EFFICIENT EXPRESSION OF CFTR FUNCTION WITH ADENOASSOCIATED VIRUS VECTORS THAT CARRY SHORTENED CFTR GENES

Adeno-associated virus (AAV)-based vectors have been shown to be effec tive in transferring the cystic fibrosis gene (CFTR) into airway epith elial cells in animal models and in patients. However, the level of CF TR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. Tn this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporatio n of an effective promoter with the CFTR gene into AAV vectors. We eng ineered and tested 20 CFTR mini-genes containing deletions that were t argeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs tone with combined d eletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and r egulation by cAMP. By using an AAV vector with a P5 promoter, we trans duced these short forms of CFTR genes into target cells and demonstrat ed high levels of CFTR expression. We also demonstrated that smaller A AV/CFTR vectors with a P5 promoter expressed the CFTR gene more effici ently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene wi th combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively t ransferred CFTR function into target cells. These new vectors should c ircumvent the limitations of AAV vector for CFTR expression. Our strat egy also may be applicable to other genes, the sizes of which exceed t he packaging limit of an AAV vector.