CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo

Lynda S Ostedgaard; Joseph Zabner; Daniel W Vermeer; Tatiana Rokhlina; Philip H Karp; Arlene A Stecenko; Christoph Randak; Michael J Welsh

doi:10.1073/pnas.261714599

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CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo

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CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo

Lynda S Ostedgaard, Joseph Zabner, Daniel W Vermeer, Tatiana Rokhlina, Philip H Karp, Arlene A Stecenko, Christoph Randak and Michael J Welsh

Proceedings of the National Academy of Sciences - PNAS, Vol.99(5), pp.3093-3098

03/05/2002

DOI: 10.1073/pnas.261714599

PMCID: PMC122478

PMID: 11854474

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Abstract

In developing gene therapy for cystic fibrosis (CF) airways disease, a transgene encoding a partially deleted CF transmembrane conductance regulator (CFTR) Cl − channel could be of value for vectors such as adeno-associated virus that have a limited packaging capacity. Earlier studies in heterologous cells indicated that the CFTR R (regulatory) domain is predominantly random coil and that parts of the R domain can be deleted without abolishing channel function. Therefore, we designed a series of CFTR variants with shortened R domains (between residues 708 and 835) and expressed them in well-differentiated cultures of CF airway epithelia. All of the variants showed normal targeting to the apical membrane, and for the constructs we tested, biosynthesis was like wild type. Moreover, all constructs generated transepithelial Cl − current in CF epithelia. Comparison of the Cl − transport suggested that the length of the R domain, the presence of phosphorylation sites, and other factors contribute to channel activity. A variant deleting residues 708–759 complemented CF airway epithelia to the same extent as wild-type CFTR and showed no current in the absence of cAMP stimulation. In addition, expression in nasal mucosa of CF mice corrected the Cl − transport defect. These data provide insight into the structure and function of the R domain and identify regions that can be deleted with retention of function. Thus they suggest a strategy for shortening the transgene used in CF gene therapy.

Biological Sciences

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Title: Subtitle: CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo
Creators: Lynda S Ostedgaard - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242; and
Joseph Zabner - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242; and
Daniel W Vermeer - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242; and
Tatiana Rokhlina - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242; and
Philip H Karp - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242; and
Arlene A Stecenko - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242; and
Christoph Randak - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242; and
Michael J Welsh - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242; and
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.99(5), pp.3093-3098
DOI: 10.1073/pnas.261714599
PMID: 11854474
PMCID: PMC122478
NLM abbreviation: Proc Natl Acad Sci U S A
ISSN: 0027-8424
eISSN: 1091-6490
Publisher: National Academy of Sciences
Language: English
Date published: 03/05/2002
Academic Unit: Neurology; Molecular Physiology and Biophysics; Pulmonary, Critical Care, and Occupational Medicine; Pulmonary Medicine; Stead Family Department of Pediatrics; Neurosurgery; Internal Medicine
Record Identifier: 9984020785302771

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