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 (Ad(GV)CFTR.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 Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) 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.

Figure 1

Schematic of the Ad_GVCFTR.10 recombinant adenovirus vector. The E1a region and the majority of the E1b and E3 regions have been deleted. The expression cassette includes (right to left): the CMV enhancer/promoter, splice sequences, normal human CFTR cDNA, and the SV40 early polyadenylation signal. The deleted regions of E1 and E3 are indicated in map units.

Figure 2

Strategy used to spray the Ad_GVCFTR.10 vector onto the airway epithelium. The vector is diluted to the required dose, and the solution is drawn into the catheter. The catheter is inserted in the channel of a fiberoptic bronchoscope previously placed in the lobar bronchus. The catheter/nozzle delivers the vector in a volume of 100 μL as a spray (droplet size, 190 μm) to the target area of the bronchi with the aid of an electronic actuating system that drives a syringe barrel over 2 seconds, pushing a column of air (1–2 mL) that generates the spray uniformly coating the epithelium over 3–4 cm.

Figure 3

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.

Figure 4

Quantitative assessment of the airway epithelium for the percentage of exogenous CFTR mRNA (derived from the Ad_GVCFTR.10 vector) compared with endogenous CFTR mRNA (individual’s own CFTR gene expression) as a function of dose and time (baseline, days 3 and 30) after endobronchial spray of the first administration (cycle 1) of the Ad_GVCFTR.10 vector. The dashed line represents the 5% level of exogenous vector-derived CFTR mRNA. Each symbol represents a different individual (see Table 1). (a) Function of dose at day 3. (b) Function of time. The data for the 10⁶ and 10^6.5 doses (undetectable at each time point for all subjects) are not shown in b. “Pre” is before first vector administration.

Figure 5

Quantitative assessment of the airway epithelium for the percentage of exogenous CFTR mRNA compared with endogenous CFTR mRNA as a function of dose and time (baseline, days 3 and 30) after endobronchial spray of the second dose (cycle 2) of the Ad_GVCFTR.10 vector. The dashed line represents the 5% level of exogenous vector-derived CFTR mRNA. Each symbol represents a different individual (see Table 1). (a) Function of dose at day 3. (b) Function of time. “Pre” is day 90 of the study. The data for the 10⁶ and 10^6.5 doses (undetectable at each time point for all subjects) are not shown in b.

Figure 6

Quantitative assessment of the airway epithelium for the percentage of exogenous CFTR mRNA compared with endogenous CFTR mRNA as a function of dose and time (baseline, days 3 and 30) after endobronchial spray of the third dose (cycle 3) of the vector. The dashed line represents the 5% level of exogenous vector-derived CFTR mRNA. Each symbol represents a different individual (see Table 1). (a) Function of dose at day 3. (b) Function of time. “Pre” is day 180 of the study. The data for the 10⁶ and 10^6.5 doses (undetectable at each time point for all subjects) are not shown in b.

Figure 7

Comparison of the quantitative assessment of the exogenous and endogenous CFTR mRNA at day 3 (all cycles) to the serum anti-Ad5 neutralizing antibody titer at day 1 of each cycle (the time of administration of the vector). Each symbol represents a different individual (see Table 1). The dose (in pfu) is next to each symbol (or group of symbols, if relevant); the number above each symbol represents the cycle number. The horizontal dashed line represents the 5% level of exogenous vector-derived CFTR mRNA. The vertical dashed line represents the detectable limit of neutralizing anti-Ad antibodies. The pretherapy neutralizing antibody data has been published previously (10).