RESUMO
Interleukin-1alpha (IL-1alpha) is a multifunctional cytokine that stimulates myelopoiesis in macaque. However, daily systemic injections of IL-1alpha are associated with severe side effects. We therefore investigated the feasibility of a gene therapy strategy aimed at increasing the IL-1alpha local production in bone marrow with limited release of the vector into the blood circulation. Intra-medullar administration of adenoviral vector containing human IL-1alpha (huIL-1alpha) gene resulted in enhanced neutrophil, monocyte and platelet counts during the two first weeks after injection. The DNA vector, the transgene expression and the huIL-1alpha production was detected in treated bone marrow without significant detection of huIL-1alpha in the peripheral blood. Associated with huIL-1alpha production, we observed concomitant plasma C reactive protein and IL-1Ra peaks in the acellular fraction of treated bone marrow at days 3 and 7. No abnormal clinical side effects were observed in any of the animals following the adenoviral vector injection.
Assuntos
Células da Medula Óssea/metabolismo , Expressão Gênica , Terapia Genética/métodos , Interleucina-1/metabolismo , Macaca fascicularis/metabolismo , Adenoviridae , Animais , Proteína C-Reativa/metabolismo , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Técnicas de Transferência de Genes , Vetores Genéticos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-1/genética , Masculino , Maurício , Técnicas de Amplificação de Ácido Nucleico , Transgenes/genéticaRESUMO
Effects of interleukin 1-alpha (IL-1alpha), a proinflammatory cytokine with pleiotropic activity, in the myelopoietic setting, is mainly linked to its ability to increase haematopoietic growth factor production by bone marrow stromal cells. In order to minimize systemic effects of IL-1alpha therapy, we proposed a model of retroviral IL-1alpha gene transfer within bone marrow stromal cells in the macaque cynomolgus. Invitro, 10-15% of bone marrow stromal cells was effectively transduced by retroviral vector (murine Moloney leukaemia virus-derived) expressing IL-1alpha/LacZ, or LacZ alone as control marker, as assessed by betaGal staining. IL-1alpha gene expression was upregulated [semiquantitative reverse transcription polymerase chain reaction (RT-PCR)] within the transduced cells and the cell supernatant showed an increased production of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF (enzyme-linked immunosorbent assay) and an increased clonogenic activity (colony-forming cell assay). Ex vivo autologous expanded IL-1alpha/LacZ transduced bone marrow stromal cells were reinfused in two macaques (and two control animals for LacZ alone as controls), without clinical systemic toxicity; LacZ expression by RT-PCR was detected in one animal of each group between d 4 and 9. A slight increase of the peripheral blood leucocyte counts (both polymorphonuclear cells and monocytes) of the two animals transduced with IL-1alpha/LacZ was observed within 10 d, indicating stimulation of myelopoiesis.
