Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus Ben-Gary Harvey,1 Philip L. Leopold,1 Neil R. Hackett,1,2 Tina M. Grasso,3 P. Mickey Williams,4 Ayly L. Tucker,4 Robert J. Kaner,1 Barbara Ferris,1 Igor Gonda,4 Theresa D. Sweeney,4 Ramachandran Ramalingam,1 Imre Kovesdi,3 Steven Shak,4 and Ronald G. Crystal1,2 1Division
of Pulmonary and Critical Care Medicine, and Gene Therapy Core Facility, Weill Medical College of Cornell University–New York Presbyterian Hospital, New York, New York 10021, USA 3GenVec Inc., Rockville, Maryland 20852, USA 4Genentech Inc., South San Francisco, California 94080, USA 2Belfer
Igor Gonda’s present address is: Aradigm Inc., Hayward, California 94545, USA. Address correspondence to: Ronald G. Crystal, Weill Medical College of Cornell University–New York Presbyterian Hospital, 520 East 70th Street, ST 505, New York, New York 10021, USA. Phone: (212) 746-2258; Fax: (212) 746-8383; E-mail:
[email protected]. Received for publication July 22, 1999, and accepted in revised form September 22, 1999.
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. J. Clin. Invest. 104:1245–1255 (1999).
Introduction Cystic fibrosis (CF) is a common, recessive hereditary disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1–3). The major manifestations are on the epithelial surface of the airways, with thick and sticky mucus, recurrent infections, and neutrophil-dominated chronic inflammation (4). The disease is associated with a progressive decline in lung function, with more than 90% of deaths secondary to pulmonary complications, at an average age of 31 years (5). There is extensive evidence that the pulmonary abnormalities in CF are initiated by a deficiency of CFTR function in the airway epithelium (1–6). With the demonstration that the normal CFTR cDNA could be transferred and expressed in the airway epithelium of experimental animals in vivo The Journal of Clinical Investigation
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(7), it was logical to hypothesize that this could be accomplished in the respiratory epithelium of individuals with CF (8). A number of clinical trials have demonstrated that this is feasible using adenovirus (Ad) (8–16), liposome/plasmid complexes (17–20), and adenoassociated virus vectors (21). Now that the feasibility of human transfer of the normal CFTR cDNA to individuals with CF has been demonstrated, the next logical step in developing gene therapy for CF is to quantify the levels of gene transfer that can be achieved and for how long they persist. Analysis of CFTR mutation genotype/phenotype correlations in humans and mice suggests that persistent levels of 5–10% of normal CFTR expression evenly distributed throughout the airways should be sufficient to compensate for the deficiency of CFTR function result-
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ing from the parental CFTR mutations (22–27). Based on these considerations, the present study is directed toward determining whether it is possible to safely transfer and express the normal human CFTR cDNA delivered by an E1–, E3– Ad vector to the airway epithelium at levels greater than 5% of the endogenous CFTR mRNA levels. Using a study design in which an Ad vector expressing the normal CFTR cDNA (AdGVCFTR.10) is repetitively administered to individuals with CF by endobronchial spray every 3 months (for 3 cycles), 6 questions are addressed: (a) Is the vector dispersed evenly throughout the epithelium? (b) Are the levels of vector-derived CFTR mRNA achieved in the airway epithelium dosedependent, and what dose of the AdGVCFTR.10 vector is necessary to achieve greater than 5% levels of normal CFTR mRNA in the airway epithelium after a single administration? (c) How long does the vector-derived CFTR cDNA expression persist? (d) Is repetitive administration safe? (e) Can vector-derived CFTR cDNA expression be achieved with repetitive administration, and how long does it persist? (f) Is there a correlation of the levels of airway epithelial expression of the vectorderived CFTR mRNA to the level of systemic anti-Ad neutralizing antibodies at the time of administration?
Methods Study population. Fourteen individuals (12 male, 2 female, age 30 ± 9 years [mean ± SEM; range, 17–48 years]) were enrolled in the study. All had CF by conventional clinical criteria, including a positive sweat chloride test (4). Of the 14 individuals, 2 were ∆F508 homozygotes, 10 were compound heterozygotes with one ∆F508 allele, and 2 had mutations other than ∆F508 in both alleles. All had mild to moderate lung disease typical of CF, with an average forced expiratory volume in 1 second of 57 ± 16% predicted (mean ± SEM; range, 33–79% predicted). Adenovirus vector. The AdGVCFTR.10 vector, based on the subgroup C, serotype 5 genome, is missing E1a, most of E1b, and the majority of E3 sequences (Figure 1). AdGVCFTR.10 contains an expression cassette that includes (5′ to 3′): the cytomegalovirus early/immediate promoter/enhancer, an artificial splice sequence, the normal human CFTR cDNA, and SV40 stop/polyA sequences. The vector was produced, purified, and stored as described previously (7, 8). The final preparation (1.6 × 1011 plaque-forming units [pfu] mL–1, particle (pu) to pfu ratio 9, and 3.7 × 1010 pfu mL–1, pu to pfu of 6) was demonstrated to have less than 1 replication-competent adenovirus in 2 × 109 pfu (the maximal dose used) (8).
Table 1
Safety of endobronchial spray administration of the AdGVCFTR.10 vector Study participant
1
2
3
4
5
6
7
8 7.5 8
9
10
11
12
13
14
108
108
108.5
108.5
109
109
3
3
3
Dose (pfu)A
106
106
106.5
106.5
107
107
107.5
SymbolB
❍
●
❏
■
e
◆
❏
3
3
1
3
1
3
3
1
2
3
3
Systemic
0
0
0
0
2E, 1F
0
0
0
0
0
1K
0
0
0
Hematologic
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Coagulation
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Hepatic
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Renal
0
0
0
0
1G
0
0
0
0
0
0
0
0
0
0
1H
0
0
0
0
0
0
0
0
0
10 10
Number of administrationsC Safety
parametersD
Gastrointestinal
0
0
0
Neurologic
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Cardiovascular
0
0
0
0
0
0
0
0
0
1J
0
0
0
0
0
1I
0
0
0
0
0
0
Pulmonary
0
0
0
0
0
0
AFor
listed, except for the 109 pfu dose, where the actual dose was 2 times that listed (see Methods for details). that listed, except for the 109 pfu dose, where the actual dose was 2 times that listed (see Metheach study individual, the same symbol is used in all figures. CAll administrations were to the subsegmental bronchi of the lower lobe; all individuals received BFor each study individual, the same symbol is used in all figures. C All administrations were to the subsegmental bronchi of ods for details). 3 administrations of the vector, except for individuals 3, 5, 8, and 9 (see results for details). DNo adverse events were attributable to the vector per se; the numECycle 1, No the right loweron lobe; all individuals received 3 administrations of the3vector, for individuals 3,developed 5, 8, andleukocytoclastic 9 (see results vasculitis for details). bers are baseed a graded toxicity scale: 0 = none; 1 = mild, 2 = moderate, = severe,except 4 = intolerable. withDrash and microscopic hematuria 11 days following administration of vector. Individual had been receiving multiple antibiotics and had been bitten by a spider adverse events wre attributable to the vector per se; the numbers are based on a graded toxicity scale: 0 = none, 1 = mild, 2 = moderate, 3 =2 days prior vector administration. wasleukocytoclastic completely resolved with prednisone therapy within 3 months. No anti-Ad were detected at any ECycle 1,Syndrome developed vasculitis with rash and microscopic hematuria 11antibodies days following administrasevere, 4 to = intolerable. actual dose was 3 times that AForeach eachcycle, cycle,thethe actual dose was 3 times
BFor
tion of vector. Individual had been receiving multiple antibiotics and had been bitten by a spider 2 days prior to vector administration. Syndrome was completely resolved with prednisone therapy within 3 months. Noanti-Ad antibodies were detected at any time. FTransient leukocytosis and anemia associated with fever related to bronchoscopic procedure. GMicroscopic hematuria associated with vasculitis. HBloody stool associated with prednisone treatment for vasculitis. IHypoxemia associated with narcotics used during bronchoscopy, resolved within 6 hours. JOccasional premature ventricular contractions noted on EKG monitor during bronchoscopy at 3 days post vector administration in cycle 2; no hemodynamic compromise; ectopies related to procedure discontinued after bronchoscopy was determined. KTransient leukocytosis and associated fever related to bronchscopy. 1246
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Figure 1 Schematic of the AdGVCFTR.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.
Study design. The clinical study, approved by the local Institutional Review Boards and Biosafety Committees, the National Institutes of Health Recombinant DNA Advisory Committee, and The Food and Drug Administration, included 7 cohorts of 2 individuals each. The study started with a baseline period to ensure that the inclusion criteria were fulfilled, to establish baseline parameters for safety evaluation, to collect baseline airway epithelial cells, and to quantify serum anti-Ad5 neutralizing antibody titers (see below for details). This was followed by vector administration periods that were grouped into 3 cycles, each of 90 days, beginning at days 1, 91, and 181, respectively. Each of the 7 dose cohorts were assigned a dose of the AdGVCFTR.10 vector 106–109 pfu (in half-log increments). The vector was administered at days 1, 91, and 181 as a spray through a fiberoptic bronchoscope (see later here) to 2 sites within the lobar bronchus of the right lower lobe; e.g., the 2 individuals assigned the 3 × 106 pfu dose received 106 pfu in 3 administrations to 2 sites within the lobar bronchus for a total dose of 3 × 106 pfu at each cycle beginning at 1, 91, and 181 days. For each cycle, airway epithelial cells (CFTR mRNA levels) and serum (for neutralizing antibodies) were obtained before vector administration and at days 3 and 30 after each vector administration (10). Assessment of the airway epithelial cells for dispersion of the AdGVCFTR.10 vector was carried out only after the first cycle of the 3 × 108.5 pfu dose. Safety parameters, including hematological, serological, and urine studies, chest roentgenogram, pulmonary function tests, electrocardiogram (EKG), and assessment of shedding of vector (nasal, pharyngeal, rectal, blood and urine samples), were obtained at multiple times after vector administration at all dose levels (see later here). Vector administration. An endobronchial spray delivery system was designed to ensure that the vector was deposited only on the airway epithelial surface and to an identifiable region that could be sampled over time (Figure 2). The vector was diluted in the storage buffer (3% glycerol, 10 mM Tris-HCl, 10 mM MgC12, 150 mM NaCl [pH 7.8]) to the appropriate dose such that the dose to be delivered was in 100 µL. The fluid containing the vector was drawn into a catheter with a spray nozzle at one end (PW-6P washing pipe; Olympus America Inc., Melville, New York, USA). The actual volume drawn into The Journal of Clinical Investigation
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the catheter was 120 µL; 100 µL was delivered, and 20 µL remained in the dead space. The catheter containing the vector was advanced through the working channel of a FB-15× fiberoptic bronchoscope (Pentax, Orangeburg, New York, USA) positioned just distal to the lobar bronchi of the right lower lobe. The formulation containing the vector was rapidly pushed through the spray nozzle with a 2-second, 55-psi pulse of air (1–2 mL) delivered through the proximal end of the catheter by an electronic actuating system (KDS824 Shot Meter; Kahnetics, Bloomington, California, USA) driving a syringe barrel; as the liquid is rapidly moved through the spray nozzle, the bulk liquid is broken into droplets. Ex vivo studies in casts of human bronchi, in vivo studies in rabbits, and in vivo studies in humans (normals and individuals with CF) demonstrated that this system delivers the vector in a spray (droplet size distribution centered around 190 ± 9 µm volume median diameter; < 1% particles < 10 µm diameter) to the targeted segmental bronchi in a circumferential fashion over an area from just distal to the nozzle for up to 2 bifurcations, i.e., 3–4 cm of the distal bronchi (28). Videotapes were made of
Figure 2 Strategy used to spray the AdGVCFTR.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.
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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.
each delivery site, along with appropriate notes as to anatomic location; these were reviewed before each bronchoscopy to ensure that correct locations were identified. Quantitative assessment of CFTR mRNA. Airway epithelial cells were collected with multiple brushings through a fiberoptic bronchoscope as described previously (8, 29). Bronchoscopies for collection of airway epithelial cells were performed at baseline (before vector administration) and after each vector administration, at days 3 and 30. During each bronchoscopy, a brush was advanced through the working channel of the bronchoscope, and airway epithelial cells from areas that received the vector were obtained by gliding the brush several times over the airway surface. The cells obtained were immediately flicked in a 15-mL conical tube containing LHC-8 medium (BioFluids, Rockville, Maryland, USA) and kept at 4°C until processing. Cell number was quantified in a hemocytometer, and differential 1248
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cell counts were obtained using modified Giemsa stains (Dade International, Miami, Florida, USA) of cytospin preparations (29). Of 120 samples analyzed, 8.4 × 106 ± 0.5 × 106 total cells (mean ± SEM; range, 1.4 × 106 to 2.5 × 107) were obtained on the average, with 65 ± 2% bronchial epithelial cells (range, 22–97, except for 5 samples that yielded
