In vitro and in vivo functional characterization of gutless recombinant SV40-derived CFTR vectors

Abstract

In cystic fibrosis (CF) respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa. Despite considerable improvement in patient survival, conventional therapies are mainly supportive. Recent progress towards gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy. As alternative gene therapy vectors for CF we examined the feasibility of using SV40-derived vectors (rSV40s) which may circumvent some of these obstacles. To accommodate the large CFTR cDNA, we removed not only SV40 Tag genes, but also all capsid genes. We therefore tested whether “gutless” rSV40s could be packaged and were able to express a functional human CFTR cDNA. Results from our in vitro analysis determined that rSV40-CFTR was able to successfully result in the expression of CFTR protein which localized to the plasma membrane and restored channel function to CFTR deficient cells. Similarly in vivo experiments delivering rSV40-CFTR to the lungs of Cftr−/− mice resulted in a reduction of the pathology associated with intra-tracheal pseudomona aeruginosa challenge. rSV40-CFTR treated mice had had less weight loss when compared to control treated mice as well as demonstrably reduced lung inflammation as evidence by histology and reduced inflammatory cytokines in the BAL. The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways. These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted.

Cre Recombinase Mediates the Removal of Bacterial Backbone to Efficiently Generate rSV40

“…30 , 37 , 38 The wide host range renders it a promising vector not only for the liver but also for organs like the brain and the lung using local administration. 39 , 40 Upon systemic injection, rSV40 mainly targets the hepatocytes, and the dispersion to other tissues can be reduced further by administration into the hepatic artery or portal vein. 41 Although SV40 gives good expression in human cell lines in vivo in mice and rat, the expression levels provided by rSV40 are clearly lower compared to AAV.…”

Gene Therapy in Cystic Fibrosis

“…65 This study also examined CFTR null mice transfected with rSV40/CFTR after Pseudomonas aeruginosa challenge and reported improvements in weight and inflammatory markers such as bronchoalveolar lavage fluid neutrophil counts, interleukin (IL)-1β, and IL-8. Simian vacuolating virus is a circular DNA virus with the ability to integrate into the host cell genome.…”

Gene and cell therapy for cystic fibrosis: From bench to bedside

“…In a recent study, Mueller et al [48] demonstrated that a novel minimal ('gutless') SV40 CFTR expression vector significantly reduced pathology in CFTR knockout mice challenged with Pseudomonas aeruginosa. The authors argue that SV40 is an attractive vector candidate because it offers stable expression through integration and is not immunogenic in rodents.…”