RESUMO
Posterior capsule opacification is the main complication of cataract surgery. Using adenovirus-mediated gene transfer, we recently reported that it was feasible to prevent PCO by overexpressing pro-apoptotic molecules such as pro-caspase 3 or Bax in the residual lens epithelial cells post-cataract surgery. However, this approach is feasible only if gene transfer can be restricted to the residual cells responsible for PCO. Initially, we tested an adenovirus (human serotype 5, HAd5), a lentivirus (HIV) and an oncoretrovirus (MLV) vector for the their in vivo transduction efficiency of rabbit lens cells. We found that HAd5 vectors were the most efficient (>90% of the cells could be transduced). Six potential lens-specific promoters were then cloned into HAd5 vectors and assayed for their ability to target expression to a specific population of cells, using in vitro, ex vivo and in vivo rabbit tissues and human lens capsular bags. We found that the LEP503, MIP and Filensin promoters induced strong lens-specific expression of a reporter gene, in human lens cells. Following this ex vivo assay, we showed in a rabbit PCO model that gene transfer could be spatially restricted to the capsular bag by confining the vector with Matrigel. Our combined approach using a lens-specific promoter and a biocompatible gel should render feasible a novel therapeutic strategy for PCO that targets the remaining lens cells.
Assuntos
Catarata/prevenção & controle , Cristalinas/genética , Terapia Genética/métodos , Cápsula do Cristalino/metabolismo , Regiões Promotoras Genéticas , Transdução Genética/métodos , Adenoviridae/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Câmara Anterior , Aquaporinas/genética , Catarata/metabolismo , Catarata/patologia , Colágeno , Proteínas de Ligação a DNA/genética , Combinação de Medicamentos , Proteínas do Olho/genética , Expressão Gênica , Marcação de Genes , Engenharia Genética , Vetores Genéticos/administração & dosagem , Humanos , Imuno-Histoquímica/métodos , Injeções , Proteínas de Filamentos Intermediários/genética , Laminina , Cápsula do Cristalino/patologia , Glicoproteínas de Membrana/genética , Microscopia de Contraste de Fase , Modelos Animais , Proteoglicanas , Coelhos , Recidiva , beta-Galactosidase/genéticaRESUMO
Posterior capsule opacification (PCO) is a common complication of cataract surgery. Using adenovirus(Ad)-mediated gene transfer, we overexpressed the proapoptotic molecules p53, procaspase 3, Bax, and TRAIL to induce therapeutic programmed cell death of residual lens cells to prevent PCO. Overexpressed TRAIL did not induce apoptosis in cultured rabbit lens cells or in human lens cells. Overexpressed p53 induced apoptosis of lens cells in vitro and ex vivo, but was unable to prevent PCO in vivo. Overexpressed procaspase 3 was associated with engagement of many components of the apoptotic pathway, including cleavage of intracellular caspase targets such as PARP and inter-nucleosome DNA fragmentation. Even when only slightly overexpressed, Bax caused apoptosis of transduced rabbit and human lens cells by engaging the mitochondrial pathway, including catalytic activation of the caspases. A single in vivo injection of Ad vectors expressing either Bax or procaspase 3 into the capsular bag at the end of phacoemulsification prevented PCO in rabbits. These experiments show that Ad-mediated Bax or procaspase 3 overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo in residual lens cells and preventing PCO in a rabbit model of PCO. Manipulation of proapoptotic molecule expression could be a novel gene therapy approach for prevention of PCO.
