RESUMO
Familial hypercholesterolemia (FH) is an inherited disease of lipoprotein metabolism caused by a defect in the LDL receptor (LDLR) leading to severe hypercholesterolemia, and associated with an increased risk of coronary heart disease and myocardial infarction. We have developed a gene therapy protocol for FH using AAV2, AAV9 and lentiviral vectors and tested safety and efficacy in LDL receptor deficient Watanabe Heritable Hyperlipidemic rabbits. We show that LV-LDLR produced a significant long-lasting decrease in total serum cholesterol whereas AAV9-LDLR resulted only in a transient decrease and AAV2-LDLR failed to reduce serum cholesterol levels. A significant pathological side effect, bile-duct proliferation, was seen in the liver of AAV2-LDLR rabbits associated with an increased expression of Cyr61 matricellular protein. Special attention should be given to liver changes in gene therapy applications when genes affecting cholesterol and lipoprotein metabolism are used for therapy.
Assuntos
Ductos Biliares/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Fígado/metabolismo , Parvovirinae/genética , Receptores de LDL/genética , Animais , Ductos Biliares/patologia , Biomarcadores , Colesterol/metabolismo , Dependovirus , Expressão Gênica , Técnicas de Transferência de Genes/efeitos adversos , Imuno-Histoquímica , Metabolismo dos Lipídeos , Fígado/patologia , CoelhosRESUMO
OBJECTIVES: Ovarian cancer is highly dependent on tumor microvessels and angiogenesis regulated by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) and angiopoietins (Ang) and their Tie receptors. We studied the efficacy of adenoviral (Ad) gene therapy with soluble VEGFR2 and Tie2 combined with paclitaxel and carboplatin for the treatment of ovarian cancer. METHODS: An intraperitoneal human ovarian cancer xenograft model in nude mice (n = 44) was used in this study. Gene therapy was given intravenously when the presence of sizable tumors was confirmed in magnetic resonance imaging. The study groups were as follows: AdCMV as a control (group I), AdCMV with chemotherapy (group II), AdsVEGFR2 and AdsTie2 (group III), and AdsVEGFR2 and AdsTie2 with chemotherapy (group IV). Antitumor effectiveness was assessed by overall tumor growth, ascites, immunohistochemistry, microvessel density, and sequential magnetic resonance imaging analyses. RESULTS: AdsVEGFR2 and AdsTie2 gene therapy (group III) significantly reduced tumor weights as compared with group II (P = 0.007). Accumulation of ascites was significantly reduced when the mice were treated with AdsVEGFR2 and AdsTie2 gene therapy or with combined gene therapy and chemotherapy as compared with controls (P = 0.029 and P = 0.010, respectively). Vascular endothelial growth factor and Ang2 levels in ascites fluid were elevated after the gene therapy. CONCLUSIONS: Combined inhibition of VEGF/VEGFR2 and Ang/Tie2 pathways provided efficient therapy for ovarian cancer in mice. In addition, antiangiogenic gene therapy has potential as a treatment for the accumulation of ascites.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Terapia Genética/métodos , Neoplasias Ovarianas/terapia , Receptor TIE-2/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Adenoviridae/genética , Animais , Ascite/tratamento farmacológico , Ascite/genética , Ascite/patologia , Ascite/terapia , Carboplatina/administração & dosagem , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Distribuição Aleatória , Receptor TIE-2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Plasmid-based Sleeping Beauty (SB) transposon vectors were developed and used to deliver genes for low-density lipoprotein and very-low-density lipoprotein receptors (LDLR and VLDLR, respectively) or lacZ reporter into liver of an LDLR-deficient mouse model of familial hypercholesterolemia (FH). SB transposase, SB100x, was used to integrate the therapeutic transposons into mice livers for evaluating the feasibility of the vectors in reducing high blood cholesterol and the progression of atherosclerosis. Hydrodynamic gene delivery of transposon-VLDLR into the livers of the mice resulted in initial 17-19% reductions in plasma cholesterol, and at the later time points, in a significant stabilization of the cholesterol level for the 6.5-month duration of the study compared to the control mice. Transposon-LDLR-treated animals also demonstrated a trend of stabilization in the cholesterol levels in the long term. Vector-treated mice had slightly less lipid accumulation in the liver and reduced aortic atherosclerosis. Clinical chemistry and histological analyses revealed normal liver function and morphology comparable to that of the controls during the follow-up with no safety issues regarding the vector type, transgenes, or the gene transfer method. The study demonstrates the safety and potential benefits of the SB transposon vectors in the treatment of FH.
Assuntos
Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hiperlipoproteinemia Tipo II/genética , Fígado/metabolismo , Receptores de LDL/genética , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Linhagem Celular , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Terapia Genética , Hiperlipoproteinemia Tipo II/metabolismo , Hiperlipoproteinemia Tipo II/terapia , Lipídeos/sangue , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Gene therapy stimulating the growth of blood vessels is considered for the treatment of peripheral and myocardial ischemia. Here we aimed to achieve angiogenic synergism between vascular endothelial growth factor-A (VEGF-A, VEGF) and fibroblast growth factor 4 (FGF4) in murine normoperfused and ischemic limb muscles. METHODS: Adeno-associated viral vectors (AAVs) carrying ß-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A) or two angiogenic genes (AAV-FGF4-IRES-VEGF-A) were injected into the normo-perfused adductor muscles of C57Bl/6 mice. Moreover, in a different experiment, mice were subjected to unilateral hindlimb ischemia by femoral artery ligation followed by intramuscular injections of AAV-LacZ, AAV-VEGF-A or AAV-FGF4-IRES-VEGF-A below the site of ligation. Post-ischemic blood flow recovery was assessed sequentially by color laser Doppler. Mice were monitored for 28 days. RESULTS: VEGF-A delivered alone (AAV-VEGF-A) or in combination with FGF4 (AAV-FGF4-IRES-VEGF-A) increased the number of capillaries in normo-perfused hindlimbs when compared to AAV-LacZ. Simultaneous overexpression of both agents (VEGF-A and FGF4) stimulated the capillary wall remodeling in the non-ischemic model. Moreover, AAV-FGF4-IRES-VEGF-A faster restored the post-ischemic foot blood flow and decreased the incidence of toe necrosis in comparison to AAV-LacZ. CONCLUSIONS: Synergy between VEGF-A and FGF4 to produce stable and functional blood vessels may be considered a promising option in cardiovascular gene therapy.
