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  • 1
    ISSN: 1546-170X
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Simian immunodeficiency virus (SIV) infection of nonhuman primates is one of the most relevant animals models of HIV infection in humans. To test a potential anti-HIV gene therapy strategy in this model, CD4-enriched lymphocytes from three rhesus macaques were subjected to retrovi-rally mediated ...
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  • 2
  • 3
    Publication Date: 2000-07-01
    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.
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  • 4
    Publication Date: 1999-03-15
    Description: We have determined the 2905 nucleotide sequence of the rhesus macaque factor IX complementary DNA (cDNA) and found it to be greater than 95% identical to that of the human factor IX cDNA. The cDNA has a large 3′ untranslated region like the human cDNA, but unlike the human cDNA has two polyadenylation sites 224 nucleotides apart that are used for transcription of the messenger RNA. The deduced amino acid sequence is greater than 97% identical to that of human factor IX, differing in only 11 of 461 amino acids in the complete precursor protein. We found a single silent polymorphism in the nucleotide sequence at the third position of the codon for asparagine at position 167 in the secreted protein (AAC/AAT). All residues subject to posttranslational modifications in the human protein are also found in the rhesus factor IX sequence. The high degree of homology between the rhesus and human factor IX proteins suggested the possibility that the human factor IX protein might be nonimmunogenic in the rhesus. We tested the immunogenicity of human factor IX in three rhesus macaques by repeated intravenous injections of monoclonal antibody–purified, plasma-derived human factor IX over the course of more than a year and assessed the recovery and half-life of the infused protein, as well as in vitro indicators of antihuman factor IX antibodies. Human factor IX recovery and half-life remained unchanged over the course of a year in the three animals studied, and aPTT mixing studies showed no evidence for neutralizing antihuman factor IX antibodies. An outbred, nonhuman primate model that permits assessment of the level and duration of factor IX expression as well as vector safety would complement the use of other (mouse and canine) hemophilia B animal models in current use for the development of gene therapy for hemophilia B.
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  • 5
    Publication Date: 2004-11-16
    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.
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  • 6
    Publication Date: 2013-11-15
    Description: Dendritic cells (DCs) play a central role in innate immunity and adaptive immunity. DCs are antigen presenting cells capable of inducing primary T cell responses or facilitating self-tolerance. Myeloid DCs (mDC) express CD11c. They are further divided into Type 1 myeloid DCs (mDC1) which are CD1c+CD141+ and Type 2 mDCs (mDC2) which are CD1c-CD141+, and plasmacytoid DC which are CD11c- and express CD303. Plasmacytoid DCs are the main source of type 1 interferon upon infection. CD34+ cells are a heterogeneous population of cells which contain both hematopoietic progenitors and stem cells. The microarray signature of CD34+CXCR4 (CD184)+ cells in both human and non-human primates suggest that this cell population plays a role in innate immunity. The signaling pathway for the Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) is the most up-regulated pathway in both human and non-human primate sorted CD34+CD184+ cells. The TREM family is involved in the amplification and regulation of inflammatory responses. Upon sorting both human and rhesus CD34+ cells into CD34+CD184+ and CD34+CD184-, we observe that CD34+CD184+ cells are both adherent and non-adherent in nature (Figure 1) and while maintained in Flt3-L, IL-3, and SCF form progeny which appear dendritic in nature (Figure 2). In addition, we have found the CD34+CD184+ subpopulation to be resistant to lentiviral vector transduction. Upon culturing in defined, serum free media supplemented with cytokines, the progeny of the CD34+CD184+ population using both flow cytometry and confocal microscopy are CD11c+ and contain both CD1c+ and CD1c- subpopulations (Figure 3) with a predominately CD80(B7-1)+, CD123(IL-3R)+, CD197 (CCR7)+ , and HLA-Dr+ immunophenotype, having variability in CD86(B7-2) +/-, CD141(BDCA-3/Thrombomodulin)+/- , and CD144 (VE-cadherin)+/- expression, and being negative for CD303(BDCA-2)and CD309(VEGFR). Of special interest is that the CD34+CD184+ progeny contain both CD1c-CD16+ and CD1c+CD16- subpopulations of mDC. CD1c-CD16+ mDC have been shown to have strong pro-inflammatory activity, whereas CD1c+CD16- mDC are mainly inducers of chemotaxis. The progeny of the CD34+CD184+ cells can be stimulated by LPS and IFNγ to produce IL-12 based on an enzyme-linked immunosorbent assay. These results demonstrate the importance of the CD34+CXCR4+ progenitor in mDC development and allow one to speculate on how this mDC progenitor might prove of therapeutic benefit in vaccine development and cancer therapy.Figure 1Human CD34+CD184+ Sorted PopulationFigure 1. Human CD34+CD184+ Sorted PopulationFigure 2Cultured Progeny CD34+CD184+Figure 2. Cultured Progeny CD34+CD184+Figure 3Non-Human Primate CD34+CD184+ Confocal MicroscopyFigure 3. Non-Human Primate CD34+CD184+ Confocal Microscopy Disclosures: No relevant conflicts of interest to declare.
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  • 7
    Publication Date: 2010-11-19
    Description: Abstract 350 Introduction: Leukapheresis is a widely utilized modality for collecting hematopoietic stem cells (HSCs). While collection of CD34+ cells with stem-cell activity is the primary goal of most mobilization and leukapheresis procedures, these cells only represent ∼1% of most leukapheresis products. The profile of the non-CD34+ cells is likely influenced by the choice of mobilization strategy, and has the potential to profoundly impact the post-transplant immune milieu of the transplant recipient. Two of the most critical of the CD34-negative cell populations that are collected during leukapheresis include effector and regulatory T cells. Thus, in evaluating mobilization regimens, the impact on these regimens on the mobilization of each of these T cell populations into the peripheral blood should be rigorously evaluated. Methods: We used a rhesus macaque model to determine the impact that mobilization with AMD3100 (a.k.a., Plerixafor or Mozobil®)+ G-CSF (“A+G”) had on peripheral blood CD4+ and CD8+ effector T cell populations as well as on FoxP3+/CD4+ T cells. Three rhesus macaques were mobilized with 10ug/kg SQ of G-CSF for five consecutive days prior to leukapheresis. AMD3100 was administered at 1mg/kg SQ in combination with the last dose of G-CSF two hours prior to leukapheresis. Leukapheresis procedures were performed for two hours using a modified CS3000 Plus cell separator. A peripheral blood sample was taken before cytokine therapy, just prior to leukapheresis following mobilization, one hour during leukapheresis, and at the end of the procedure. These samples were analyzed by multicolor flow cytometry using a BD LSRII flow cytometer. Results: Bulk, effector, and regulatory T cell subpopulations were analyzed flow cytometrically. The proportion of total CD3+ T cells remained stable during mobilization and apheresis: Thus, CD3+ T cells represented 77% of peripheral blood lymphocytes prior to mobilization, and 69% post-apheresis). The balance of CD4+ to CD8+ T cells was also relatively stable. Thus, for one of the three animals tested, the CD4+ and CD8+ proportions remained unchanged after apheresis. For two animals, the average CD4+ % decreased from 67% prior to mobilization to 52% post-apheresis. In these two animals, there was a reciprocal increase in the % of CD3+ T cells that were CD8+ (28% pre-G+A to 40% post-apheresis). The CD28+/CD95- naïve (Tn), CD28+/CD95+ central memory (Tcm) and CD28-/CD95+ effector memory (Tem) subpopulation balance of CD4+ and CD8+ T cells was also determined, by comparing the relative percentages of each subpopulation post-apheresis with their relative percentages prior to mobilization. Compared to their pre-G+A percentages, the post-apheresis CD4+ percentages of Tn, Tcm and Tem were 92%, 93% and 160%, respectively. Thus, the relative proportions of Tn and Tcm CD4+ cells decreased post-apheresis, while the relative proportion of CD4+ Tem increased compared to cytokine administration. For CD8+ T cell subpopulations, the post-apheresis proportions of Tn, Tcm, and Tem compared to their pre-G-CSF proportions were 99%, 70% and 130%, respectively–thus demonstrating the same direction of change as observed for CD4+ T cells. The most striking change in T cell subpopulations occurred in the CD4+/FoxP3+ compartment. The proportion of CD4+ T cells expressing FoxP3 increased by an average of 600% when post-apheresis samples were compared to pre-mobilization samples (FoxP3+ cells were 9.6% of CD4+ T cells post-apheresis versus 1.5% pre-GCSF). An average of 32% of these FoxP3+ CD4+ T cells expressed high levels of CXCR4. CXCR4 expression has been previously documented on human FoxP3+ T cells (Zou et al., Cancer Res, 2004), but this is the first observation of high level expression of CXCR4 on macaque FoxP3+ CD4 T cells, or of their ability to be efficiently mobilized with AMD3100. Discussion: These results suggest that treatment with AMD3100 and G-CSF may mobilize T cell subsets into the peripheral blood that could have beneficial effects during allo-transplantation. The combination of an increase in Tem cells, which have been observed to have decreased ability to cause GvHD (Zheng et al., Blood 2008), along with FoxP3+/CD4+ T cells, which may have regulatory functions, suggests that A+G mobilization could produce an apheresis product with a beneficial CD34-negative cell profile for allogeneic transplantation. Disclosures: No relevant conflicts of interest to declare.
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  • 8
    Publication Date: 2010-11-19
    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.
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  • 9
    Publication Date: 2003-03-15
    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.
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  • 10
    Publication Date: 1999-10-01
    Description: We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated β-galactosidase gene (β-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.
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