Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus
Ben-Gary Harvey, … , Steven Shak, Ronald G. Crystal
Ben-Gary Harvey, … , Steven Shak, Ronald G. Crystal
Published November 1, 1999
Citation Information: J Clin Invest. 1999;104(9):1245-1255. https://doi.org/10.1172/JCI7935.
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Article

Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus

Abstract

We sought to evaluate the ability of an E1–, E3– adenovirus (Ad) vector (AdGVCFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered AdGVCFTR.10 at doses of 3 × 106 to 2 × 109 plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.

Authors

Ben-Gary Harvey, Philip L. Leopold, Neil R. Hackett, Tina M. Grasso, P. Mickey Williams, Ayly L. Tucker, Robert J. Kaner, Barbara Ferris, Igor Gonda, Theresa D. Sweeney, Ramachandran Ramalingam, Imre Kovesdi, Steven Shak, Ronald G. Crystal

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Competitive PCR analysis used to quantify the amount of exogenous (vecto...
Competitive PCR analysis used to quantify the amount of exogenous (vector-derived) CFTR mRNA and endogenous CFTR mRNA in airway epithelial cells. Using nested RT-PCR using fluorescent primers, RNA samples prepared from bronchial brushings were spiked with standard RNAs at concentrations comparable to the endogenous or exogenous levels estimated from preliminary analyses. The spike for the endogenous RNA is derived from in vitro transcription of a plasmid containing the CFTR mRNA with a 78-bp deletion. The spike of the exogenous RNA is derived from in vitro transcription of a plasmid containing the cloned Ad-encoded CFTR mRNA with a 50-bp deletion. These competitor RNAs were shown to amplify with equal efficiency as the target of interest (data not shown). The RNA mixtures were subjected to nested PCR as described in Methods with 1 of the second-round primers labeled with FAM. The PCR products were identified from fluorescent electropherograms by their mobility relative to rhodamine-labeled molecular weight standard and quantified using the GeneScan software (Perkin-Elmer Applied Biosystems). The upper panel shows detection of the endogenous CFTR mRNA and spike, and the lower panel shows an example of detection of exogenous CFTR mRNA and spike.