Cystic fibrosis transmembrane regulator missing the first four transmembrane segments increases wild type and DeltaF508 processing.

We previously generated an adenoassociated viral gene therapy vector, rAAV-Delta264 cystic fibrosis transmembrane conductance regulator (CFTR), missing the first four transmembrane domains of CFTR. When infected into monkey lungs, Delta264 CFTR increased the levels of endogenous wild type CFTR protein. To understand this process, we transfected Delta264 CFTR plasmid cDNA into COS7 cells, and we noted that protein expression from the truncation mutant is barely detectable when compared with wild type or DeltaF508 CFTR. Delta264 CFTR protein expression increases dramatically when cells are treated with proteasome inhibitors. Cycloheximide experiments show that Delta264 CFTR is degraded faster than DeltaF508 CFTR. VCP and HDAC6, two proteins involved in retrograde translocation from endoplasmic reticulum to cytosol for proteasomal and aggresomal degradation, coimmunoprecipitate with Delta264 CFTR. In cotransfection studies in COS7 cells and in transfection of Delta264 CFTR into cells stably expressing wild type and DeltaF508 CFTR, Delta264 CFTR increases wild type CFTR protein and increases levels of maturation of immature band B to mature band C of DeltaF508 CFTR. Thus the adenoassociated viral vector, rAAV-Delta264 CFTR, is a highly promising cystic fibrosis gene therapy vector because it increases the amount of mature band C protein both from wild type and DeltaF508 CFTR and associates with key elements in quality control mechanism of CFTR.