RESUMO
Development of curative approaches for HIV-1 infected patients requires novel approaches aimed at eliminating viral reservoirs and replacing potential target cells with infection-resistant immune cell populations. We have previously shown that autologous transplantation of genetically modified hematopoietic stem cells (HSCs) with lentiviral vectors encoding the mC46-fusion inhibitor results in a significant reduction in viral pathogenesis following challenge with the highly pathogenic dual tropic, SHIV89.6P strain. In this study, we used a combinatorial approach in which following engraftment of genetically modified HSCs, pigtailed macaques were vaccinated with a previously developed vaccinia-based vaccine expressing SIV-Gag, Pol. Using this dual therapy approach, lower viremia was detected in both the acute and chronic phase of disease with levels reaching near the lower limits of detection. In comparison with macaques receiving HSCT only, the combination approach resulted in a further log decrease in plasma viremia. Similar to our previous studies, positive selection of all CD4(+) T-cell subsets was observed; however, higher gene-modified CD4(+) T-cell levels were observed during the chronic phase when vaccination was included suggesting that combining vaccination with HSCT may lower the necessary threshold for achieving viremic control.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Vacinas contra a SAIDS/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Animais , Linfócitos T CD4-Positivos/imunologia , Macaca nemestrina , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Subpopulações de Linfócitos T/imunologia , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologia , Carga ViralRESUMO
Foamy virus (FV) vectors are promising for hematopoietic stem cell (HSC) gene therapy but preclinical data on the clonal composition of FV vector-transduced human repopulating cells is needed. Human CD34(+) human cord blood cells were transduced with an FV vector encoding a methylguanine methyltransferase (MGMT)P140K transgene, transplanted into immunodeficient NOD/SCID IL2Rγ(null) mice, and selected in vivo for gene-modified cells. The retroviral insertion site profile of repopulating clones was examined using modified genomic sequencing PCR. We observed polyclonal repopulation with no evidence of clonal dominance even with the use of a strong internal spleen focus forming virus promoter known to be genotoxic. Our data supports the use of FV vectors with MGMTP140K for HSC gene therapy but also suggests additional safety features should be developed and evaluated.
Assuntos
Imunodeficiência Combinada Severa/genética , Spumavirus/genética , Integração Viral/genética , Animais , Testes Genéticos/métodos , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Condicionamento Pré-TransplanteRESUMO
The 'Berlin Patient', who maintains suppressed levels of HIV viremia in the absence of antiretroviral therapy, continues to be a standard bearer in HIV eradication research. However, the unique circumstances surrounding his functional cure are not applicable to most HIV(+) patients. To achieve a functional or sterilizing cure in a greater number of infected individuals worldwide, combinatorial treatments, targeting multiple stages of the viral life cycle, will be essential. Several anti-HIV gene therapy approaches have been explored recently, including disruption of the C-C chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) coreceptor loci in CD4(+) T cells and CD34(+) hematopoietic stem cells. However, less is known about the efficacy of these strategies in patients and more relevant HIV model systems such as non-human primates (NHPs). Combinatorial approaches, including genetic disruption of integrated provirus, functional enhancement of endogenous restriction factors and/or the use of pharmacological adjuvants, could amplify the anti-HIV effects of CCR5/CXCR4 gene disruption. Importantly, delivering gene disruption molecules to genetic sites of interest will likely require optimization on a cell type-by-cell type basis. In this review, we highlight the most promising gene therapy approaches to combat HIV infection, methods to deliver these therapies to hematopoietic cells and emphasize the need to target viral replication pre- and post-entry to mount a suitably robust defense against spreading infection.
Assuntos
Terapia Antirretroviral de Alta Atividade , Infecções por HIV/genética , Infecções por HIV/terapia , Replicação Viral/genética , Antígenos CD34/genética , Antígenos CD34/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Terapia Genética , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Células-Tronco Hematopoéticas/citologia , Humanos , Terapia de Alvo Molecular , Receptores CCR5/genética , Receptores CXCR4/genéticaRESUMO
Allogeneic hematopoietic cell transplantation (HCT) is the only known cure for patients with Fanconi anemia (FA) who develop aplasia or leukemia. However, transplant regimens typically contain high-dose alkylators, which are poorly tolerated in FA patients. Furthermore, as many patients lack human leukocyte antigen (HLA)-matched family donors, alternative donors are used, which can increase the risk of both graft rejection and graft-versus-host disease (GVHD). To improve on these three concerns, we developed a multi-institutional clinical trial using a fludarabine (FLU)-based conditioning regimen with limited alkylators/low-dose radiation, HLA-haploidentical marrow, followed by reduced-dose cyclophosphamide (CY) to treat three FA patients with aplasia. All three patients engrafted with 100% donor CD3 chimerism at 1 month. One patient died early from disseminated toxoplasmosis infection. Of the two survivors, one had significant pretransplant co-morbidities and inadequate immunosuppression, and developed severe acute GVHD. The other patient had only mild acute and no chronic GVHD. With a follow-up of 2 and 3 years, respectively, both patients are doing well, are transfusion-independent, and maintain full donor chimerism. The patient with severe GVHD has resolving oral GVHD and good quality of life. We conclude that using low-intensity conditioning, HLA-haploidentical marrow, and reduced-dose CY for in vivo T-cell depletion can correct life-threatening aplasia in FA patients.
Assuntos
Anemia de Fanconi/terapia , Rejeição de Enxerto/prevenção & controle , Doença Enxerto-Hospedeiro/prevenção & controle , Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas , Depleção Linfocítica , Linfócitos T/imunologia , Vidarabina/análogos & derivados , Adolescente , Antineoplásicos/uso terapêutico , Criança , Terapia Combinada , Anemia de Fanconi/imunologia , Feminino , Seguimentos , Humanos , Quimeras de Transplante/imunologia , Condicionamento Pré-Transplante , Transplante Homólogo , Vidarabina/uso terapêuticoRESUMO
Gammaretroviral vectors are an efficient means to effect gene therapy. However, genotoxicity from insertion at nonrandom sites can confer a competitive advantage to transduced cells, resulting in clonal proliferation or neoplasia. Six pig-tailed macaques (Macaca nemestrina) underwent total body irradiation and reconstitution with autologous stem cells genetically modified by a gammaretroviral vector overexpressing HOXB4. Two animals were euthanized owing to irradiation- or transplantation-associated toxicity, whereas the other 4 had successful reconstitution. Of the 4 macaques with successful reconstitution, 1 has no long-term follow-up information; 1 was euthanized owing to infection with simian varicella virus infection 18 months post-total body irradiation; and the 2 others are described herein as case Nos. 1 and 2. After being stable for 3 years, case No. 1 developed pancytopenia and petechiation, and after 2 years of stability case No. 2 developed anemia and thrombocytopenia. Despite therapy, the animals deteriorated and were euthanized. Gross findings included emaciation; case No. 1 also had hemorrhage, peritonitis, and cholecystitis. Histologically, bone marrow was hypercellular with predominately blast cells of all hematopoietic lineages, though with myeloid predominance, and with maturation arrest and blast cell dysplasia (myelodysplasia). Myelodysplasia was likely from a combination of insertional mutagenesis by the retroviral vector and overexpression of HOXB4. Consequences of myelodysplasia included the blood dyscrasias and, in case No. 1, hemorrhage, bacterial cholecystitis, hepatitis, and peritonitis.
Assuntos
Proteínas de Homeodomínio/genética , Macaca nemestrina , Doenças dos Macacos/patologia , Síndromes Mielodisplásicas/veterinária , Fatores de Transcrição/genética , Animais , Evolução Fatal , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Terapia Genética/veterinária , Vetores Genéticos , Masculino , Doenças dos Macacos/genética , Mutagênese Insercional/métodos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologiaRESUMO
Large animal models have been instrumental in advancing hematopoietic stem cell (HSC) gene therapy. Here we review the advantages of large animal models, their contributions to the field of HSC gene therapy and recent progress in this field. Several properties of human HSCs including their purification, their cell-cycle characteristics, their response to cytokines and the proliferative demands placed on them after transplantation are more similar in large animal models than in mice. Progress in the development and use of retroviral vectors and ex vivo transduction protocols over the last decade has led to efficient gene transfer in both dogs and nonhuman primates. Importantly, the approaches developed in these models have translated well to the clinic. Large animals continue to be useful to evaluate the efficacy and safety of gene therapy, and dogs with hematopoietic diseases have now been cured by HSC gene therapy. Nonhuman primates allow evaluation of aspects of transplantation as well as disease-specific approaches such as AIDS (acquired immunodeficiency syndrome) gene therapy that can not be modeled well in the dog. Finally, large animal models have been used to evaluate the genotoxicity of viral vectors by comparing integration sites in hematopoietic repopulating cells and monitoring clonality after transplantation.
Assuntos
Terapia Genética/tendências , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Modelos Animais , Animais , Dano ao DNA , Cães , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Infecções por HIV/genética , Infecções por HIV/terapia , Células-Tronco Hematopoéticas/virologia , Humanos , Camundongos , PrimatasRESUMO
Highly active antiretroviral therapy has greatly reduced the morbidity and mortality from human immunodeficiency virus (HIV) infection, but AIDS continues to be a serious health problem worldwide. Despite enormous efforts to develop a vaccine, there is still no cure, and alternative approaches including gene therapy should be explored. In this study we developed and compared combinatorial foamy virus (FV) anti-HIV vectors that also express a mutant methylguanine methyltransferase (MGMTP140K) transgene to increase the percentage of gene-modified cells after transplantation. These FV vectors inhibit replication of HIV-1 and also the simian immunodeficiency virus/HIV-1 (SHIV) chimera that can be used in monkey AIDS gene therapy studies. We identified a combinatorial FV vector that expresses 3 anti-HIV transgenes and inhibits viral replication by over 4 logs in a viral challenge assay. This FV anti-HIV vector expresses an HIV fusion inhibitor and two short hairpin RNAs (shRNAs) targeted to HIV-1 tat and rev, and can be produced at high titer (3.8 x 10(7) transducing units ml(-1)) using improved helper plasmids suitable for clinical use. Using a competitive repopulation assay, we show that human CD34(+) cells transduced with this combinatorial FV vector efficiently engraft in a mouse xenotransplantation model, and that the percentage of transduced repopulating cells can be increased after transplantation.
Assuntos
Terapia Genética/métodos , Vetores Genéticos , HIV-1 , Vírus da Imunodeficiência Símia , Vírus Espumoso dos Símios/genética , Animais , Metilases de Modificação do DNA/deficiência , Enzimas Reparadoras do DNA/deficiência , Técnicas de Transferência de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Transdução Genética , Transgenes , Proteínas Supressoras de Tumor/deficiência , Replicação ViralRESUMO
One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.
Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética/métodos , Lentivirus/genética , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Anemia de Fanconi/patologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mitomicina/farmacologia , Transdução GenéticaRESUMO
C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti-CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL-4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before infusion. The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. The cytokine-induced killer cells generated as described here could have an important impact on bone marrow purging for autologous bone marrow transplantation as well as for adoptive immunotherapy.
Assuntos
Citotoxicidade Imunológica , Síndromes de Imunodeficiência/imunologia , Interferon gama/farmacologia , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Linfoma de Células B/imunologia , Animais , Anticorpos Monoclonais , Antígenos CD/análise , Antígenos CD19 , Antígenos de Diferenciação de Linfócitos B/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos B/imunologia , Medula Óssea/imunologia , Medula Óssea/patologia , Complexo CD3 , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Transplante de Neoplasias , Receptores de Antígenos de Linfócitos T/análise , Proteínas Recombinantes/farmacologia , Transplante Heterólogo , Ensaio Tumoral de Célula-TroncoRESUMO
PURPOSE: We used total body irradiation (TBI) as conditioning for cord blood transplantation studies in pigtailed macaques. In these studies, different doses of TBI were explored to obtain optimal myelosuppression with acceptable radiation-related side effects. METHODS: Four macaques received TBI ranging from 800 to 1320 cGy, followed by standard post-transplant care. Hematopoietic recovery was monitored by CBC and donor contribution by variable number of tandem repeats analysis. RESULTS: Animals receiving 800 or 1020 cGy TBI tolerated the irradiation well with autologous recovery of neutrophils within 24 days. At a dose of 1200 cGy, neither autologous recovery nor extramedullary toxicity was observed. A fourth animal received 1320 cGy of TBI and suffered significant toxicity necessitating termination of the study. CONCLUSIONS: We conclude that previously considered myeloablative doses of TBI allowed for autologous recovery in the pigtailed macaque, and that a dose of 1200 cGy may be most appropriate, providing both myeloablation and acceptable non-hematopoietic toxicities.
Assuntos
Medula Óssea/efeitos da radiação , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Irradiação Corporal Total , Animais , Plaquetas/efeitos da radiação , Medula Óssea/patologia , Relação Dose-Resposta à Radiação , Feminino , Hematopoese , Macaca nemestrina , Neutrófilos/efeitos da radiação , Contagem de Plaquetas , Doses de Radiação , Lesões por Radiação/patologiaRESUMO
We have previously compared the repopulation ability of gene-modified baboon CD34+ cells in an autologous transplantation versus a xenotransplant model in irradiated nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Baboon CD34-selected marrow cells were transduced with a gammaretrovirus vector and infused into irradiated baboons and NOD/SCID mice. A limited integration-site analysis could only detect two common retrovirus integration sites in the NOD/SCID and monkey. Here, we performed locus-specific PCR on 30 clones recovered from NOD/SCID beta2-microglobulin mice reconstituted with transduced baboon CD34+ cells. We identified five common integrants in the baboon early after transplant (2-6 weeks) but none during the long-term follow-up (6 and 12 months). These results confirm that repopulating cells in the NOD/SCID mouse contribute only to short-term repopulation in a clinically relevant large animal model.
Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Papio , Animais , Antígenos CD34 , Divisão Celular , Células Clonais , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Gammaretrovirus/genética , Terapia Genética/veterinária , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Animais , Reação em Cadeia da Polimerase/métodos , Tempo , Transdução Genética/métodos , Transplante Autólogo , Transplante HeterólogoRESUMO
Therapies based on immune cells have been applied for diseases ranging from cancer to diabetes. However, the viral and electroporation methods used to create cytoreagents are complex and expensive. Consequently, we develop targeted mRNA nanocarriers that are simply mixed with cells to reprogram them via transient expression. Here, we describe three examples to establish that the approach is simple and generalizable. First, we demonstrate that nanocarriers delivering mRNA encoding a genome-editing agent can efficiently knock-out selected genes in anti-cancer T-cells. Second, we imprint a long-lived phenotype exhibiting improved antitumor activities into T-cells by transfecting them with mRNAs that encode a key transcription factor of memory formation. Third, we show how mRNA nanocarriers can program hematopoietic stem cells with improved self-renewal properties. The simplicity of the approach contrasts with the complex protocols currently used to program therapeutic cells, so our methods will likely facilitate manufacturing of cytoreagents.Current widely used viral and electroporation methods for creating therapeutic cell-based products are complex and expensive. Here, the authors develop targeted mRNA nanocarriers that can transiently program gene expression by simply mixing them with cells, to improve their therapeutic potential.
Assuntos
Técnicas de Reprogramação Celular , RNA Mensageiro/química , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Feminino , Edição de Genes/métodos , Técnicas de Inativação de Genes , Impressão Genômica , Células-Tronco Hematopoéticas/citologia , Humanos , Células Jurkat , Células K562 , Leucócitos Mononucleares , Camundongos Endogâmicos NOD , Nanopartículas/uso terapêutico , Estudo de Prova de Conceito , Linfócitos T/citologia , Fatores de Transcrição/genética , Transfecção/métodosRESUMO
We developed a haploidentical transplantation protocol with post-transplant cyclophosphamide (CY) for in vivo T-cell depletion (TCD) using a novel adapted-dosing schedule (25 mg/kg on days +3 and +4) for Fanconi anemia (FA). With median follow-up of 3 years (range, 37 days to 6.2 years), all six patients engrafted. Two patients with multiple pre-transplant comorbidities died, one from sepsis and one from sepsis with associated chronic GVHD. Four patients without preexisting comorbidities and early transplant referrals are alive with 100% donor chimerism and excellent performance status. We conclude that adjusted-dosing post-transplant CY is effective in in vivo TCD to promote full donor engraftment in patients with FA.
Assuntos
Ciclofosfamida/administração & dosagem , Anemia de Fanconi/terapia , Depleção Linfocítica/métodos , Transplante Haploidêntico/métodos , Criança , Pré-Escolar , Esquema de Medicação , Anemia de Fanconi/mortalidade , Feminino , Humanos , Imunossupressores/administração & dosagem , Masculino , Linfócitos TRESUMO
A total of 21 patients with severe aplastic anemia (SAA) underwent marrow transplantation from HLA-identical siblings following a standard conditioning regimen with cyclophosphamide (50 mg/kg/day × 4 days) and horse antithymocyte globulin (30 mg/kg/day × 3 days). Post-grafting immunosuppression consisted of a short course of methotrexate (MTX) combined with cyclosporine (CSP). The transplant protocol tested the hypothesis that the incidence of chronic GvHD could be reduced by limiting the marrow grafts to ⩽2.5 × 108 nucleated marrow cells/kg. None of the patients rejected the graft, all had sustained engraftment and all are surviving at a median of 4 (range 1-8) years after transplantation. Chronic GvHD developed in 16% of patients given ⩽2.5 × 108 nucleated marrow cells/kg. Post-grafting immunosuppression has been discontinued in 20 of the 21 patients. In conclusion, limiting the number of transplanted marrow cells may have resulted in minimal improvement in the incidence and severity of chronic GvHD.
Assuntos
Anemia Aplástica/terapia , Transplante de Medula Óssea/métodos , Contagem de Células , Doença Enxerto-Hospedeiro/prevenção & controle , Adolescente , Adulto , Anemia Aplástica/complicações , Criança , Pré-Escolar , Feminino , Sobrevivência de Enxerto , Teste de Histocompatibilidade , Humanos , Terapia de Imunossupressão/métodos , Masculino , Pessoa de Meia-Idade , Irmãos , Resultado do Tratamento , Adulto JovemRESUMO
We have characterized a chromosomal translocation in a cell line (SU-DUL5) established from a patient with lymphoblastic lymphoma in which the c-myc gene on chromosome 8 was juxtaposed to a t(14;18). Cytogenetic analysis of this cell line showed 14q+, 18q-, and 8p+q+ marker chromosomes in the absence of t(14;18). Genomic Southern blot analysis showed juxtaposition of the immunoglobulin heavy chain joining region (JH) with chromosome 18 near the minor breakpoint cluster region (mcr) of the bcl-2 gene. There was also a rearranged c-myc gene detectable with a 5' c-myc probe. Molecular cloning studies showed that the c-myc gene was joined to chromosome 18 DNA. Nucleotide sequence analysis of cloned breakpoint DNA revealed that the crossover between chromosomes 8 and 18 occurred at the 3' end of the bcl-2 gene resulting in replacement of the bcl-2 gene on the 14q+ chromosome with the c-myc gene. As a result of this translocation the SU-DUL5 cell line contains no detectable bcl-2 mRNA or protein but has abundant levels of c-myc mRNA. Our data suggest that bcl-2 inactivation occurred simultaneously with c-myc translocation in a B cell lymphoblastic lymphoma.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes/genética , Translocação Genética/genética , Adulto , Sequência de Bases , Northern Blotting , Linhagem Celular , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 8 , Humanos , Cariotipagem , Masculino , Dados de Sequência Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Green fluorescent proteins (GFPs) have been widely used to monitor gene transfer and expression after lentiviral and oncoretroviral transduction of hematopoietic cells. Studies have shown a complete disappearance of GFP-containing cells after transplantation of GFP-transduced repopulating cells in nonhuman primates that was further shown to be mediated by transgene-specific immune responses. We wished to evaluate whether cyclosporine could prevent immune responses to GFP. We first determined whether an immune response to GFP was responsible for the disappearance of gene-modified cells in dogs. We performed immune assays in two dogs transplanted with lentivirally transduced CD34+ cells. Blood samples were obtained twice per week for up to 800 days and the GFP transgene product was measured by flow cytometry in blood leukocytes. Peripheral blood leukocytes were stimulated in vitro for 5 days, using a panel of GFP peptides. Intracellular levels of tumor necrosis factor alpha (TNF-alpha), measured by flow cytometry, and T cell proliferation after GFP peptide stimulation were measured. Dogs that exhibited a decrease in GFP marking developed potent immune responses in vitro to the transgene product GFP as shown by an increase in GFP-specific TNF-alpha production (p < 0.05) when compared with nontransplanted controls. T cells from dogs with low GFP marking exhibited a significant increase in proliferation in response to GFP peptide stimulation in vitro (p < 0.05). To study whether cyclosporine could inhibit the development of GFP-specific immune responses, we treated five dogs with cyclosporine after transplantation of GFP-transduced hematopoietic cells. Dogs treated with cyclosporine after hematopoietic stem cell transplantation showed stable GFP marking in blood leukocytes over 800 days. Our data suggest that cyclosporine prevents immunoactivation against transgene products after transplantation of GFP-transduced hematopoietic stem cells as indicated by stable GFP marking.
Assuntos
Ciclosporina/farmacologia , Proteínas de Fluorescência Verde/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/imunologia , Animais , Citocinas/metabolismo , Cães , Proteínas de Fluorescência Verde/efeitos dos fármacos , Proteínas de Fluorescência Verde/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Imunossupressores/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução GenéticaRESUMO
OBJECTIVE: To test gibbon ape leukemia virus (GALV) pseudotype vector transduction of marrow subpopulations that contribute to hematopoietic reconstitution in vivo. MATERIALS AND METHODS: Autologous CD34(+) Lin(-), CD34(+) Lin(+), and CD34(-) Lin(-) marrow cells, transduced by coculture with PG13/LN, PG13/LNX, and PG13/LNY vector-producing cells, respectively, were transplanted in three female baboons. Two female baboons also were transplanted with fresh allogeneic CD34(-)Lin(-) marrow cells from MHC-matched male siblings and, to ensure survival, with autologous CD34(+)Lin(-) and CD34(+)Lin(+) marrow cells transduced with PG13/LN and PG13/LNX, respectively. The LN, LNX, and LNY vectors are identical except for different length sequences at the 3' end of the bacterial neomycin phosphotransferase (neo) gene. RESULTS: LN(+) and LNX(+) cells from CD34(+)Lin(-) and CD34(+)Lin(+) cells, respectively, but no LNY(+) from CD34(-)Lin(-) cells were detectable in blood and marrow of all animals after transplant. LN(+), CD34(+)Lin(-) cells contributed to reconstitution of the T, B, and myeloid lineages. LNX(+), CD34(+)Lin(+) cells contributed only to B and myeloid lineages. Male cells, CD34(-)Lin(-), were detected by polymerase chain reaction in blood and marrow of the two allogeneic transplanted animals at estimated frequencies of =0.001% 1 month after transplant in both animals. Male cells became undetectable in one animal and have remained detectable, with declining frequency, in the other for more than 15 months. In this animal, no male CD34(+) or colony-forming cells have been detected. CONCLUSIONS: CD34(+)Lin(-) and CD34(+)Lin(+) marrow cells can serve as targets for GALV pseudotype retrovirus-mediated gene transfer. CD34(+)Lin(-) cells contribute to reconstitution of all hematopoietic lineages. Autologous CD34(-)Lin(-) cells were either not transduced by GALV pseudotype retrovirus vectors using current approaches or did not contribute significantly to reconstitution, as suggested by allogeneic transplants.
Assuntos
Antígenos CD34/análise , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Transplante Homólogo/imunologia , Irradiação Corporal Total , Animais , Linfócitos B/imunologia , Células da Medula Óssea/citologia , Células Cultivadas , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Substâncias de Crescimento/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Canamicina Quinase/genética , Vírus da Leucemia do Macaco Gibão , Masculino , Papio , Linfócitos T/imunologiaRESUMO
We studied the effect of recombinant canine stem cell factor (rcSCF) on hematopoietic recovery, incidence of graft failure, graft-vs.-host disease (GVHD), and survival after marrow transplantation from dog leukocyte antigen (DLA)-identical canine littermates. Ten animals received 100 microg rcSCF/kg/day b.i.d. by subcutaneous injection on days 1 through 10 after 920 cGy total body irradiation and transplantation of a mean of 3.7x10(8) marrow cells/kg body weight. None of the dogs received GVHD prophylaxis. All animals showed hematopoietic engraftment. The median number of days to achieve 1000 neutrophils/mm3 was 9; 100 monocytes/mm3 were reached after 15 days, 500 lymphocytes/mm3 after 21 days, and 20,000 platelets/mm3 after 16 days. One animal developed GVHD involving skin, gut, and liver and died of bacterial pneumonia 21 days after transplantation. The remaining nine dogs were observed for a median of 37 days (range 29-84 days) posttransplantation until they were killed. Facial edema was seen in three dogs during the first 2-3 days of rcSCF administration. These results show that within the limits of this study it appears to be safe to administer SCF after DLA-identical littermate marrow transplants in dogs. Comparison with previously published data in the same model showed that neutrophil and monocyte recovery was significantly faster in dogs receiving SCF treatment compared with dogs without growth factor treatment (recovery to achieve 1000 neutrophils/mm3: median 9 days vs. 13 days, p = 0.002; recovery to 100 monocytes/mm3: median 15 days vs. 105 days, p = 0.0002). Otherwise, no significant differences were seen. Results obtained with SCF treatment were similar to those previously obtained in the same model with recombinant human granulocyte colony-stimulating factor (rhG-CSF) treatment except that recovery of lymphocytes to 500/mm3 appeared to be more rapid in G-CSF-treated dogs (median 15 days vs. 21 days, p = 0.03).
Assuntos
Transplante de Medula Óssea/veterinária , Hematopoese/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Animais , Transplante de Medula Óssea/imunologia , Cães , Feminino , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/etiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Histocompatibilidade , Masculino , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
High transduction frequency of hematopoietic stem/progenitor cells is essential to derive clinical benefits for treating certain inherited and acquired diseases. We demonstrate here stable gene transfer into human bone marrow-derived CD34+ progenitors using cationic lipids to facilitate GaLV and amphotroic pseudotyped retroviral-mediated transductions. Furthermore, the transgene was detected only in the progeny of flow cytometer sorted CD34+ population transduced by the LAPSN (PG13) viral vector in the presence of cationic lipids but not when transduction was facilitated with conventional polycations Polybrene or protamine sulfate. Thus, a combination of GaLV pseudotyped vectors and cationic lipids results in increased transduction frequencies of the CD34+ cells without a requirement of extended in vitro culture, or co-cultivation with producer cell lines. These improvements may result in the production of therapeutically significant quantities of genetically modified hematopoietic cells.
Assuntos
Antígenos CD34/análise , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células-Tronco Hematopoéticas , Vírus da Leucemia do Macaco Gibão/genética , Lipídeos , Fosfatase Alcalina/genética , Células da Medula Óssea , Cátions , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Genes Reporter/genética , Células-Tronco Hematopoéticas/química , Brometo de Hexadimetrina , Humanos , Vírus da Leucemia Murina de Moloney/genética , Espermina/análogos & derivadosRESUMO
We studied hematopoietic progenitors from fetal baboon blood, marrow, and liver at four time points (125, 140, 160, and 175 days) during the third trimester (gestation approximately 180 days) to determine if fetal baboons might be an appropriate model for in utero gene therapy of hematopoietic stem cells (HSCs). Cells were studied for expression of CD34, CD33, CD38, and HLA-DR, for progenitor content in colony-forming cell assays, and for susceptibility of CD34+ progenitors to retrovirus-mediated gene transfer. Throughout the third trimester, the frequency of CD34+ progenitors in blood and marrow appears to remain unchanged at approximately 0.6 and 5.0%, respectively. In liver, progenitors progressively decrease to undetectable levels by day 175. The proportion of fetal baboon bone marrow and liver CD34+ cells expressing CD38 and HLA-DR appears to increase with increasing fetal age, similar to changes reported for human cord blood CD34+ cells. In fetal baboon blood the proportion of CD34+ cells expressing CD33 appears to decrease with increasing gestational age, also similar to changes reported for human cord blood cells. Progenitors from human cord blood and baboon fetal tissues were similarly susceptible to transduction by the gibbon ape leukemia pseudotyped retroviral vector LAPSN(PG13) containing the genes for human placental alkaline phosphatase (AP) and the bacterial neomycin phosphotransferase (neo). Fetal baboon and human hematopoietic progenitor cells undergo similar phenotypic changes during the third trimester of fetal development and are similarly susceptible to retrovirus-mediated gene transfer. The fetal baboon may be a model in which approaches to mobilization and gene transfer into fetal HSCs can be studied.