A Controlled Study of Adenoviral-VectorMediated Gene Transfer in the Nasal Epithelium of Patients with Cystic Fibrosis
Michael R. Knowles, M.D., Kathy W. Hohneker, R.N., Zhaoqing Zhou, Ph.D., John C. Olsen, Ph.D., Terry L. Noah, M.D., Ping-Chuan Hu, Ph.D., Margaret W. Leigh, M.D., John F. Engelhardt, Ph.D., Lloyd J. Edwards, Ph.D., Kim R. Jones, M.D., Ph.D., Mariann Grossman, B.S., James M. Wilson, M.D., Ph.D., Larry G. Johnson, M.D., and Richard C. Boucher, M.D.
Background Cystic fibrosis is a monogenic disease that derangesmultiple systems of ion transport in the airways, culminatingin chronic infection and destruction of the lung. The introductionof a normal copy of the cystic fibrosis transmembrane conductanceregulator (CFTR) gene into the airway epithelium through genetransfer is an attractive approach to correcting the underlyingdefects in patients with cystic fibrosis. We tested the feasibilityof gene therapy using adenoviral vectors in the nasal epitheliumof such patients.
Methods An adenoviral vector containing the normal CFTR complementaryDNA in four logarithmically increasing doses (estimated multiplicityof infection, 1, 10, 100, and 1000), or vehicle alone, was administeredin a randomized, blinded fashion to the nasal epithelium of12 patients with cystic fibrosis. Gene transfer was quantitatedby molecular techniques that detected the expression of CFTRmessenger RNA and by functional measurements of transepithelialpotential differences (PDs) to assess abnormalities of ion transportspecific to cystic fibrosis. The safety of this treatment wasmonitored by nasal lavage and biopsy to assess inflammationand vector replication.
Results The adenoviral vector was detected in nasal-lavage fluidby culture, the polymerase chain reaction (PCR), or both ina dose-dependent fashion for up to eight days after vector administration.There was molecular evidence of gene transfer by reverse-transcriptasePCR assays or in situ hybridization in five of six patientstreated at the two highest doses. However, the percentage ofepithelial cells transfected by the vector was very low (<1percent), and measurement of PD across the epithelium revealedno significant restoration of chloride transport or normalizationof sodium transport. At the lower doses of vector, there wereno toxic effects. However, at the highest dose there was mucosalinflammation in two of three patients.
Conclusions In patients with cystic fibrosis, adenoviral-vectormediatedtransfer of the CFTR gene did not correct functional defectsin nasal epithelium, and local inflammatory responses limitedthe dose of adenovirus that could be administered to overcomethe inefficiency of gene transfer.
Cystic fibrosis is a recessive genetic disease caused by mutationsin the cystic fibrosis transmembrane conductance regulator (CFTR)gene.1,2,3,4 The normal CFTR gene codes for a protein (CFTR)that plays a key part in epithelial transport of salt and water.5,6Mutations in CFTR result in abnormal secretions that obstructand ultimately damage epithelium in many areas of the body.7
The principal cause of death among patients with cystic fibrosisis lung disease. Patients who are homozygous for mutations inthe CFTR gene have defective cyclic AMP (cAMP)regulatedsecretion of chloride8,9 and elevated absorption of sodium10in the airway epithelium, which thicken airway secretions, impairmucociliary clearance, and produce chronic bacterial infectionof the airways.5,6,7,11 Carriers of a mutation in the CFTR genedo not have lung disease, which indicates that a single copyof the normal CFTR gene is sufficient for normal defense ofthe lung. The transfer of a single copy of normal CFTR intoall epithelial cells affected by cystic fibrosis might be expectedto correct airway function.
We performed a double-blind, vehicle-controlled study to assessthe efficacy and safety of gene transfer to treat disease ofthe airways associated with cystic fibrosis. An adenoviral vectorwas selected for this study because of its reportedly high efficiencyin gene transfer in both animal models12 and preclinical studiesof human airway epithelium in vitro.13 The study used a dose-escalationprotocol, in which the dose was defined with reference to previousstudies14,15 by the estimated multiplicity of infection (thenumber of infectious adenoviral vectors delivered per airwayepithelial cell). We confined our treatment to the nasal epithelium,which has morphologic features16 and cystic fibrosisspecificdefects of ion transport similar to those of the lower airways,17because of concern about safety generated in preclinical studiesin which lung tissue was treated with adenoviral vector,18,19,20and also because efficacy can be tested accurately and repeatedlyat this site.
Methods
This protocol was approved by the Committee for Protection ofthe Rights of Human Subjects, the Recombinant Advisory Committeeof the National Institutes of Health, and the Food and DrugAdministration.21 Additional information about the methods usedis available from the National Auxiliary Publications Service(*).
Study Subjects
Twelve patients with stable pulmonary disease (mean [±SE]forced expiratory volume in one second, 65.0±6.9 percentof the predicted value) and pancreatic exocrine insufficiencywere studied (Table 1). All were seropositive for adenovirus.22The women had negative pregnancy tests and used contraceptivemethods. Informed consent was obtained from all patients.21
Table 1. Study Design and Dosage of Adenoviral Vector.
Gene-Transfer Vector
We used a replication-defective adenovirus serotype 5 vectorwith a strong enhancer (cytomegalovirus)promoter (chicken-actin [CB]) (Ad5-CB-CFTR, recently designated H5.020CBCFTR)21to express the CFTR complementary DNA (cDNA). Three lots ofthe adenoviral vector with particle:plaque-forming unit ratiosranging from 20:1 to 50:1 were produced by Good ManufacturingPractices at the Institute for Human Gene Therapy (Universityof Pennsylvania) and shipped frozen (-70°C) to the Universityof North Carolina at Chapel Hill. Each lot was efficacious forCFTR transduction by the Western blot assay and for chloridesecretion in vitro, and vector was titered and shown to be efficacious(by the Western blot assay) after dilution with 2 ml of 3 percentglycerol in phosphate-buffered saline (vehicle).
Study Design
Dosage
The adenoviral vector was administered to four cohorts of threepatients each, in logarithmically increasing doses. The doseof a biologic vector is complex and may be described in termsof the concentration (in plaque-forming units per milliliter),the total dose (in plaque-forming units), and the estimatednumber of vectors administered per epithelial cell (the estimatedmultiplicity of infection) (Table 1). Two milliliters of vehicleor adenoviral vector was infused (for 30 minutes) under directvision onto the inferior and medial surfaces of the inferiorturbinate and the nasal floor (approximately 8 cm2, or 2x107surface epithelial cells23) of the right nostril, with the subjectin a right lateral recumbent position. After treatment, thesubject remained in that position for an additional 20 minutes.The subject was then repositioned in the left lateral recumbentposition, and the left nostril was treated with the alternativesolution. Dosing-simulation studies indicated that 58±4percent of the instilled solution remained in the nose for 50minutes. The identity of the instilled solutions was known toonly one investigator.
Detection of Vector
We monitored for residual adenoviral vector in urine samplesand samples obtained with a swab from the nose, pharynx, andrectum by viral culture of 293 cells21 and by a nested polymerasechain reaction (PCR) using primers that amplify the L3 regionof the adenovirus and sequences of CFTR specific to the vector.
Safety
We monitored symptoms, vital signs, and blood counts and inspectedthe nose visually each day. Chest radiography, blood-chemistrytesting, and spirometry were performed at the beginning andend of the study. Serum titers of adenoviral antibody were measuredbefore and 21 days after treatment.22 Nasal-lavage fluid wasanalyzed daily for total cell counts (by hemocytometry), differentialcell counts (in cytospin preparations), and the presence ofcytokines (interleukin-1, interleukin-6, interleukin-8, andinterleukin-10) and albumin.24,25 Six days after treatment,biopsy specimens from the inferior turbinates were obtained,snap-frozen (in liquid nitrogen), and analyzed for inflammatorycell infiltrates.26
Efficacy of Gene Transfer
A reverse-transcriptase PCR assay of DNase-treated RNA fromscrape-biopsy specimens of nasal epithelium obtained 6 and 21days after dosing was performed27; vector-specific CFTR messengerRNA (mRNA) was amplified with primers across the junction of3' CFTR and untranslated viral sequences. In situ hybridizationwas performed with riboprobes specific for expressed vectorsequences.28
The functional efficacy of the vector treatment was assayedby measuring the potential difference (PD), or voltage, acrossthe nasal epithelium on 3 separate days before treatment, thendaily for up to 14 days, and on day 21 after treatment.16,17,29The protocol measured the basal PD (an index of sodium transportand mucosal integrity) and indexes of sodium transport (thebasal PD and the degree of inhibition by a sodium-channel blocker,amiloride [10-4 M]); basal chloride permeability (the responseto perfusion with a chloride-free solution containing amiloride);cAMP-regulated (CFTR) secretion of chloride (the response toisoproterenol [10-5 M] in the perfusate in the presence of achloride-free solution); and the capacity of the epitheliumto secrete chloride (the effect of ATP [10-4 M] in the chloride-freeperfusate on calcium-mediated chloride secretion29). PD wasmeasured under the inferior turbinate (normal epithelium), thenasal floor, and the medial surface (metaplastic epithelium)of the inferior turbinate.16
Statistical Analysis
The primary analysis of the effects of treatment on nasal PDused a mixed model to fit repeated measures of the values obtainedfrom days 1 through 6 after treatment with vehicle or adenoviralvector (the post-treatment values), using the base-line values(those obtained from day 5 before treatment to the day of treatment)as predictive variables in each cohort.30 The secondary analysiscompared the mean base-line and post-treatment values by pairedt-tests in each cohort. Each analysis yielded the same outcome.Mean base-line and post-treatment concentrations of inflammatorycells and mediators in nasal-lavage fluid were compared by pairedt-tests. Scores for inflammatory-cell infiltrates in the biopsyspecimens treated with vehicle were compared with those in thespecimens treated with adenoviral vector by paired t-tests.P values of less than 0.05 were considered to indicate statisticalsignificance.
Results
Detection of Vector
Culture
Vector was routinely cultured from samples obtained 20 minutesafter dosing from nostrils dosed with adenoviral vector, andfor up to four days after higher doses (estimated multiplicityof infection, >100). One patient (Patient 11; estimated multiplicityof infection, 1000) had positive cultures from the vehicle-dosednostril on day 1 and from rectal samples obtained one and twodays after dosing. Additional data on individual patients areavailable from *.
PCR
Adenoviral-vector DNA was detected by PCR in samples obtainedfrom the dosed nostrils for two to eight days after the administrationof higher doses (estimated multiplicity of infection, >100).Vector DNA was detected in the vehicle-dosed nostrils of twopatients (Patients 4 and 10) one and four days, respectively,after dosing, and from the pharynx of two patients (Patients8 and 10) two days after dosing. In urine samples, two patients(Patients 2 and 8) had PCR products compatible with sheddingof wild-type adenoviral DNA (L3/E1a-positive, CFTR-negative).31,32One patient (Patient 4) was positive by PCR for L3 but not forE1a or CFTR on day 1 after treatment, making it impossible todistinguish between wild-type adenoviral DNA and the degradedvector.
Molecular Assessment of Efficacy
Reverse-Transcriptase PCR Assay
Figure 1 shows the PCR product (210 base pairs [bp]) of vector-expressedCFTR mRNA from inferior-turbinate epithelium obtained six daysafter the administration of adenoviral vector; vector-specificproducts were observed in several samples containing reversetranscriptase. The summary data (Table 2) show that only subjectswho received the higher doses expressed vector-specific mRNA.No subject had a positive reverse-transcriptase PCR productfrom samples obtained three weeks after treatment. Samples fromthe side of the nose on which vehicle was administered werenegative.
Figure 1. Reverse-Transcriptase PCR Assays for Adenoviral-VectorMediated Expression of CFTR mRNA in the Nasal Epithelium of Patients with Cystic Fibrosis.
Nasal epithelial cells were obtained by curette biopsy. RNA was extracted, treated with DNase, and exposed to reverse transcriptase (+) or buffer (-). A nested PCR was performed, and the products were resolved on 3 percent NuSieve agarose gels and stained with ethidium bromide. The results in five patients who received various doses of vector (Patients 4 and 6 from cohort 2, Patients 7 and 9 from cohort 3, and Patient 12 from cohort 4) are shown. Viral DNA (from the adenoviral vector) and water were added as a template for the positive and blank controls, respectively. The 210-base-pair (bp) vector-specific PCR product for CFTR mRNA is shown. M denotes the molecular-weight marker.
Table 2. Presence or Absence of Adenoviral-Vector CFTR mRNA in Samples of Nasal Epithelium Obtained Six Days after Treatment.
In Situ Hybridization
Biopsy specimens from the six subjects given the higher doses(cohorts 3 and 4; estimated multiplicity of infection, >100)were studied. Previous studies and the positive controls usedin this study (CFPAC cells) indicated that in situ signals couldbe detected in single cells probed for CFTR mRNA expressed inresponse to the strong CB promoter.33 Approximately 14,000 cellswere examined in two sections from a biopsy specimen. No insitu signal was detected in specimens from five of six subjects.In one patient who received the highest dose (Patient 11), fourpatchy areas (i.e., containing approximately 15 to 20 cells)of gene transfer were detected; two were in regions of intactairway epithelium, and two were in areas of damage, includingexpression in the submucosa. No signal was detected in the nostrilstreated with vector when the samples were treated with RNaseor studied with sense probes, nor was an antisense signal detectedin the nostrils treated with vehicle.
Assessment of Biologic Efficacy by Measurements of PD
The measurements of PD under the inferior surface of the turbinate(in normal ciliated epithelium) are summarized below.
Basal PD
A total of 1440 PD measurements were made (Figure 2). Therewas no change in basal PD among patients who were exposed tovector at an estimated multiplicity of infection of 1, 10, or100 (cohorts 1, 2, and 3). In contrast, the mean basal PD decreasedby 15.1±1.6 mV after the administration of vector atthe highest dose (cohort 4, including Patients 10, 11, and 12;estimated multiplicity of infection, 1000). There was no significantchange in the PD of epithelium treated with vehicle in any cohort.
Figure 2. Basal Transepithelial Potential Difference (PD) before and after the Administration of Adenoviral Vector or Vehicle in Each Cohort.
Individual measurements of the basal PD were made three times before the administration of adenoviral vector or vehicle (dashed vertical lines, day 0) and serially for up to 21 days after treatment. The three patients in each cohort are designated by three symbols (, , and ); the data for the nostrils in which vehicle was administered are designated by open symbols, and those for the nostrils in which vector was administered are designated by solid symbols. Horizontal lines (dashed for vehicle, solid for vector) indicate the mean values before treatment and on days 1 through 6 after treatment (before biopsy). Arrows indicate the time of biopsy. P = 0.02 for the difference in the mean values obtained before and after treatment with vector in the highest-dose cohort (cohort 4).
Inhibition by Amiloride
There was no change in the percentage of inhibition of the basalPD with amiloride, a sodium-channel blocker, after treatmentwith either vector or vehicle in any cohort (inhibition in cohort4, 69.5±2.7 percent before treatment with vector and68.5±2.1 percent afterward; estimated multiplicity ofinfection, 1000).
Basal Chloride Permeability
There were no systematic changes in PD after chloride substitution,even in the highest-dose cohort (change in PD in nostrils treatedwith vector in cohort 4, 5.8±0.7 mV before treatmentand 4.5±0.4 mV afterward; in nostrils treated with vehicle,4.7±0.6 and 4.5±0.5 mV, respectively).
Isoproterenol-Regulated Chloride Secretion
We found no evidence of isoproterenol (cAMP)-induced increasesin PD (chloride secretion), even in the highest-dose cohort(change in nostrils treated with vector in cohort 4, 0.2±0.3mV before treatment and -0.2±0.6 mV afterward; in nostrilstreated with vehicle, 0.8±0.4 and 0.3±0.3 mV,respectively).
Combined Response to Chloride Substitution and Isoproterenol
The change in nasal PD in amiloride-treated epithelium in responseto both chloride substitution and treatment with isoproterenolbest discriminates patients with cystic fibrosis from normalsubjects29,34 (Figure 3). The individual data points, relativeto previously published values from normal subjects29 (shadedarea in Figure 3), are shown for each cohort. There were nosignificant changes in PD, even in the highest-dose cohort (changein PD with vector in cohort 4, 4.9±0.3 mV before treatmentand 2.0±1.7 mV afterward; with vehicle, 4.6±1.6and 3.8±1.4 mV, respectively).
Figure 3. Assay of Nasal PD to Determine the Extent of CFTR-Mediated Chloride Transport before and after the Administration of Adenoviral Vector or Vehicle in Each Cohort.
The combined change in transepithelial PD in amiloride-pretreated nasal epithelium after chloride-free perfusion (with replacement by gluconate) and the administration of isoproterenol is shown plotted against time in each cohort. The intervals measured, the time of administration (vertical dashed lines), symbols denoting patients and type of treatment (open for vehicle, solid for vector), arrows indicating the time of biopsy, and horizontal lines (dashed for vehicle, solid for vector) are as described in the legend to Figure 2. The mean (±SD) changes in the PD of normal subjects29 in response to the chloride-substitutionisoproterenol maneuver are shown at the top of each panel as solid horizontal lines and shaded areas, respectively.
ATP-Regulated Chloride Secretion
Large ATP-induced increases in PD, reflecting calcium-mediatedchloride secretion, were detected in all cohorts before treatment(change in PD with vehicle, -19.9±2.7 mV; with vector,-20.6±2.4 mV), and they persisted after treatment (-20.8±2.3and -18.7±2.4 mV, respectively).
Data similar to those given above for normal epithelium wereobtained in each cohort for measurements of PD on the medialsurface of the inferior turbinate (metaplastic epithelium).
Safety
No patient had significant changes in vital signs, completeblood count, blood chemistry, chest radiographs, or resultsof spirometric analysis. No patient receiving doses of adenoviralvector with an estimated multiplicity of infection of 1, 10,or 100 (cohorts 1, 2, and 3) had local symptoms or signs. Twopatients (Patients 10 and 12) in cohort 4 (the highest-dosegroup) had symptoms and signs of toxic effects within 12 to24 hours of the administration of the dose. One patient (Patient10) had an earache and an inflamed tympanic membrane; the other(Patient 12) had jaw pain and mandibular-angle (nodal) tenderness;these symptoms were all ipsilateral to the site of administrationof vector. Visual inspection by an ear, nose, and throat specialistwho was unaware of the dosing schedule and the symptoms of thepatients revealed unilateral induration of the nasal mucosaand increases in mucosal sensitivity and secretions that wereconfined to the nostril treated with vector. Maximal symptomsand signs occurred after 48 to 96 hours, and there was completeresolution within three weeks. Further information on individualpatients is available from *.
Nasal Lavage and Measurements of Cells and Cytokines
No difference in the total cell count or in the quantity ofinterleukin-1, interleukin-6, interleukin-8, and interleukin-10in nasal-lavage fluid was noted on days 1 through 6 after theadministration of vector or vehicle in any cohort. The nostrilstreated with vector in the highest-dose cohort had a greaterincrease in the total number of cells, neutrophils, interleukin-6,and interleukin-8 in response to the nasal biopsy than did thenostrils treated with vehicle, suggesting that the adenoviralvector primed the epithelium for an inflammatory response.
Albumin in Nasal-Lavage Fluid
There was no change in the albumin concentration in nasal-lavagefluid in Patients 1 through 9 (cohorts 1, 2, and 3) after theadministration of either vector or vehicle on days 1 through6, as compared with the base-line values (Figure 4). In contrast,in the highest-dose cohort (cohort 4), the albumin concentrationdoubled in the nostrils treated with vector, but not in thosetreated with vehicle. This increase was approximately threefoldin the two symptomatic patients (Patients 10 and 12).
Figure 4. Mean (±SE) Change from Base Line in the Albumin Content of Nasal-Lavage Fluid during the Six Days after the Administration of Vehicle (Open Bars) or Vector (Solid Bars), Expressed as the Percentage of Change from the Values before Treatment.
The values for cohorts 1, 2, and 3 (Patients 1 through 9) and cohort 4 (Patients 10, 11, and 12) are shown separately. The two patients (Patients 10 and 12) in cohort 4 who had signs and symptoms of nasal inflammation or damage are shown so that their data may be compared with the mean data for cohort 4. The base-line levels of albumin in nasal-lavage fluid in each group shown were as follows: cohorts 1, 2, and 3, 12.0 µg per milliliter in the vehicle-treated nostril and 12.2 µg per milliliter in the vector-treated nostril; cohort 4, 2.4 and 6.4 µg per milliliter; and Patients 10 and 12, 2.5 and 4.2 µg per milliliter.
Histopathological Analysis of Nasal-Biopsy Specimens
There was no difference in the number of inflammatory cellsin the epithelium or submucosa between the biopsy specimensfrom the nostrils treated with adenoviral vector and those treatedwith vehicle in any cohort.
Serum Antibodies to Adenovirus
One patient (Patient 10) in the highest-dose cohort had an increase(from 1:80 to 1:1280) in the titer of neutralizing antibody21 days after the administration of the vector.
Discussion
In this double-blinded, vehicle-controlled trial of adenoviralvector in patients with cystic fibrosis, we found molecularevidence of low-efficiency gene transfer and expression of thenormal CFTR mRNA in nasal epithelium, but there was no significantfunctional correction of abnormalities in ion transport. Theabsence of adenoviral-vector tropism for surface columnar cells,35which normally express CFTR,36 explains this failure. This problemcannot be overcome by simply increasing the dose of the vector,because the highest dose we used (with an estimated multiplicityof infection of 1000) was associated with inflammatory responsesin two of three patients; in addition, studies in animals indicatea wide spectrum of toxic effects at higher doses.18,19,20
Careful attention was paid to the design, production, and administrationof the vector we used. With its strong promoter, this vectorwas completely effective in correcting cystic fibrosisassociateddefects of chloride35 and sodium transport in the airway epitheliumin vitro.37 Three separate lots of vector were produced21,38and tested for the titer and for the efficacy of CFTR gene transferat two independent sites before their use. The adequacy of vectoradministration and the viability of the vector were confirmedby its detection in the nostril treated with vector up to eightdays after the administration of the dose.
Two outcome variables were measured with respect to efficacyand were grouped on the basis of dosage. In the two lower-dosecohorts (cohorts 1 and 2; estimated multiplicity of infection,1 and 10, respectively), we found little evidence of adenoviral-vectormediatedgene transfer. Only one of six patients was positive for genetransfer by the reverse-transcriptase PCR assay. This assayis sensitive to as few as 5 cells expressing vector CFTR mRNAamong 500,000 cells not expressing this mRNA (0.001 percent);thus, the negative PCR data in these cohorts indicated thatvirtually no gene transfer occurred.39 The lack of functionalcorrection of PD is consistent with this conclusion.
In the two higher-dose cohorts (cohorts 3 and 4; estimated multiplicityof infection, 100 and 1000, respectively), there was more evidenceof gene transfer by the reverse-transcriptase PCR assay (fourof six patients were positive). However, in situ hybridization,which is about one order of magnitude less sensitive than thePCR assay, detected expression in only one of these six patients,and the percentage of epithelial cells expressing CFTR mRNAin this one patient was low (less than 1 percent of the totalnumber). Previous studies have shown that CFTR expression in3 to 6 percent of the cells in the cystic fibrosis epitheliumis required to correct the defect in chloride secretion.35,40The PD protocols that are sensitive measures of CFTR-mediatedchloride transport (e.g., the combined response to chloridesubstitution and isoproterenol) detected no systematic evidenceof gene transfer, a finding consistent with an efficiency ofgene transfer of less than 1 percent (Figure 3).
Three data points slightly above the range for cystic fibrosisin the highest-dose cohort could reflect patchy gene transferor nonspecific effects of isoproterenol to activate a calcium-regulatedchloride pathway in inflamed tissues. The relation between theefficiency of correction and the restoration of sodium transportis different from that for chloride, with virtually all cellsrequiring functional correction in order for the normalizationof sodium transport to occur. There was no change in the percentageof inhibition of PD by amiloride in cohorts 3 and 4 to suggesta correction of sodium transport. Thus, in these cohorts vector-mediatedgene transfer was detected, but its efficiency was too low foreither the chloride or the sodium-transport defect associatedwith cystic fibrosis to be corrected.
No evidence of systemic toxic effects was noted in any patient,and no local toxic effects were noted in the cohorts receivingthe three lowest doses. However, two of the three patients inthe highest-dose cohort had symptoms and signs of local toxiceffects in the mucosa, reduced basal PDs, and an increased fluxof albumin into the fluid subsequently obtained by lavage fromthe nostrils treated with vector observations consistentwith epithelial damage.29,41,42 The syndrome was not associatedwith increased numbers of white cells or concentrations of cytokinesin nasal-lavage fluid or with the cellular infiltrates associatedwith more chronic toxic effects (lasting 3 to 21 days) thathave been identified in studies in animals.18,19,20 The rapidonset of the syndrome, like the severe pulmonary inflammatoryreaction to Ad-CFTR, a similar adenoviral vector, in a patientdescribed by Crystal et al.,15 coupled with recent studies inanimals,43 suggests a neurogenic inflammatory cause.
Three observations were made that may pertain to the safetyof the vector we used. First, there was dissemination of thevector for example, to the pharynx and stool at higher doses. Second, we detected vector DNA in the nasalcavity for up to eight days. The precise location of the vectorin the epithelium is not known, and therefore the possibilityof transmission or recombination of vector with wild-type adenoviruscannot be discounted.21,44 Finally, we observed an increasein the serum antibody titer in one patient receiving the highestdose of vector, which may have implications for the safety ofthe vector (e.g., enhanced immunologic responses on subsequenttreatment with vector), implications for the efficacy of repeatedadministration of vector, or both.19
Two unblinded trials of Ad-CFTR in patients with cystic fibrosishave been reported. We could not confirm the correction of thecystic fibrosisassociated nasal defect of chloride transportreported by Zabner et al.14 Our study paralleled their studywith respect to the site of deposition, and we used a vectorwith a stronger promoter and used that vector in higher concentrations(1.5 to 3 logs). The discrepancy probably relates to the PDprotocols used to measure efficacy. The protocol used by Zabneret al. does not readily discriminate between patients with cysticfibrosis and normal subjects, making it difficult to interpretthe results. The chloride-free PD protocols used in this studyare highly sensitive in such discrimination29,34 and can detectfull or even partial correction of chloride transport. Thisdiscrepancy highlights the need for standardized PD protocolsand for complementary molecular and morphologic methods of assessinggene transfer.
Crystal et al.15 reported no quantitative nasal bioelectricdata, but they did report positive results of immunocytochemicalanalysis for CFTR in one of four patients after bronchial brushingof a region that had received transbronchoscopic doses of vector.The evidence of transduction in bronchial cells may possiblyreflect an increased efficiency of gene transfer in lower airwayepithelium as compared with nasal epithelium, as has been reportedin rodents.12,18 Alternatively, damage to airway epitheliumcan substantially enhance gene transfer by abrading the relevanttarget cells (i.e., the surface columnar cells) and exposingthe vector to basal cells, which are more easily transducedbut do not normally express CFTR.35 Thus, data pertaining totransduced epithelium from previously traumatized areas maynot be representative of gene transfer to intact, undamagedepithelium, such as that treated in our study.
In summary, adenoviral-vectormediated gene transfer tonasal epithelium affected by cystic fibrosis is inefficient.One potential strategy to overcome this problem would be totarget adenoviral vectors to basal cells. Another would involveattempting to differentiate basal cells into columnar cells.A third would be to modify the vector itself so that it becomestropic for columnar cells. If the nasal epithelium is typicalof all human airway regions with respect to the observed inefficiencyof adenoviral-vectormediated gene transfer, it wouldappear prudent also to accelerate the development of alternativevectors,45 or to modify the adenoviral vectors, if gene transferis to be successful in treating lungs in patients with cysticfibrosis.
Supported by grants from the Cystic Fibrosis Foundation (R026and R881), the National Institute of Diabetes and Digestiveand Kidney Diseases (P30 DK47757), the National Heart, Lung,and Blood Institute (HL42384 and HL51818), and the NationalInstitutes of Health (RR00046).
Dr. Wilson and Ms. Grossman hold equity in Genova, Inc., a companyinvolved in the development of gene-transfer technology.
We are indebted to K. Burns, C. Foy, J. Robinson, J. Winders,C.-H. Wong, and I. Wortman for expert technical assistance,to L. Dudus and H. Ye for assistance with the in situ hybridizationstudies, to P. Noone and W. Bennett for assistance with thedosing-simulation studies, to K. Kozarsky for assistance inthe serologic testing for adenovirus by the Western blot assay,to L. Brown for editorial assistance, to the nursing staff ofthe General Clinical Research Center for expert patient care,and to the patients who participated in this challenging study.
* See NAPS document no. 05245 for seven pages of supplementarymaterial. Order from NAPS c/o Microfiche Publications, P.O.Box 3513, Grand Central Station, New York, NY 10163-3513.
Source Information
From the Department of Medicine, Cystic FibrosisPulmonary Research and Treatment Center (M.R.K., K.W.H., Z.Z., J.C.O., K.R.J., L.G.J., R.C.B.), and the Departments of Pediatrics (T.L.N., P.-C.H., M.W.L.) and Biostatistics (L.J.E.), School of Public Health, University of North Carolina, Chapel Hill; and the Institute for Human Gene Therapy and the Department of Molecular and Cellular Engineering, University of Pennsylvania, Philadelphia (J.F.E., M.G., J.M.W.).
Address reprint requests to Dr. Knowles at the Cystic FibrosisPulmonary Research and Treatment Center, 724 BurnettWomack Bldg., CB 7020, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7020.
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Gene Therapy
Alton E., Geddes D. M., Wagner J. A., Knowles M., Boucher R.
Extract |
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N Engl J Med 1996;
334:332-333, Feb 1, 1996.
Correspondence
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