Journal article
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
Human gene therapy, Vol.16(9), pp.1116-1123
09/2005
DOI: 10.1089/hum.2005.16.1116
PMID: 16149910
Abstract
We previously reported that spliceosome-mediated RNA trans-splicing (SMaRT), using recombinant adenoviral vectors expressing pre-trans-splicing molecules (PTMs), could partially restore cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity to polarized human DeltaF508 CF airway epithelia. Although these studies proved that SMaRT could correct CFTR mRNA defects, recombinant adenoviral 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 alternative, 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 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 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 membrane with rAAV2 or rAAV5 CFTR-PTM vectors in the presence of proteasome-modulating agents (doxorubicin and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal) to enhance transduction. Epithelia were then evaluated for cAMP-sensitive short-circuit currents 2 weeks postinfection. Levels of CFTR correction seen with rAAV2 (1.07 +/- 0.24 microA) and rAAV5 (0.90 +/- 0.20 microA) CFTR-PTM vectors were similar, representing 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-corrected epithelia, whereas only DeltaF508 mRNA was detected in polarized cells infected with control rAAV LacZ-PTM vectors. These results provide evidence that rAAV vectors are capable of using SMaRT to correct CFTR function 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.
Details
- Title: Subtitle
- 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
- Creators
- Xiaoming Liu - Department of Anatomy, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USAMeihui LuoLiang N ZhangZiying YanRoman ZakWei DingS Gary MansfieldLloyd G MitchellJohn F Engelhardt
- Resource Type
- Journal article
- Publication Details
- Human gene therapy, Vol.16(9), pp.1116-1123
- Publisher
- United States
- DOI
- 10.1089/hum.2005.16.1116
- PMID
- 16149910
- ISSN
- 1043-0342
- eISSN
- 1557-7422
- Grant note
- R01 HL58340 / NHLBI NIH HHS DK54759 / NIDDK NIH HHS
- Language
- English
- Date published
- 09/2005
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Radiation Oncology; Internal Medicine
- Record Identifier
- 9984025351602771
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