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.

Department of Anatomy, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Human Gene Therapy (Impact Factor: 4.02). 10/2005; 16(9):1116-23. DOI: 10.1089/hum.2005.16.1116
Source: PubMed

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.

0 Bookmarks
 · 
71 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: http://www.intechopen.com/books/gene-therapy-tools-and-potential-applications/gene-therapy-for-the-col7a1-gene
    02/2013; , ISBN: 978-953-51-1014-9
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Eukaryotic living systems avail post-transcriptional alternative pre-mRNA splicing schemes to generate diverse but closely related tissue-specific and/or cell-stage-specific protein molecules. These events involve manipulation of the splicing process through a variety of combinatorial strategies that involve a number of cis- and trans-acting components. Generally, these strategies target spliceosome assembly on a particular set of donor and acceptor splice sites present in a specific segment of the select pre-mRNA molecule. Normally, the process results in production of the required mRNA and hence the protein variant. However, occasionally the process takes an undesired twist to produce a defective mRNA and hence a corresponding abnormal protein variant. Such unusual splicing schemes have been documented as the cause of various human physiological disorders, recently termed as spliceopathies. The only way to rectify such a disorder is to revert the abnormal splicing pattern back to the normal scheme, an uphill task that can safely be referred to as Spliceotherapy and is currently being negotiated by only a few research laboratories investigating regulation of alternative pre-mRNA splicing schemes. Using published literature as a source, this article will review the alternative pre-mRNA splicing schemes; the splicing aberrations that are known to result in a spliceopathy; and the success and shortcomings of the attempts made so for , spliceotherapy, to cure the corresponding disease.
    Journal of Rashid Latif Medical College. 01/2012; 1:12-14.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cystic fibrosis (CF), the most common, life-threatening monogenetic disease in Caucasians, is caused by mutations in the CFTR gene, encoding a cAMP-and cGMP-regulated epithelial chloride channel. Symptomatic therapies treating end-organ manifestations have increased the life expectancy of CF patients towards a mean of 40 years. The recent development of CFTR-targeted drugs that emerged from high throughput screening and are capable of correcting the basic defect promises to transform the therapeutic landscape from a trial-and-error prescription to personalized medicine. This stratified approach is tailored to a specific functional class of mutations in CFTR, but can be refined further to an individual level by exploiting recent advances in ex vivo drug testing methods. These tests range from CFTR functional measurements in rectal biopsies donated by a CF patient to the use of patient-derived intestinal or pulmonary organoids. Such organoids may serve as an inexhaustible source of epithelial cells that can be stored in biobanks and allow medium- to high-throughput screening of CFTR activators, correctors and potentiators on the basis of a simple microscopic assay monitoring organoid swelling. Thus the recent breakthrough in stem cell biology allowing the culturing of mini-organs from individual patients is not only relevant for future stem cell therapy, but may also allow the preclinical testing of new drugs or combinations that are optimally suited for an individual patient.
    The international journal of biochemistry & cell biology 01/2014; · 4.89 Impact Factor

Full-text

View
4 Downloads
Available from