Of the 14 individuals enrolled, 10 completed the full study (e.g., all 3 cycles of vector administration). Of the 4 individuals not completing the study, 1 was discontinued after the second cycle because of the development of arrhythmias during a bronchoscopy procedure, and 1 was discontinued 11 days after the first vector administration because of the development of leukocytoclastic vasculitis (see later here). Two individuals dropped out for personal reasons. To follow each individual through the entire data set, the same symbol is used for the same individual throughout (see Table ).
Safety of endobronchial spray administration of the AdGVCFTR. 10 vector
The study was carried out without any significant adverse effects clearly attributable to the Ad vector (Table ). One individual (dose, 3 × 107 pfu) developed leukocytoclastic vasculitis with bilateral lower extremity rash and microscopic hematuria, 11 days after vector administration. No pulmonary abnormalities were observed. This individual was receiving a variety of medications for CF and was bitten by a spider 2 days previously. Neither replication-competent nor replication-deficient virus was observed in nasal, pharyngeal, rectal, blood, or urine samples obtained at multiple times after vector administration. No rise in anti-Ad antibodies was detected over a 3-month observation period. Although it is not possible to definitively exclude the vector as an etiological agent, the lack of anti-Ad antibodies or of Ad as a persistent antigen suggests that this event was not associated with administration of the vector.
No shedding of the vector was detected in any subject at any time during the study. Assessment of nasal, pharyngeal, urine, blood, and rectal samples at days 1–3 after vector administration showed no detectable replication-deficient or replication-competent adenovirus in a total of 345 samples.
Vector dispersion in the airway epithelium.
Cytospin preparations of bronchial brushings included intact islands of cells, single intact cells, broken cells, debris, and mucus as described previously (29
). Intact islands of epithelial cells contained well-preserved nuclei and closely apposed cell membranes. Single cells included a mixture of epithelial cells and inflammatory cells, more than 93% of which were neutrophils, with the remaining cells being alveolar macrophages. A significant proportion of single cells were not viable (41 ± 2%; range, 8–70). Based on the concept that the epithelial cells in the islands provided a reasonable representation of the epithelium in situ and could provide an estimate of the dispersion of the vector in the intact epithelial sheet, all FISH analyses were carried out in the island population of epithelium. Intact islands of cells in cytospin preparations of bronchial brushings were positive for cytokeratin-18, showing that the islands corresponded to nondissociated patches of airway epithelium.
All probes used in the analysis of Ad genome in patient samples were tested for specificity by conducting control experiments in parallel with each analysis of patient samples. Controls, including human A549 lung epithelial carcinoma cells infected with AdNull in vitro, normal human airway epithelial cells from bronchial brushings infected with AdNull in vitro, and naive cell samples for each cell type, were positive for human chromosome 7 and, when infected with AdNull, were positive for Ad vector. In A549 cells, the assay was able to detect at least 2 copies of Ad genome per cell.
Despite these controls, we were unable to identify definitively the Ad vector genome in the clinical samples using FISH analysis. Although signal of the appropriate wavelength (red channel) was observed in a dispersed fashion in the epithelial cells throughout the sample, the signals were low level and could not be definitively identified as different from low-level autofluorescence that was observed specifically at red wavelengths in the brushed airway epithelial clinical samples from both CF and normal individuals. Although these data are strongly consistent with the concept that the vector is not localized at high levels in small foci of cells, the autofluorescence did not permit a definitive statement regarding demonstration that the vector was dispersed evenly throughout the epithelium.
To evaluate the level of vector-derived CFTR mRNA in the airway epithelium of naive individuals (e.g., first-dose Ad vector) after endobronchial spray administration of AdGVCFTR.10, airway epithelial samples were evaluated before, and at days 3 and 30 after, vector administration. No vector-derived CFTR mRNA was detected in any pretherapy samples except for individual 8 (cycle 1, 3 × 107.5 pfu dose). This was the only false positive in a total of 40 pretherapy samples for all cycles (n = 14 for cycle 1; n = 11 for cycle 2; n = 10 for cycle 3), and it was concluded that the “pre” and “+3 day” samples for this individual had been, most likely, inadvertently mixed. However, because this could not be definitely proved, all samples for cycle 1 of individual 8 were eliminated from the analysis.
Evaluation of the cycle 1, +3 day samples showed a dose dependence in vector-derived CFTR mRNA expression in the airway epithelium (Figure a). No vector-derived expression was observed in any individuals at 3 days at the 3 × 106
to 3 × 106.5
pfu dose range. In contrast, positive vector expression was observed in all individuals at the 3 × 108.5
to 3 × 109
pfu dose range. In the mid-dose range (3 × 107
to 3 × 108
pfu), some individuals demonstrated vector-derived CFTR mRNA expression and some did not. Strikingly, not only was dose-dependent expression observed, but at the higher dose ranges, most of the samples demonstrated levels of vector-derived CFTR mRNA at or above the 5% level of exogenous CFTR mRNA compared with the level of endogenous CFTR mRNA believed to be in the therapeutic range (22
). Re-evaluation of the airway epithelium of the same individuals 30 days after the first vector administration demonstrated no exogenous gene expression in any of the study individuals (Figure b). Because airway epithelial samples were not gathered at time points between 3 and 30 days, it is not possible to come to a conclusion regarding the persistence of expression over this period. However, we can conclude from these studies that: (a) at high doses, vector-derived expression 3 days after administration is at or above normal levels; and (b) the expression does not persist for 30 days, and thus, in its current form, Ad vectors are incapable of maintaining normalizing levels of vector-derived CFTR mRNA levels in the airway epithelium on a persistent basis after a single administration.
Figure 4 Quantitative assessment of the airway epithelium for the percentage of exogenous CFTR mRNA (derived from the AdGVCFTR.10 vector) compared with endogenous CFTR mRNA (individual’s own CFTR gene expression) as a function of dose and time (baseline, (more ...) Second administration.
At the lower doses (3 × 106 to 3 × 106.5 pfu) of the AdGVCFTR.10 vector, repeat administration at the second cycle (initiated 90 days after the first administration) demonstrated no expression (Figure ). Interestingly, for the intermediate doses, expression was observed, with levels at or above the 5% “protective” level (Figure a). However, in marked contrast to the first administration, the 3 × 109 pfu dose yielded no expression with the second administration. Thus, although expression is achieved at some doses with the second administration, it is not dose-dependent, as with the first administration. Rather, it appears that midlevel doses yield repeat expression, but repeated high-level doses do not yield measurable expression. As observed in the first cycle of vector administration, for those dose levels at which expression was observed 3 days after the second administration, expression was back to baseline by day 30 (Figure b). Thus, although samples were not available to determine whether expression persisted at 4–29 days, it clearly does not persist to 30 days.
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 AdGVCFTR.10 (more ...) Third administration.
Unlike observations after the first and second administrations, administration of the vector the third time resulted in no expression of vector-derived CFTR mRNA in the airway epithelium (Figure ). This was true at all dose levels (Figure a) and at both days 3 and 30 after the third vector administration (Figure 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. (more ...) Systemic anti-Ad antibodies.
One explanation for the inability to have successful gene transfer by the third administration (Figure ), and the inability to have successful gene transfer at the highest doses after the second administration (Figure ), is that the intrabronchial administration of the Ad vector induces systemic anti-Ad5 neutralizing antibodies that prevent the vector from successfully infecting the airway epithelial target cells. On the basis of studies in experimental animals (36
), we expected that serum neutralizing antibodies would be observed, at least at higher doses and/or with repeat administration of the vector. As previously reported as part of an analysis of systemic anti-Ad neutralizing antibody titers in association with several human gene therapy applications, there were no major increases in serum anti-Ad5 neutralizing antibodies in any individual in the present study (10
). An occasional sample was observed with a 2- to 4-fold increase over the baseline level, but this was not dose-dependent. In some instances, the anti-Ad5 neutralizing antibody levels actually decreased after vector administration. Although these observations do not yield insight regarding antibody levels on the airway epithelial surface, they clearly demonstrate that repetitive endobronchial administration of an Ad5 vector to individuals with CF does not induce significant levels of systemic anti-Ad5 neutralizing antibodies.
Interestingly, a comparison of vector-mediated expression at day 3 after vector administration to the level of serum anti-Ad5 neutralizing antibodies at the time of vector administration (Figure ) showed that at anti-Ad titers of ≤1:40, there was no relationship between the level of expression from the vector and the serum anti-Ad neutralizing antibody titer. However, at anti-Ad titers ≥1:80, no vector-derived expression was observed, regardless of dose or whether it was the first, second, or third administration.
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 (more ...)