RESUMO
We designed experiments to evaluate the therapeutic potential of glial cell line derived neurotrophic factor (GDNF) to rescue photoreceptors from genetically determined cell death. Gene transfer of the neurotrophic factor to the retina was achieved via a recombinant adeno-associated virus (rAAV) vector containing the chicken beta-actin promoter/immediate early cytomegalovirus enhancer (CBA) driving the human GDNF gene. We delivered AAV-CBA-GDNF to the retinas of an animal model of retinitis pigmentosa, the TgN S334ter-4 rhodopsin line of transgenic rats. Immunohistochemical studies localized AAV-CBA-GDNF-derived recombinant protein to cell bodies, inner segments, and outer segments of photoreceptor cells as well as to retinal pigment epithelial cells. We assessed the effect of viral delivery by morphometric and electroretinographic analysis. These experiments showed that GDNF vector treatment leads to increased rod photoreceptor survival as indicated by morphometric analysis of outer nuclear layer thickness. AAV-CBA-GDNF-treated retinas also demonstrated functional improvement by the substantially increased amplitude of electroretinograms. AAV-CBA-GDNF delivery had a significant rescue effect on photoreceptor degeneration in this animal model.
Assuntos
Dependovirus/genética , Terapia Genética , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/genética , Retinose Pigmentar/terapia , Actinas/genética , Animais , Animais Geneticamente Modificados , Western Blotting , Primers do DNA/química , Eletrorretinografia , Genes Reporter , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Proteínas de Fluorescência Verde , Técnicas Imunoenzimáticas , Proteínas Luminescentes/metabolismo , Modelos Animais , Proteínas do Tecido Nervoso/biossíntese , Células Fotorreceptoras/fisiologia , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Retinose Pigmentar/metabolismo , Retinose Pigmentar/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Viral delivery of neurotrophins or other therapeutic genes is an attractive option for treating retinal degeneration. Regulated expression of these genes in the retina is needed to aid in dose delivery and to promote safety. To evaluate whether tetracycline (tet)-inducible transgenes encapsidated in recombinant adeno-associated viruses (rAAV) can provide controlled gene expression in vitro and in the rat retina, two viruses were constructed: a silencer/activator vector and an inducible doxycycline (dox)-responsive GFP vector. Combinations of these two viruses were subretinally injected into wild-type rats and dox was orally administered through the drinking water. Retinal GFP expression was monitored in vivo with a noninvasive fluorescence imaging method. Eyes were also examined by histology, Western analysis, and electroretinography. Subretinal injection of rAAV efficiently delivers inducible genes to both photoreceptors and retinal pigment epithelial cells. GFP expression was initially observed 1 week postinduction, and GFP protein was undetectable after removal of dox. In uninduced animals, GFP expression was negligible. The dox dosage was varied in vivo and showed a correlation to the level of GFP expression. Thus, transduction of retinal cells with tet-inducible vectors allows for tight regulation of gene expression.
