk of adverse outcomes resulting from insertional oncogenesis is a major concern. We are investigating whether inclusion of the herpes simplex virus thymidine kinase (HSVtk) gene into integrating vectors into rhesus macaque HSCs confers ganciclovir (GCV) sensitivity allowing ablation of vector-containing cells from the blood and other hematopoietic compartments, as an approach to increasing safety of gene therapy procedures. HSVtk suicide genes have been studied in detail in transduced mature T cells, but never in stem and progenitor cells. We infused autologous CD34+ cells transduced ex vivo with gammaretrovirus vectors encoding the HSVtk as suicide gene along with marker genes into 4 rhesus macaques, following myeloablative irradiation. In the first animal, a vector consisting of the MND backbone driving the sr39 high affinity tk mutant, and IRES and a truncated NGFR marker gene was used. A stable marking level of 5% NGFR+ circulating cells was observed for 6 months following transplantation, confirmed by q-PCR. The drug GCV was infused at 5 mg/Kg BID for 21 days. This animal had complete elimination of vector-containing cells in all peripheral blood lineages as assessed by flow cytometry and qPCR, and remains negative now 4 months after GCV discontinuation. Three additional animals were transplanted with autologous CD34+ cells transduced with a vector containing a standard HSVtk gene and GFP as a marker. These animals had lower stable marking levels of approximately 1% at 4 months post-transplant, and after 21 days of GCV, had a clear decrease in the level of GFP+ cells, but not complete ablation, likely due to lower drug-sensitivity of the tk protein expressed by this vector. Cells with a lower level of GFP expression were not eliminated, supporting this hypothesis. Additional animals receiving cells transduced with the sr39 tk retroviral vector and with a lentiviral vector containing a codon-optimized HSVtk are in progress. These data suggest that inclusion of a suicide gene in integrating vectors may be an effective way to address genotoxicity concerns, should clonal outgrowth occur, and increase safety of HSC-targeted gene therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral MDR1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analyses on the clonal level and a long-term follow-up in a model with more direct relevance to human HSC biology were lacking. Methods: In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were transduced either with a MDR1 gene-retroviral vector (HaMDR1-vector) or a NeoR-retroviral vector (G1Na-vector). After autologous transplantation granulocytes and mononuclear cells of each animal were obtained at different time points and analysed by using a highly sensitive and specific ligation-mediated PCR (LM-PCR) and by quantitative real time PCR. Results: In monkey M120 73 different cell populations (clones) were detected between 8 weeks and 4 years after transplantation; in monkey M038 49 clones could be identified between 16 weeks and 4 years after transplantation. Remarkably, 99 clones descend from the G1Na-vector, whereas only 23 clones descend from the HaMDR1-vector. Quantitative real time PCR analyses support these data: in both animals, the mean proportion of the G1Na-vector and the HaMDR1-vector, respectively, is 2,46%±0,014% and 0,455%±0,003%. Furthermore, single G1Na clones identified with LM-PCR 4 years after transplantation were quantified, showing a copy number between 1.43±0,52 and 7.1±0.34, according to 0.001%±0.0003% and 0.004%±0.0003% of the granulocytes fraction. In the MNC fraction, the G1Na clones were not detectable. At the same time point, 2 HaMDR1 clones were quantified, one in the granulocyte and one in the MNC fraction, respectively. Here, the copy numbers were 2.87±0.56 and 3.52±1.11, according to 0.002%±0.0003% and 0.002%±0.0008% of the granulocyte and MNC fraction, respectively. Conclusions: We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

Description: There is increasing evidence that insertional activation of proto-oncogenes by retroviral vectors is a significant safety issue that must be addressed before clinical gene therapy, particularly targeting hematopoietic stem and progenitor cells, can be further developed. The risk of insertional mutagenesis for replication-incompetent retroviral vectors has been assumed to be low until the occurence of T cell leukemias in children treated with HSC-directed gene therapy for X-SCID, and recent evidence that retroviral integration is more common in the promoter region of transcriptionally-active genes. The occurence of “common integration sites” in a particular gene also suggests a non-random insertion pattern, and/or immortalization or other change in the behavior of a clone harboring an insertion in these particular genes. We have previously reported a highly non-random occurence of 14 unique vector integrations in the first two introns of the MDS1/EVI1 proto-oncogene out of a total of 702 identified from myeloid cells of 9 rhesus macaques at least 6 months post-transplantion of retrovirally-transduced CD34+ cells.(Calmels et al, 2005). This same gene locus was found frequently activated by insertions in murine bone marrow cells immortalized in long-term in vitro culture after transduction with retroviral vectors.(Du et al Blood, 2005) To begin to investigate the factors contributing to this worrisome finding, particularly given the very recent report of a marked over-representation of MDS1/EVI1 insertions in a human clinical gene therapy trial for chronic granulomatous disease, we asked whether continued ex vivo expansion of transduced CD34+ cells prior to transplantation would further select for clones with insertions in MDS1/EVI1 or other proto-oncogenes. Rhesus CD34+ cells were transduced with the G1Na standard retroviral vector, identical to that used in the prior studies, using our standard 96 hour transduction protocol in the presence of Retronectin and SCF, FLT3L and thrombopoietin. At the end of transduction, all cells were continued in culture for an additional 7 days under the same culture conditions, and then reinfused into the donor animal following 1200 rads TBI. At 1 month post-transplant there were no CIS and no MDS1/EVI1 insertions identified. However, at 6 months post-transplantation 5 out of 27 (19%) of the unique insertions identified in granulocytes were within the first two introns of MDS1/EVI1, very significantly higher than the 2% of MDS1/EVI1 insertions (14 of 702) identified in animals that were transplanted with cells not subjected to additional ex vivo expansion.(p

Description: Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34+ cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34+ cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF–mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.