HUMAN GENE THERAPY 16:1116–1123 (September 2005)
© Mary Ann Liebert, Inc.
Spliceosome-Mediated RNA Trans-Splicing with Recombinant
Adeno-Associated Virus Partially Restores Cystic Fibrosis
Transmembrane Conductance Regulator Function to Polarized
Human Cystic Fibrosis Airway Epithelial Cells
XIAOMING LIU,1,2MEIHUI LUO,1LIANG N. ZHANG,1ZIYING YAN,1,2ROMAN ZAK,1WEI DING,1
S. GARY MANSFIELD,3LLOYD G. MITCHELL,3and JOHN F. ENGELHARDT1,2
We previously reported that spliceosome-mediated RNA trans-splicing (SMaRT), using recombinant adeno-
viral vectors expressing pre-trans-splicing molecules (PTMs), could partially restore cystic fibrosis trans-
membrane conductance regulator (CFTR) chloride channel activity to polarized human ?F508 CF airway
epithelia. Although these studies proved that SMaRT could correct CFTR mRNA defects, recombinant ade-
noviral infection from the basolateral surface was required because of inefficient infection from the apical
membrane. Hence, applications of SMaRT technology for CF gene therapy require further testing with al-
ternative, more clinically viable, vector systems. Furthermore, because recombinant adeno-associated virus
(rAAV) vectors have packing limitations with respect to the size of the CFTR transgene insert, SMaRT cor-
rection of CFTR has the added attraction of a smaller transgene cassette. In the present study, we investi-
gated whether rAAV vectors could effectively rescue CFTR chloride conductance in polarized human CF air-
way epithelial cells, using a SMaRT approach. AAV vectors were generated to carry 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 mem-
brane with rAAV2 or rAAV5 CFTR-PTM vectors in the presence of proteasome-modulating agents (dox-
orubicin and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal) to enhance transduction. Epithelia were then eval-
uated for cAMP-sensitive short-circuit currents 2 weeks postinfection. Levels of CFTR correction seen with
rAAV2 (1.07 ? 0.24 ?A) and rAAV5 (0.90 ? 0.20 ?A) CFTR-PTM vectors were similar, representing con-
ductance 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-corrected epithe-
lia, whereas only ?F508 mRNA was detected in polarized cells infected with control rAAV LacZ-PTM vec-
tors. These results provide evidence that rAAV vectors are capable of using SMaRT to correct CFTR func-
tion after 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.
1Department of Anatomy and Department of Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242.
2Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, Carver College of Medicine, University of Iowa, Iowa City, IA
3Intronn Inc., Gaithersburg, MD 20878.
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Address reprint requests to:
Dr. John F. Engelhardt
Departments of Anatomy and Cell Biology
University of Iowa
School of Medicine
51 Newton Road, Room 1-111 BSB
Iowa City, IA 52242
Received for publication May 17, 2005; accepted after revision
July 6, 2005.
Published online: August 5, 2005.