RESUMEN
The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression.
Asunto(s)
Ganglios Basales/inmunología , Dependovirus/inmunología , Terapia Genética , Vectores Genéticos/inmunología , Enfermedad de Parkinson/terapia , Transactivadores/inmunología , Animales , Descarboxilasas de Aminoácido-L-Aromático/genética , Regulación de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Inmunidad Humoral , Masculino , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Elementos de Respuesta/efectos de los fármacos , Tetraciclina/farmacología , Transactivadores/genética , Transgenes/efectos de los fármacosRESUMEN
Inflammatory processes are thought to underlie the dopamine (DA) neuron loss seen in Parkinson's disease (PD). However, it is not known if the inflammation precedes that loss, or is a consequence of it. We injected tumor necrosis factor alpha (TNFalpha) and interleukin 1 beta (IL-1beta) into the median forebrain bundle to determine if these pro-inflammatory cytokines could induce DA neuron loss in the substantia nigra (SN) by themselves. The magnitude of the DA cell loss as well as the decreases in striatal DA, were both dose and time to sacrifice dependent. Injecting both cytokines together produced greater cell losses and DA reductions than that seen when the cytokines were injected alone. The DA neuron loss seen was more pronounced in the lateral nigra and its ventral tier and similar to that seen when other toxins are injected. These data suggest that TNFalpha and IL-1beta can induce DA neuron loss by themselves and could produce DA neuron loss independent of other inflammatory events.
