CFTR with a partially deleted R domain and uses thereof
a technology of cftr and r domain, which is applied in the field of dna molecules encoding partially deleted cftr, can solve the problems of inefficient gene transfer from the apical surface of differentiated airway epithelia, small size of aav vector that can be inserted, and inefficient gene transfer
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example 1
Construction of CFTR Variants
[0057]DNA molecules encoding exemplary embodiments of CFTR proteins comprising partial deletions in the R domain were made in pTM1-CFTR4 by PCR deletion mutagenesis (Quik Change Mutagenesis™, Stratagene, La Jolla, Calif.) and confirmed by sequencing. Constructs were ligated into an adenovirus serotype 5 vector in which the CMV promoter drives cDNA expression. The exemplary CFTR proteins were named by the residues that were deleted; for example in Δ708-835, residues between and including aa 708 and 835 are deleted. An identical adenovirus expressing green fluorescent protein (GFP) was used as a negative control. FIG. 1A shows the eight variants constructed which include, Δ708-835 (SEQ ID NO:9), Δ708-759 (SEQ ID NO: 10), Δ708-723 / 749-783 / 832-835 (SEQ ID NO:11), Δ708-723 / 749-783 / 819-835 (SEQ ID NO:12), Δ708-759 / 819-835 (SEQ ID NO:13), Δ760-835 (SEQ ID NO: 14), Δ708-783 (SEQ ID NO:15), and Δ708-783 / 823-835 (SEQ ID NO: 16). FIG. 1A indicates the deletions by ...
example 2
[0058]To confirm protein size and phosphorylation, HeLa cells were infected with 200 MOI of recombinant adenovirus in Eagles minimal essential media (EMEM) for 45 min. Cells were lysed 18-24 hr later, CFTR immunoprecipitated, and phosphorylated with γ-32P-ATP and the catalytic subunit of PKA as described previously. Baldursson et al., 2001, J. Biol. Chem. 276:1904-1910. For pulse chase studies, HeLa cells were infected as above, and after 18-24 hr cells were methionine starved, labeled with 35S-methionine, and pulse-chase studies carried out as described previously (Ostedgaard, Zeiher & Welsh, 1999, J. Cell Sci. 112:2091-2098. Proteins were separated on 8% SDS-PAGE, stained, destained, dried and exposed to phosphorscreens. After phosphorimaging, counts in bands B (immature) and C (mature) were quantitated. FIGS. 4A&B show that two representative CFTR proteins of the present invention comprising partially deleted R domains, namely Δ708-759 and Δ708-723 / 749-783 / 832...
example 3
Well-Differentiated CF Airway Epithelia
[0059]Cultures of human airway epithelia were obtained from CF bronchus (μF508 / μF508 or μF508 / other genotypes) and cultured at the air-liquid interface as previously described (Karp et al., 2002, Epithelial Cell Culture Protocols, ed. Wice (Human, Totowa, N.J.) 188:115-137, incorporated herein by reference. Epithelia were used at least 14 days after seeding when they were well-differentiated with a surface consisting of ciliated cells, goblet cells and other non-ciliated cells. They also retained the functional properties of airway epithelia including transepithelial electrolyte transport and resistance. FIG. 2 shows the short circuit current in well-differentiated airway epithelia in the presence of wild-type CFTR and GFP, demonstrating that wild-type CFTR can provide a functional chloride ion channel in CF airway epithelia. Bars at top of FIG. 2 indicate additions to solutions (detailed below in Example 5). Zero current level is shown by dash...
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