RESUMEN
The nuclear replication and retention functions of the Epstein-Barr virus (EBV) have been utilized here to maintain retroviral constructs episomally within human cell-based retroviral packaging lines. These hybrid EBV/retroviral constructs are capable of producing helper-free recombinant retrovirus as soon as 48 hr and for at least 30 days after transfection into 293T-based ecotropic and/or amphotropic retroviral packaging cells. Viral titers greater than 10(7) TU/ml were obtained after puromycin selection of transfected retroviral packaging cells. This episomal approach to retroviral production circumvents some limitations inherent in transient and chromosomally stable retroviral producer systems, affording reproducibly rapid, large-scale, stable, and high-titer retrovirus production.
Asunto(s)
ADN Recombinante/genética , Vectores Genéticos/biosíntesis , Herpesvirus Humano 4/genética , Virus de la Leucemia Murina de Moloney/genética , Plásmidos/biosíntesis , Células 3T3 , Animales , Replicación del ADN , ADN Viral/genética , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Virus Helper , Herpesvirus Humano 4/fisiología , Humanos , Ratones , Virus de la Leucemia Murina de Moloney/fisiología , Plásmidos/genética , Plásmidos/fisiología , Puromicina/farmacología , Transfección , Replicación ViralRESUMEN
Therapeutic gene delivery in severe genetic skin disease may require production of a uniformly corrected population of cells capable of regeneration of normal skin elements when returned to the host. To achieve this, we have used lamellar ichthyosis (LI), a disorder of epidermal differentiation recently associated with defects in keratinocyte transglutaminase (TGase1), as a prototype. We have used a high-efficiency retroviral delivery approach to uniformly restore normal levels of TGase1 expression to primary keratinocytes from severely affected LI patients previously lacking TGase1. Delivered TGase1 was correctly targeted to membrane association and restored patient cell transglutaminase activity levels to normal. Corrected primary LI patient keratinocytes also demonstrated restoration of previously defective involucrin cross-linking and in vitro measures of cornification to levels found in normal cells. These results indicate that efficient TGase1 delivery to early passage keratinocytes can produce a population of corrected LI patient cells. The capability to produce such cells may provide a basis for future efforts at gene therapy for genetic skin disease.