RESUMO
Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) targeted gene therapy; however, RIC may be insufficient for efficient engraftment and inducing immunological tolerance to transgenes. We previously established long-term gene marking in our rhesus macaque autologous HSC transplantation model following 10 Gy total body irradiation (TBI). In this study, we evaluated RIC transplantation with 4 Gy TBI in two rhesus macaques that received equal parts of CD34(+) cells transduced with green fluorescent protein (GFP)-expressing lentiviral vector and empty vector not expressing transgenes. In both animals, equivalently low gene marking between GFP and empty vectors was observed 6 months post-transplantation, even with efficient transduction of CD34(+) cells in vitro. Autologous lymphocyte infusion with GFP marking resulted in an increase of gene marking in lymphocytes in a control animal with GFP tolerance, but not in the two RIC-transplanted animals. In vitro assays revealed strong cellular and humoral immune responses to GFP protein in the two RIC-transplanted animals, but this was not observed in controls. In summary, 4 Gy TBI is insufficient to permit engraftment of genetically modified HSCs and induce immunological tolerance to transgenes. Our findings should help in the design of conditioning regimens in gene therapy trials.
Assuntos
Antígenos CD34/metabolismo , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Sobrevivência de Enxerto/imunologia , Sobrevivência de Enxerto/efeitos da radiação , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/imunologia , Irradiação Corporal Total/métodos , Animais , Células Cultivadas , Terapia Combinada , Relação Dose-Resposta à Radiação , Células-Tronco Hematopoéticas/efeitos da radiação , Lentivirus/genética , Macaca mulatta , Modelos Animais , Transdução Genética , Transgenes , Condicionamento Pré-TransplanteRESUMO
SUMMARY: Two male first cousins with mild haemophilia A had baseline factor VIII levels of 12-15% and experienced bleeding requiring coagulation factor infusion therapy with trauma and surgical procedures. Both the patients with haemophilia A also had electrocardiographically documented symptomatic paroxysmal atrial fibrillation (PAF) for several years that had become resistant to pharmacological suppression. Radiofrequency ablation was considered in both the cases but deferred considering refusal of consent by the patients to undergo the procedure. Remission of arrhythmias has been reported in patients with iron-overload syndromes. Body iron stores assessed by serum ferritin levels were elevated in both men but neither had the C282Y or H63D genes for haemochromatosis. Calibrated reduction of iron stores by serial phlebotomy, avoiding iron deficiency, was followed by remission of symptomatic PAF in both cases. Iron reduction may be an effective treatment for arrhythmias apart from the classic iron-overload syndromes and deserves further study particularly in patients with bleeding disorders who might be at risk for arrhythmias and other diseases of ageing.
Assuntos
Fibrilação Atrial/etiologia , Fibrilação Atrial/terapia , Hemofilia A/complicações , Sobrecarga de Ferro/complicações , Sobrecarga de Ferro/terapia , Flebotomia , Fator VIII/administração & dosagem , Ferritinas/sangue , Hemofilia A/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Adult marrow-derived cells have been shown to contribute to various nonhematologic tissues and, conversely, primitive cells isolated from nonhematopoietic tissues have been shown to reconstitute hematopoiesis. Circulating endothelial progenitor cells (EPCs) have been reported to be at least partially donor derived after allogeneic bone marrow transplantation, and shown to contribute to neovascularization in murine ischemia models. However, it is unknown whether these EPCs are actually clonally derived from the same population of stem and progenitor cells that reconstitute hematopoiesis, or from another cell population found in the marrow or mobilized blood that is transferred during transplantation. To approach this question, we characterized circulating EPCs and also endothelial cells from large vessels harvested at autopsy from rhesus macaques previously transplanted with retrovirally transduced autologous CD34-enriched peripheral blood stem cells (PBSCs). Endothelial cells were grown in culture for 21-28 days and were characterized as CD31(+) CD14(-) via flow cytometry, as acLDL(+) UEA-1(+) via immunohistochemistry, and as Flk-1(+) by reverse transcriptase-polymerase chain reaction (RT-PCR). Animals had stable vector marking in hematopoietic lineages of 2-15%. Neither cultured circulating EPCs collected in steady state (n = 3), nor endothelial cells grown from large vessels (n = 2), had detectable retroviral marking. EPCs were CD34(+) and could be mobilized into the circulation with granulocyte colony-stimulating factor. Under ex vivo culture conditions, in which CD34(+) cells were optimized to transduce hematopoietic progenitor and stem cells, there was a marked depletion of EPCs. Transduction of EPCs was much more efficient under conditions supporting endothelial cell growth. Further elucidation of the origin and in vivo behavior of EPCs may be possible, using optimized transduction conditions and a vascular injury model.
Assuntos
Endotélio Vascular/metabolismo , Células 3T3 , Análise de Variância , Animais , Antígenos CD34/genética , Proteínas de Bactérias/metabolismo , Linhagem da Célula , Células Cultivadas , Células Clonais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Células Precursoras Eritroides/metabolismo , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Proteínas Luminescentes/metabolismo , Macaca mulatta , Camundongos , Modelos Animais , Retroviridae/genética , Transdução GenéticaRESUMO
BACKGROUND: Defining the optimum regimen and time for repeat peripheral blood progenitor cell mobilization would have important clinical applications. STUDY DESIGN AND METHODS: Remobilization with SCF and G-CSF at 2 weeks after an initial mobilization in mice and at 2 or 4 weeks after an initial mobilization in nonhuman primates was examined. In mice, competitive repopulation assays were used to measure long-term progenitor cell-repopulating activity. In monkeys, mobilization of hematopoietic progenitor CFUs was used as a surrogate marker for progenitor cell-repopulating ability. RESULTS: Efficacy of progenitor cell remobilization differed in the two animal species. In mice, peripheral blood progenitor cell-repopulating ability with repeat mobilization at 2 weeks was 70 percent of that with the initial mobilization. In monkeys, there was no significant difference in peripheral blood progenitor cell mobilization between the initial and the repeat mobilizations at 2 weeks. In mobilizations separated by 4 weeks, however, peripheral blood progenitor cell mobilization was higher than that with initial mobilizations. CONCLUSION: In animal models, mobilization of peripheral blood progenitor cells with remobilization after a 2-week interval is similar to or moderately decreased from that with the initial mobilization. Progenitor cell collection at this time point may be useful in certain clinical circumstances. A 4-week interval between remobilizations may be preferable. Clinical trials in humans would be useful to clarify these issues.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/citologia , Macaca mulatta/sangue , Camundongos/sangue , Fator de Células-Tronco/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Feminino , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/metabolismo , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Transformação Celular Viral , Células Cultivadas , Citocinas/farmacologia , Primers do DNA/genética , Fibronectinas/farmacologia , Terapia Genética , Vetores Genéticos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Humanos , Macaca mulatta , Fragmentos de Peptídeos/farmacologia , Retroviridae/genética , Fator de Células-Tronco/farmacologia , Transdução GenéticaRESUMO
Three species of fleas, Oropsylla montana (Baker), Hoplopsyllus anomalus (Baker), and Echidnophaga gallinacea (Westwood), occur seasonally on the California ground squirrel, Spermophilus beecheyi (Richardson). Few studies have focused on the biology and ecology of these fleas despite their importance in the epidemiology of sylvatic plague. To best duplicate a natural parasite-host relationship in the laboratory, a novel nest box was developed that facilitated housing wild-caught S. beecheyi, was conducive to rearing fleas, and met modern standards for laboratory animal hygiene. This flexible system allowed adult fleas with different feeding strategies to be colonized successfully while providing sufficient flea eggs for both colony maintenance and biological research. The techniques described could be adapted to work with other species of rodents and their fleas.
Assuntos
Sciuridae/parasitologia , Sifonápteros/crescimento & desenvolvimento , Animais , CaliforniaRESUMO
Nonhuman primate model systems of autologous CD34+ cell transplant are the most effective means to assess the safety and capabilities of lentivirus vectors. Toward this end, we tested the efficiency of marking, gene expression, and transplant of bone marrow and peripheral blood CD34+ cells using a self-inactivating lentivirus vector (CS-Rh-MLV-E) bearing an internal murine leukemia virus long terminal repeat derived from a murine retrovirus adapted to replicate in rhesus macaques. In vitro cytokine stimulation was not required to achieve efficient transduction of CD34+ cells resulting in marking and gene expression of the reporter gene encoding enhanced green fluorescent protein (EGFP) following transplant of the CD34+ cells. Monkeys transplanted with mobilized peripheral blood CD34+ cells resulted in EGFP expression in 1 to 10% of multilineage peripheral blood cells, including red blood cells and platelets, stable for 15 months to date. The relative level of gene expression utilizing this vector is 2- to 10-fold greater than that utilizing a non-self-inactivating lentivirus vector bearing the cytomegalovirus immediate-early promoter. In contrast, in animals transplanted with autologous bone marrow CD34+ cells, multilineage EGFP expression was evident initially but diminished over time. We further tested our lentivirus vector system by demonstrating gene transfer of the human common gamma-chain cytokine receptor gene (gamma(c)), deficient in X-linked SCID patients and recently successfully used to treat disease. Marking was 0.42 and.001 HIV-1 vector DNA copy per 100 cells in two animals. To date, all EGFP- and gamma(c)-transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.
Assuntos
Citocinas/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , HIV-1/genética , Células-Tronco Hematopoéticas/metabolismo , Macaca mulatta/metabolismo , Receptores de Superfície Celular/genética , Animais , Antígenos CD34/metabolismo , Biomarcadores , Citometria de Fluxo , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/farmacologia , Proteínas de Fluorescência Verde , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucaférese , Proteínas Luminescentes , Linfócitos/metabolismo , Macaca mulatta/genética , Reação em Cadeia da Polimerase , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Fator de Células-Tronco/farmacologia , Fatores de Tempo , Transdução GenéticaRESUMO
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 of MDR1-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 either MDR1 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 both MDR 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 neo vector-containing cells. There was no evidence for expansion of MDR1 vector-transduced cells. Long-term engraftment with MDR1 vector- and neo vector-transduced cells occurred despite prolonged culture.
Assuntos
Resistência Microbiana a Medicamentos/genética , Genes MDR/genética , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Animais , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Dosagem de Genes , Terapia Genética/normas , Vetores Genéticos/efeitos adversos , Vetores Genéticos/normas , Transplante de Células-Tronco Hematopoéticas/normas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Macaca mulatta , Modelos Animais , Neomicina , Transdução Genética/métodos , Transdução Genética/normasRESUMO
Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell (HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells (STR), or the fibronectin fragment CH-296 (FN), and various cytokines such as flt3 ligand (FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and clinically feasible peripheral blood (PB) progenitor cell transduction methods. First, we compared transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor (SCF), and FLT (our best cytokine combination in prior studies) with a combination of megakaryocyte growth and development factor (MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10-15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.
Assuntos
Antígenos CD34/genética , Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas/metabolismo , Retroviridae/genética , Animais , Southern Blotting , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Fibronectinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Macaca mulatta , Proteínas de Membrana/farmacologia , Reação em Cadeia da Polimerase , Fator de Células-Tronco/farmacologia , Células Estromais/metabolismo , Trombopoetina/farmacologiaRESUMO
Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.
Assuntos
Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Tolerância Imunológica , Animais , Sequência de Bases , Transfusão de Sangue Autóloga , Primers do DNA/genética , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Canamicina Quinase/genética , Canamicina Quinase/imunologia , Transfusão de Linfócitos , Linfócitos/imunologia , Linfócitos/metabolismo , Macaca mulatta , Modelos Biológicos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transdução Genética , Transplante AutólogoRESUMO
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.
Assuntos
Linfócitos B/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Linfócitos T/citologia , Animais , Antígenos CD34/sangue , Diferenciação Celular , Ensaio de Unidades Formadoras de Colônias , Técnicas de Transferência de Genes , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos , Mobilização de Células-Tronco Hematopoéticas , Humanos , Canamicina Quinase/genética , Macaca mulatta , Retroviridae , Transfecção , Irradiação Corporal TotalRESUMO
Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34(+) cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted.
Assuntos
Antígenos CD34/análise , Expressão Gênica , Vetores Genéticos , HIV-1/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Linfócitos T/metabolismo , Animais , Citomegalovirus/genética , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas/virologia , Humanos , Vírus da Leucemia Murina/genética , Leucopoese , Transplante de Fígado/imunologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Ativação Linfocitária , Macaca mulatta , Camundongos , Camundongos SCID , Regiões Promotoras Genéticas , Linfócitos T/imunologia , Linfócitos T/virologia , Timo/imunologia , Timo/transplante , Transdução Genética , Replicação ViralRESUMO
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. (Blood. 2000;95:445-452)
Assuntos
Citocinas/farmacologia , Granulócitos/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Proteínas Luminescentes/genética , Tetra-Hidrofolato Desidrogenase/genética , Transfecção/métodos , Animais , DNA Complementar/administração & dosagem , Fibronectinas/farmacologia , Proteínas de Fluorescência Verde , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Humanos , Proteínas Luminescentes/biossíntese , Subpopulações de Linfócitos/imunologia , Linfócitos/citologia , Linfócitos/imunologia , Macaca mulatta , Fragmentos de Peptídeos/farmacologia , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/farmacologia , Tetra-Hidrofolato Desidrogenase/biossínteseRESUMO
We used a first-generation adenovirus vector (AVC3FIX5) to assess whether human factor IX could be expressed and detected in the rhesus macaque, which we have shown does not make high-titer antibodies to human factor IX protein. Three animals received 1 x 10(10) to 1 x 10(11) plaque-forming units per kilogram by intravenous injection. Human factor IX was present within 24 hours of vector administration and peaked 4 days later at 4,000 ng/mL in the high-dose recipient, and lower levels were seen in the intermediate-dose recipient. No human factor IX was detected in the low-dose recipient's plasma. Serum cytokine analysis and early hypoferremia suggested a dose-dependent acute-phase response to the vector. Human factor IX was detectable in rhesus plasma for 2 to 3 weeks for the high- and intermediate-dose recipients, but disappeared concomitant with high-titer antihuman factor IX antibody development. There was substantial, dose-dependent, dose-limiting liver toxicity that was manifest as elevated serum transaminase levels, hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting times. Of particular interest was prolongation of the thrombin clotting time, an indicator of decreased fibrinogen or fibrinogen dysfunction. All evidence of liver toxicity resolved except for persistent hypofibrinogenemia in the high-dose recipient, indicating possible permanent liver damage. Our data suggest a narrow therapeutic window for first-generation adenovirus-mediated gene transfer. The development of antihuman factor IX antibodies and abnormalities of fibrinogen in the rhesus macaque is of concern for application of adenovirus (or other viral) vectors to hemophilia gene therapy.
Assuntos
Adenoviridae , Fator IX/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Relação Dose-Resposta a Droga , Fator IX/biossíntese , Fator IX/toxicidade , Técnicas de Transferência de Genes/efeitos adversos , Vetores Genéticos/efeitos adversos , Humanos , Macaca mulattaRESUMO
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 beta-galactosidase gene (beta-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.
Assuntos
Dependovirus/genética , Vetores Genéticos , Retroviridae/genética , Linfócitos T/citologia , Animais , Resistência Microbiana a Medicamentos/genética , Genes Reporter , Transfusão de Linfócitos , Macaca mulatta , Neomicina , Reação em Cadeia da Polimerase , Linfócitos T/imunologia , Transfecção , beta-Galactosidase/genéticaRESUMO
The ability to engraft significant numbers of genetically modified hematopoietic stem and progenitor cells without the requirement for fully myeloablative conditioning therapy is a highly desirable goal for the treatment of many nonmalignant hematologic disorders. The aims of this study were to examine, in nonhuman primates (rhesus), (1) the effects of pretreatment of host animals with cytokines (G-CSF and SCF), i.e., before nonmyeloablative irradiation, on the degree and duration of neo gene marking of circulating leukocytes after autologous cell reinfusion and (2) to compare transduction of primitive hematopoietic target cells in the presence of our standard transduction cytokine combination of IL-3, IL-6, and stem cell factor (SCF) and in the presence of an alternative combination containing SCF, G-CSF, and the thrombopoietin analog MGDF. Cytokine-mobilized rhesus peripheral blood progenitor/stem cells (PBSCs) were enriched for CD34+ cells and transduced with neo vectors (either G1Na or LNL6) for 96 hr in cultures containing rhIL-3, rhIL-6, and rhSCF or MGDF, rhSCF, and rhG-CSF and cryopreserved. Four animals underwent minimal myeloablative conditioning with 500 cGy irradiation with or without pretreatment with SCF and G-CSF, followed by reinfusion of the cryopreserved cells on the subsequent day. Neutrophil nadirs (< or =500/mm3) were 0-3 days in duration; there were no significant periods of severe thrombocytopenia. Marking of circulating granulocytes and mononuclear cells was extensive and durable in all animals (exceeding 12% in the mononuclear cells of one animal) and persisted beyond the final sampling time in all animals (up to 33 weeks). No difference in extent or duration of marking was attributable to either cytokine presensitization of recipients prior to irradiation, or to the substitution of MDGF and G-CSF for IL-3 and IL-6 during transduction.
Assuntos
Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/efeitos da radiação , Transplante de Células-Tronco Hematopoéticas , Retroviridae/genética , Condicionamento Pré-Transplante , Animais , Antibacterianos/farmacologia , Antígenos CD34/metabolismo , Resistência a Medicamentos/genética , Técnicas de Transferência de Genes , Marcadores Genéticos , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos da radiação , Células-Tronco Hematopoéticas/virologia , Leucócitos Mononucleares/imunologia , Macaca mulatta , Neomicina/farmacologia , Reação em Cadeia da Polimerase , Doses de Radiação , Fator de Células-Tronco/farmacologia , Transdução GenéticaRESUMO
The transduction efficiencies of immunoselected rhesus macaque (Macaca mulatta) CD34+ cells and colony-forming progenitor cells based on polymerase chain reaction (PCR) analysis were comparable for an amphotropic Moloney murine leukemia virus (MLV) retroviral vector and a retroviral vector derived from the gibbon ape leukemia virus (GaLV) packaging cell line, PG13. On performing autologous transplantation studies using immunoselected CD34+ cells transduced with the GaLV envelope (env) retroviral vector, less than 1% of peripheral blood (PB) contained provirus. This was true whether bone marrow (BM) or cytokine-mobilized PB immunoselected CD34+ cells were reinfused. This level of marking was evident in two animals whose platelet counts never fell below 50,000/microliter and whose leukocyte counts had recovered by days 8 and 10 after having received 1.7 x 10(7) or greater of cytokine-mobilized CD34+ PB cells/kg. Reverse transcriptase(RT)-PCR analysis of CD34+ subsets for both the GaLV and amphotropic receptor were performed. The expression of the GaLV receptor was determined to be restricted to CD34+ Thy-1+ cells, and both CD34+ CD38+ and CD34+ CD38dim cells, while the amphotropic receptor was present on all CD34+ cell subsets examined. Our findings suggest that, in rhesus macaques, PG13-derived retroviral vectors may only be able to transduce a subset of CD34+ cells as only CD34+ Thy-1+ cells express the GaLV receptor.
Assuntos
Antígenos CD34 , Antígenos CD , Vetores Genéticos/genética , Transplante de Células-Tronco Hematopoéticas , Vírus da Leucemia do Macaco Gibão/genética , Vírus da Leucemia Murina de Moloney/genética , Transfecção/métodos , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos de Diferenciação , Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Macaca mulatta , NAD+ Nucleosidase , Antígenos Thy-1RESUMO
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.
Assuntos
Modelos Animais de Doenças , Fator IX/genética , Terapia Genética , Hemofilia B/terapia , Sequência de Aminoácidos , Animais , Anticorpos/sangue , Anticorpos Monoclonais , Antígenos/administração & dosagem , Antígenos/imunologia , DNA Complementar/química , Fator IX/química , Fator IX/imunologia , Humanos , Macaca mulatta , Dados de Sequência Molecular , Farmacocinética , Reação em Cadeia da Polimerase , Polimorfismo Genético , Homologia de SequênciaRESUMO
The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)-mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with 0.01% in the original IL-3/IL-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Macaca mulatta/sangue , Animais , Células Cultivadas/transplante , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Filgrastim , Genes Reporter , Marcadores Genéticos , Vetores Genéticos , Sobrevivência de Enxerto , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Canamicina Quinase/genética , Proteínas Proto-Oncogênicas/farmacologia , Quimera por Radiação , Receptores Proteína Tirosina Quinases/farmacologia , Proteínas Recombinantes , Fator de Células-Tronco/farmacologia , Células Estromais/citologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Irradiação Corporal TotalRESUMO
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 retrovirally mediated gene transfer with a vector expressing an antisense tat/rev gene. This group of animals and three control macaques were subsequently infected with SIVmac239. Blood and lymph nodes from all macaques were sampled for more than a year to monitor the progress of infection. Although all animals became infected, the animals that received the lymphocytes engineered with the antisense vector demonstrated a significant reduction in viral load in both peripheral blood and lymph nodes, had sustained numbers of CD4+ cells, and exhibited little disruption of lymph node architecture.