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: We recently completed a longitudinal study over 12 months on T-cell immune reconstitution after total body irradiation and autologous transplantation of peripheral blood progenitor cells in 3–5 year old rhesus macaques. The focus of the study was to evaluate the source of T-cell recovery using three different sources of cells for transplantation. The starting number of CD34+ cells contained within the graft was adjusted to be the same for each of the three groups: unselected peripheral blood progenitor cells (PBPC) (n=3) vs. CD34+ selected mobilized PBPC (n=4) vs. CD34+ selected cells cultured for 4 days in vitro (Flt-3L/MDGF/SCF/retronectin) and retrovirally-transduced (n=3). Peripheral blood and lymph nodes were collected for phenotypic and TREC analysis. There was a trend for animals receiving cultured and transduced CD34+ cells to attain higher absolute naive and memory CD4+ T-cell, CD20+ B-cell and CD16+ NK-cell numbers in the first months after transplantation compared to the two other groups. There were no differences for numbers of CD8+ T-cells, monocytes or dendritic cells. The absolute TREC amount per μl blood of the CD4+ T-cells in the group that received selected-transduced cells was significantly higher than in the group that received unselected cells (p=0.0166) and for CD8+ cells significantly higher than for selected cells (p=0.0464). There was significantly less peripheral T-cell expansion as measured by Ki-67 expression in the selected-transduced group compared to the two other groups: At 1 month after transplantation the mean Ki-67 expression level for CD8+ cells in peripheral blood was 33.9% in the selected vs. 23.3% in the unselected vs. 8% in the selected-transduced group. Histology at 12 months revealed striking differences in the thymus between the 3 groups, while other organs (lymph nodes, spleen, tonsils and Peyer’s patches) showed no remarkable differences. In the CD34+ selected-transduced group the thymus showed preserved lobular architecture with well defined cortical and medullary areas, compared to atrophy with fat replacement, decreased thickness of the cortex, and cystic changes of the thymic epithelium in the CD34+ selected and unselected groups. The degree of atrophy was more pronounced in the latter group. This study demonstrates an enhanced ability of in vitro expanded and retrovirally transduced cells to repopulate the thymus compared to non-manipulated CD34+ selected cells. In vitro expansion and transduction may promote development of T-cell and NK-cells as has been described earlier for fetal thymic organ culture of cord blood CD34+ cells as well as in the SCID-hu mouse model. Our observation of an acceleration of T-cell and NK cell immune reconstitution following in vitro culture contrasts with limited prior clinical trial experiences describing poor repopulating potential of cultured progenitor cells. Our data suggest that in vitro culture and retroviral gene transfer per se does not induce an intrinsic differentation defect. Future studies are planned to examine mechanisms behind the improved repopulation ability of selected-transduced progenitor cells, which has important implications for gene therapy trials, and suggests that cultured cells may be useful for a number of clinical applications.

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.

Description: One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

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: We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34+ cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4+, CD8+, CD20+, and CD16/56+ lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP+ myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol.

Description: An understanding of the number and contribution of individual pluripotent hematopoietic stem cells (HSCs) to the formation of blood lineages has important clinical implications for gene therapy and stem cell transplantation. We have been able to efficiently mark rhesus macaque long-term repopulating stem and progenitor cells with retroviral vectors, and track their in vivo contributions to hematopoiesis using the linear amplification mediated–polymerase chain reaction (LAM-PCR) technique of insertion site analysis. We assessed the impact of busulfan on contributions of individual retrovirally marked clones to hematopoiesis. There were 2 macaques that received transplants of retrovirally transduced CD34+ cells 2 years previously that were then treated with 4 mg/kg busulfan. Despite only transient and mild suppression of peripheral blood counts, the numbers of individual stem/progenitor clones contributing to granulocyte production decreased dramatically, by 80% in the first monkey and by 60% in the second monkey. A similar impact was seen on clones contributing to T cells. The clone numbers recovered gradually back toward baseline by 5 months following busulfan in the first monkey and by 3 months in the second monkey, and have remained stable for more than one year in both animals. Tracking of individual clones with insertion-site–specific primers suggested that clones contributing to hematopoiesis prior to busulfan accounted for the majority of this recovery, but that some previously undetected clones began to contribute during this recovery phase. These results indicate that even low-dose busulfan significantly affects stem and progenitor cell dynamics. The clonal diversity of hematopoiesis was significantly decreased after even a single, clinically well-tolerated dose of busulfan, with slow but almost complete recovery over the next several months, suggesting that true long-term repopulating stem cells were not permanently deleted. However, the prolonged period of suppression of many clones suggests that transplanted HSCs may have a marked competitive advantage if they can engraft and proliferate during this time period, and supports the use of this agent in nonmyeloablative regimens

Description: Transduction of murine stem cells with a multidrug-resistance 1 gene (MDR1) retrovirus results in dramatic ex vivo and in vivo expansion of repopulating cells accompanied by a myeloproliferative disorder. Given the use ofMDR1-containing vectors in human trials, investigations have been extended to nonhuman primates. Peripheral blood stem cells from 2 rhesus monkeys were collected, CD34-enriched, split into 2 portions, and transduced with eitherMDR1 vectors or neo vectors and continued in culture for a total of 10 days before reinfusion. At engraftment, the copy number in granulocytes was extremely high from bothMDR vectors and neo vectors, but the copy number fell to 0.01 to 0.05 for both. There were no perturbations of the leukocyte count or differential noted. After 3 cycles of stem cell factor/granulocyte colony-stimulating factor, there were no changes in the levels of MDR1 vector– or neovector–containing cells. There was no evidence for expansion ofMDR1 vector–transduced cells. Long-term engraftment with MDR1 vector– and neo vector–transduced cells occurred despite prolonged culture.

Description: High-titer, HIV-1–based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34+ cells and clonogenic progenitors very poorly (〈 1%), reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier, we developed a simian immunodeficiency virus (SIV)–based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% ± 1% of rhesus bulk CD34+ cells and 75% ± 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector–mediated stem cell gene transfer in vivo, 3 rhesus macaques underwent transplantation with transduced, autologous cytokine-mobilized peripheral blood CD34+ cells following myeloablative conditioning. Hematopoietic reconstitution was rapid, and an average of 18% ± 8% and 15% ± 7% GFP-positive granulocytes and monocytes, respectively, were observed 4 to 6 months after transplantation, consistent with the average vector copy number of 0.19 ± 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models.

Description: Hematopoietic cytokines such as filgrastim are used extensively to stimulate granulocyte production or to mobilize hematopoietic progenitors into the circulation; however, their effect on more primitive hematopoietic progenitor and stem cells in vivo is unknown, particularly in large animals or humans. In particular, there is concern that chronic therapy with cytokines could result in stem cell exhaustion or clonal dominance; however, direct assessment of the dynamics of individual stem and progenitor cell clones in vivo has not been previously reported. A number of models can be proposed regarding the mechanisms by which the marrow responds to cytokine stimulation, including recruitment of previously quiescent clones, stimulation of proliferation of already active clones, or prevention of apoptosis of more mature progenitors from all clones. Using retroviral marking and comprehensive insertion site tracking of individual stem and progenitor cell clones in 2 rhesus macaques, we analyzed the effect of chronic administration of granulocyte colony-stimulating factor (G-CSF), or a combination of G-CSF plus stem cell factor (SCF). The overall number of contributing clones remained constant, and the relative output from each clone did not change significantly during or following cytokine treatments. These results suggest that individual transduced stem or progenitor cells can contribute to hematopoiesis for prolonged periods, with no evidence for an effect of G-CSF or G-CSF/SCF on the number, the lifespan, or the relative activity of individual stem or progenitor cell clones. These relevant large animal studies are reassuring regarding clinical applications of cytokines and provide new insights into their mechanisms of action.