521. Spliceosome-Mediated RNA Trans-Splicing with rAAV Partially Restores CFTR Function to Polarized Human CF Airway Epithelial Cells

Abstract

Top of pageAbstract We previously reported that Spliceosome-Mediated RNA Trans-splicing (SMaRT) using recombinant adenoviral vectors expressing pre-trans-splicing molecules (PTMs) could partially restore CFTR chloride channel activity to polarized human |[Auml]|F508 CF airway epithelia. Although these studies proved that SMaRT could correct CFTR mRNA defects, recombinant adenoviral infection from the basolateral surface was required due to inefficient infection from the apical membrane. Hence, applications of SMaRT technology for CF gene therapy require further testing with alternative, more clinically viable, vector systems. Furthermore, since rAAV vectors have packing limitations with respect to the size of the CFTR transgene insert, SMaRT correction of CFTR has the added attraction of a smaller transgene cassette. In the present study, we investigated whether rAAV vectors could effectively rescue CFTR chloride conductance in polarized human CF airway epithelial cells using a SMaRT approach. AAV vectors were generated to contain a PTM engineered to bind intron 9 of CFTR pre-mRNA and then trans-splice the normal sequence for human CFTR exons 10-24 into the endogenous pre-mRNA. Human CF polarized airway epithelia were infected from the apical membrane with rAAV2 or rAAV5 CFTR-PTM vectors in the presence of proteasome-modulating agents (Doxorubicin and LLnL) to enhance transduction. Epithelia were then evaluated for cAMP-sensitive short circuit currents at 2 weeks post-infection. Levels of CFTR correction seen with rAAV2 (1.07 |[plusmn]| 0.24 micro-Amps) and rAAV5 (0.90 |[plusmn]| 0.20 micro-Amps) CFTR-PTM vectors were similar, representing a conductance equivalent to 14.2% and 13.6% of that observed in non-CF human polarized epithelia, respectively. RT-PCR analysis demonstrated the existence of wild type CFTR transcript in CFTR-PTM correct epithelia, while only the |[Delta]|F508 mRNA was detected in polarized cells infected with control rAAV LacZ-PTM vectors. These results provided evidence that rAAV vectors are capable of utilizing SMaRT for correction of CFTR function following apical infection of human CF airway epithelia. The ability of CFTR-PTM-mediated correction to maintain endogenous CFTR regulation of the transgene product may further improve the efficacy of gene therapy for CF.