Assuntos
Cromossomos Humanos Par 1 , Cromossomos Humanos Par 22 , Exoma , Leucemia Megacarioblástica Aguda/genética , Metaloproteinase 8 da Matriz/genética , Mutação , Translocação Genética , Criança , Proteínas de Ligação a DNA/genética , Humanos , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Masculino , Proteínas de Fusão Oncogênica/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Ligação a RNA/genética , TransativadoresRESUMO
Gene transfer targeting cells of the blood and immune system was one of the first areas of investigation in the field of gene therapy. Despite the encouraging results achieved in early studies using murine bone marrow, the task of gene transfer into human hematopoietic stem cells proved to be far more difficult. As a result, progress has been disappointingly slow and initial clinical trials generally failed to achieve significant levels of gene marking. The continued application of new advances in vectorology and hematopoietic stem cell biology has now led to improvements in preclinical models that are being translated into clinical trials. The progress and remaining problems are discussed in this review article.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Adenoviridae/genética , Animais , Antígenos CD34/análise , Ensaios Clínicos como Assunto , Expressão Gênica , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Tolerância Imunológica , Camundongos , Retroviridae/genética , Transdução GenéticaRESUMO
Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) are currently the most commonly used vehicles for stable gene transfer into mammalian hematopoietic cells. But, even with reasonable transduction efficiency, expression only occurs in a low percentage of transduced cells and decreases to undetectable levels over time. We have previously reported the modified MND LTR (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) to show increased expression frequency and decreased methylation in transduced murine embryonic stem cells and hematopoietic stem cells. We have now compared expression of the enhanced green fluorescent protein (eGFP) from a vector using the MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in mature hematopoietic and lymphoid cells in the mouse bone marrow transplant (BMT) model. In primary BMT recipients, we observed a higher frequency of expression from the MND LTR (20% to 80%) in hematopoietic cells of all lineages in spleen, bone marrow, thymus, and blood compared with expression from the MoMuLV LTR (5% to 10%). Expression from the MND LTR reached 88% in thymic T lymphocytes and 54% in splenic B lymphocytes for up to 8 months after BMT. The mean fluorescence intensity of the individual cells, indicating the amount of protein synthesized, was 6- to 10-fold higher in cells expressing MNDeGFPSN compared with cells expressing LeGFPSN. Transduction efficiencies determined by DNA polymerase chain reaction of vector copy number were comparable for the 2 vectors. Therefore, the MND vector offers an improved vehicle for reliable gene expression in hematopoietic cells.
Assuntos
Transplante de Medula Óssea , Técnicas de Transferência de Genes , Vetores Genéticos , Células-Tronco Hematopoéticas/metabolismo , Linfócitos/metabolismo , Células 3T3 , Animais , Feminino , Dosagem de Genes , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde , Vírus da Leucemia Murina/genética , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Retroviridae/genética , Fatores de Tempo , Transdução GenéticaRESUMO
Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Leucemia Experimental/imunologia , Proteínas Luminescentes/genética , Linfócitos T Citotóxicos/imunologia , Animais , Antígeno B7-1/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Proteínas de Fluorescência Verde , Leucemia Experimental/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas de DNARESUMO
Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2 degreesCFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2 degreesCFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2 degreesCFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2 degreesCFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0-10% of the vector containing 2 degreesCFU-S from 8 recipients and expression in >50% of the vector-containing 2 degreesCFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo.
Assuntos
Vetores Genéticos , Células-Tronco Hematopoéticas/metabolismo , Retroviridae/genética , Animais , Sequência de Bases , Transplante de Medula Óssea , Ensaio de Unidades Formadoras de Colônias , Metilação de DNA , Primers do DNA/genética , DNA Recombinante/genética , DNA Recombinante/metabolismo , Feminino , Efeito Fundador , Expressão Gênica , Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Leucemia Murina de Moloney/genética , Sequências Repetitivas de Ácido Nucleico , Transdução Genética , Transplante Isogênico , Integração Viral/genéticaRESUMO
Pseudo-aneurysms after abdominal aortic replacement are rare, occurring in less than 1% of operated patients. Usually asymptomatic, they may present clinically as a pulsatile mass or less commonly complicated with rupture. Aortocaval fistulas are rare, usually related to ruptured aortic aneurysms or trauma. A case of aortocaval fistula secondary to a ruptured pseudoaneurysm in a 81 year old woman is reported. After an infrarenal aortic aneurysm repair, the patient remained asymptomatic for 7 years, but abdominal pain and syncope developed and promoted further investigation. She was found to have a 6 cm by 6 cm retroaortocaval false aneurysm which had ruptured into the inferior vena cava. Computed tomography with intravenous contrast suggested the diagnosis and color Doppler ultrasound failed to confirm it. The etiology and management of this rare case are discussed.