AU725816B2 - Gene therapy for cystic fibrosis - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION Name of Applicant: Address of Applicant: Genzyme Corporation One Kendall Square, Cambridge, Massachusetts 02139, United States of America Richard J. Gregory; Donna Armentano; Larry A.

Couture; and Alan E. Smith.

DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Actual Inventor(s): Address for Service: Complete Specification for the invention entitled: Gene therapy for cystic fibrosis The following statement is a full description of this invention, including the best method of performing it known to us: P:\OPERVM 57349-94CLM 3014/97 -1A GENE THERAPY FOR CYSTIC FIBROSIS The contents of European Patent Publication No. 0 446 071, published September 11, 1991 (EP Application No. 91301819.8, claiming priority of United States Serial Number 07/613,592, filed on November 15, 1990, which is a continuation-in-part application of United States Serial Number 07/589,295, filed on September 27, 1990, which is itself a continuation-in-part application of United States Serial Number 07/488,307, filed on March 5, 1990) are incorporated herein by reference. Definitions of language or terms not provided in the present application are the same as those set forth in the European publication. Any reagents or minerals used in the examples of the present application whose source is not expressly identified also is the same as those described in the European publication, AF508 CFTR gene and CFTR antibodies.

Background of the Invention Cystic Fibrosis (CF) is the most common fatal genetic disease in humans (Boat, T.F.

et al. in The Metabolic Basis of Inherited Diseases (Scriver, C.R. et al. eds., McGraw-Hill, New York (1989)). Approximately one in every 2,500 infants in the United States is bom with the disease. At the present time, there are approximately 30,000 CF patients in the United States. Despite current standard therapy, the median age of survival is only 26 years.

Disease of the pulmonary airways is the major cause of morbidity and is responsible for of the mortality. The first manifestation of lung disease is often a cough, followed by progressive dyspnea. Tenacious sputum becomes purulent because of colonization of Staphylococcus and then with Pseudomonas. Chronic bronchitis and bronchiectasis can be partially treated with current therapy, but the course is punctuated by increasingly frequent exacerbations of the pulmonary disease. As the disease progresses, the patient's activity is progressively limited. End-stage lung disease is heralded by increasing hypoxemia, pulmonary hypertension, and cor pulmonale.

The upper airways of the nose and sinuses are also involved in CF. Most patients with CF develop chronic sinusitis. Nasal polyps occur in 15-20% of patients and are common by the second decade of life. Gastrointestinal problems are also frequent in CF; infants may suffer meconium ileus. Exocrine pancreatic insufficiency, which produces symptoms of malabsorption, is present in the large majority of patients with CF. Males are almost uniformly infertile and fertility is decreased in females.

Based on both genetic and molecular analyses, a gene associated with CF was isolated as part of 21 individual cDNA clones and its protein product predicted (Kerem, B.S. et al.

(1989) Science 245:1073-1080; Riordan, J.R. et al. (1989) Science 245:1066-1073; -2- Rommens, J.M. et al (1989) Science 245:1059-1065)). European Publication No. 0 446 017 describes the construction of the gene into a continuous strand, expression of the gene as a functional protein and confirmation that mutations of the gene are responsible for CF. (See also Gregory, et al (1990) Nature 347:382-386; Rich, D.P. et al. (1990) Nature 347:358-362). This publication also discloses experiments which show that proteins expressed from wild type but not a mutant version of the cDNA complemented the defect in the cAMP regulated chloride channel shown previously to be characteristic of CF.

The protein product of the CF associated gene is called the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan, J.R. et al. (1989) Science 245:1066- 1073). CFTR is a protein of approximately 1480 amino acids made up of two repeated elements, each comprising six transmembrane segments and a nucleotide binding domain.

The two repeats are separated by a large, polar, so-called R-domain containing multiple potential phosphorylation sites. Based on its predicted domain structure, CFTR is a member of a class of related proteins which includes the multi-drug resistance (MDR) or P- 15 glycoprotein, bovine adenyl cyclase, the yeast STE6 protein as well as several bacterial amino acid transport proteins (Riordan, J.R. et al. (1989) Science 245:1066-1073; Hyde, S.C.

et al. (1990) Nature 346:362-365). Proteins in this group, characteristically, are involved in pumping molecules into or out of cells.

CFTR has been postulated to regulate the outward flow of anions from epithelial cells 20 in response to phosphorylation by cyclic AMP-dependent protein kinase or protein kina' C (Riordan, J.R. et al. (1989) Science 245:1066-1073; Welsh, 1986; Frizzell, R.A. et al. (1986) Science 233:558-560; Welsh, M.J. and Liedtke, C.M. (1986) Nature 322:467; Li, M. et al.

(1988) Nature 331:358-360; Huang, T-C. etal. (1989) Science 244:1351-1353).

Sequence analysis of the CFTR gene of CF chromosomes has revealed a variety of mutations (Cutting, G.R. et al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863-870; and Kerem, B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S. et al. (1990) Proc. Natl. Acad Sci. USA 87:8447-8451). Population studies have indicated that the most common CF mutation, a deletion of the 3 nucleotides that encode phenylalanine at position 508 of the CFTR amino acid sequence (AF508), is associated with approximately 70% of the cases of cystic fibrosis. This mutation results in the failure of an epithelial cell chloride channel to respond to cAMP (Frizzell R.A. et al. (1986) Science 233:558-560; Welsh, M.J.

(1986) Science 232:1648-1650.; Li, M. et al. (1988) Nature 331:358-360; Quinton, P.M.

(1989) Clin. Chem. 35:726-730). In airway cells, this leads to an imbalance in ion and fluid transport. It is widely believed that this causes abnormal mucus secretion, and ultimately results in pulmonary infection and epithelial cell damage.

Studies on the biosynthesis (Cheng, S.H. et al. (1990) Cell 63:827-834; Gregory, R.J.

et al. (1991) Mol. Cell Biol. 11:3886-3893) and localization (Denning, G.M. et al. (1992) J.

Cell Biol. 118:551-559 of CFTR AF508, as well as other CFTR mutants, indicate that many CFTR mutant proteins are not processed correctly and, as a result, are not delivered to the -3plasma membrane (Gregory, R.J. et al. (1991) Mol. Cell Biol. 11:3886-3893). These conclusions are consistent with earlier functional studies which failed to detect cAMPstimulated Cl- channels in cells expressing CFTR AF508 (Rich, D.P. et al. (1990) Nature 347:358-363; Anderson, M.P. et al. (1991) Science 251:679-682).

To date, the primary objectives of treatment for CF have been to control infection, promote mucus clearance, and improve nutrition (Boat, T.F. et al. in The Metabolic Basis of Inherited Diseases (Scriver, C.R. et al. eds., McGraw-Hill, New York (1989)). Intensive antibiotic use and a program of postural drainage with chest percussion are the mainstays of therapy. However, as the disease progresses, frequent hospitalizations are required.

Nutritional regimens include pancreatic enzymes and fat-soluble vitamins. Bronchodilators are used at times. Corticosteroids have been used to reduce inflammation, but they may produce significant adverse effects and their benefits are not certain. In extreme cases, lung transplantation is sometimes attempted (Marshall, S. et al. (1990) Chest 98:1488).

Most efforts to develop new therapies for CF have focused on the pulmonary i* 15 complications. Because CF mucus consists of a high concentration of DNA, derived from lysed rieutrophils, one approach has been to develop recombinant human DNase (Shak, S. et al. (:1990) Proc. Natl. Sci. Acad USA 87:9188). Preliminary reports suggest that aerosolized enzyme may be effective in reducing the viscosity of mucus. This could be helpful in clearing the airways of obstruction and perhaps in reducing infections. In an attempt to limit 20 damage caused by an excess of neutrophil derived elastase, protease inhibitors have been tested. For example, alpha-1 -antitrypsin purified from human plasma has been aerosolized to deliver enzyme activity to lungs of CF patients (McElvaney, N. et al. (1991) The Lancet 337:392). Another approach would be the use of agents to inhibit the action of oxidants derived from neutrophils. Although biochemical parameters have been successfully measured, the long term beneficial effects of these treatments have not been established.

Using a different rationale, other investigators have attempted to use pharmacological agents to reverse the abnormally decreased chloride secretion and increased sodium absorption in CF airways. Defective electrolyte transport by airway epithelia is thought to alter the composition of the respiratory secretions and mucus (Boat, T.F. et al. in The Metabolic Basis of Inherited Diseases (Scriver, C.R. et al. eds., McGraw-Hill, New York (1989); Quinton, P.M. (1990) FASEBJ. 4:2709-2717). Hence, pharmacological treatments aimed at correcting the abnormalities in electrolyte transport could be beneficial. Trials are in progress with aerosolized versions of the drug amiloride; amiloride is a diuretic that inhibits sodium channels, thereby inhibiting sodium absorption. Initial results indicate that the drug is safe and suggest a slight change in the rate of disease progression, as measured by lung function tests (Knowles, M. et al. (1990) N. Eng. J. Med. 322: 1189-1194; App, E.(1990) Am.

Rev. Respir. Dis. 141:605). Nucleotides, such as ATP or UTP, stimulate purinergic receptors in the airway epithelium. As a result, they open a class of chloride channel that is different from CFTR chloride channels. In vitro studies indicate that ATP and UTP can stimulate I -J -I.I .1 -4chloride secretion (Knowles, M. et al. (1991) N. Eng. J. Med. 325:533). Preliminary trials to test the ability of nucleotides to stimulate secretion in vivo, and thereby correct the electrolyte transport abnormalities are underway.

Despite progress in therapy, cystic fibrosis remains a lethal disease, and no current therapy treats the basic defect. However, two general approaches may prove feasible. These are: 1) protein replacement therapy to deliver the wild type protein to patients to augment their defective protein, and; 2) gene replacement therapy to deliver wild type copies of the CF associated gene. Since the most life threatening manifestations of CF involve pulmonary complications, epithelial cells of the upper airways are appropriate target cells for therapy.

The feasibility of gene therapy has been established by introducing a wild type cDNA into epithelial cells from a CF patient and demonstrating complementation of the hallmark defect in chloride ion transport (Rich, D.P. et al. (1990) Nature 347:358-363 This initial v'ork involved cells in tissue culture, however, subsequent work has shown that to deliver the gene to the airways of whole animals, defective adenoviruses may be useful (Rosenfeld, 15 (1992) Cell 68:143-155). However, the safety and effectiveness of using defective adenoviruses remain to be demonstrated.

Summary of the Invention In general, the instant invention relates to vectori for transferring selected genetic 20 material of interest DNA or RNA) to cells in vivo. In preferred embodiments, the vectors are adenovirus-based. Advantages of adenovirus-based vectors for gene therapy are that they appear to be relatively safe and can be manipulated to encode the desired gene product and at the same time are inactivated in terms of their ability to replicate in a normal lytic viral life cycle. Additionally, adenovirus has a natural tropism for airway epithelia.

Therefore, adenovirus-based vectors are particularly preferred for respiratory gene therapy applications such as gene therapy for cystic fibrosis.

In one embodiment, the adenovirus-based gene therapy vector comprises an adenovirus 2 serotype genome in which the Ela and Elb regions of the genome, which are involved in early stages of viral replication have been deleted and replaced by genetic material of interest DNA encoding the cystic fibrosis transmembrane regulator protein).

In another embodiment, the adenovirus-based therapy vector is a pseudo-adenovirus (PAV). PAVs contain no potentially harmful viral genes, have a theoretical capacity for foreign material of nearly 36 kb, may be produced in reasonably high titers and maintain the tropism of the parent adenovirus for dividing and non-dividing human target cell types.

PAVs comprise adenovirus inverted terminal repeats and the minimal sequences of a wildtype adenovirus type 2 genome necessary for efficient replication and packaging by a helper virus and genetic material of interest. In a preferred embodiment, the PAV contains adenovirus 2 sequences.

3- 2-98 1 1 !58 /7 5/ In a further embodiment, the adenovirus'-based gene therapy vector contains the 6-pen reading frarne 6 (ORF6) of adenoviral early region 4 (E4) from the E4 promoter and is deleted for all other E4 open reading frames. Optionally, this vector can iniclude deletions in the ElI and/or E3 regions, Alternatively, the aclenovirus-based gene therapy vector contains the open reading frame 3 (ORY3) of adenoviral E4 from the E4 promoter and is deleted for all other E4 open reading frame s. Again, optionally, Uhs vector can include deletions in the El and/or £3 regions. The deletion of non-essential open reading frames of E4 increases the cloning capacity by approximately 2 kb without significantly reducing dhe vriability of the virus in cell culture. In combination with deletions in the ElI and/or E3 regions of adenovirus vectors, the theoretical insert capacity of the resultant vectors is increased to 8-9 kb, The invention also relates to methods of gene therapy using the disclosed vectors and genetically engineered cells produced by the method.

Birief Descrintion of te Tables and Drawn Further understanding of the invention may be had by reference to the tables and figures wherein: Table I shows CFTR, mutants wherein the known association with CF (Y, yes or N, no), exon localization, domain location and presence or absence of bands A, B, and C of mutant CFTR species is Shown. TM6, indicates transmernbrane domain 6; NBD, riucleotide binding domain; ECD, extraccilular domain and Term, termination at 21 codons past residue 1337; and Table II shows the nucleotide sequence of Ad2/CFTR- 1.

The convention for namning mutants is, first, the amniro acid normally found at the particular residue, the residue number (Riordan, T.R. et al. (1989) Science 245:1066-1073), and the amino acid to which the residue was converted. The single letter amino acid code is used; D, aspartic acid; F, phenylalanine; G, glycine; 1, isoleucine; lysine; M, methionine; N, asparagine; Q, glutamine; R, arginine; S, senine; W, tryptophan- A, alanmne: C, cysteine; E, glutamic acid; H, histidine;- L, leucine; P,

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104 32-98;11!58 6/ -6praline; T, threonine; Y, tyrosine, and V,vatine. Thus 0551D is a mutant in which glycine 551 is converted to aspartic acid.

Figure 1 shows alignment of CFTR partial cDNA clones used in construction of cDNA conaining complete coding sequence of the CFTR; only restriction sites relevant to the DNA constructions described below are shown.

Figure 2 depicts plasmid construction of the CFTR cDNA clone pKX-CFTRI.

Figure 3 depicts plasmid construction of the CFTR cDNA clone pKX-CFTPR2.

Figure 4 depicts plasmid construction of the CFTR cDNA clone pSC-CFTR2.

Figure 5 shows a plasmid, map of the CFTR cDNA clone pSC-CFTR2, Figure 6 shows the DNA sequence of synthetic DNAs used for insertion of an intron into the CFTR cDNA sequence, with the relevant restriction endonuclease sites and nucleotide positions noted.

Figures 7A and 7B depict plasrnid construction of the CFTR cDNA clone pKK-CFTR3, Figure 8 shows a plasmid map of the CFTR cDNA pKK-CFTR3 containing an intron between nurcleoticics 1716 and 171t7.

Figure 9 shows treatment of CFTR with glycosidases.

SRA/L1 T0 3- 2-98 1 1 !58 /7 7/ -7- Figures IQOA and l OB show an analysis of CFTR expressed from COS-7 transfected cells- Figures I IlA and 1 l B show pulse-chase labeling of wild type and AF508 mutant CFTR in COS-7 transfected cells.

Figures 12A-12D show immunolocalization of wild type and AF509 mutant CFTR in COS-7 cells transfected with pMT-CFTR or Figure 13 shows an analysis of mutaint forms of CFTR.

Figure 14 shows a map of the first generation adenovirus based vector encoding CFTR (Ad2/CFTR-1).

Figure 15 shows the plasmnid construction of the Ad2/CFT,-1I vector.

Figures 16A and 16B show a map of the second generation adenovirus based vector, PAV.

Figures 1 7A and 1 7B show the plasmid construction for. a second generation adenoviral vector (Ad2E4ORF6).

Figures 1 8A and 1 8B show differential cell analyses of bronchoalveolar lavage specimens from control and infected rats. These data demonstrate that none of the rats treated with Ad2/CFTR-1I had a change in the total or differential white blood cell count 4, 10, and 14 days after infection (Figure 1 SA) and 3, 7, 14 days after infection (Figure 1 SB).

e7 RAL,

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O4 l .0V 3- 2-98:11!58 /7 8/ -8- Figures 19A arnd 19B show examples of UTV fluorescence from an agarosc gel electrophoresis, stained with ethidium bromide, of products of RT-PCR from nasal brushings of Rhesus monkeys after application of AdZ/CFTR-l or Ad2/P-Gal.

Figures 20A-20D show serum antibody titers in Rhesus monkeys after three vector admninistrations. These graphs demonstrate that all three monkeys treated with Ad2/CFTR-1 developed antibodies against adenovirus.

Figures 21 A-21II are photomicrographs of human nasal mucosa immediately before, during, and after Ad2/CFTR-1I application. These photomicrographs demonstrate that inspection of the nasal mucosa showed mild to moderate erythema, edema, and exudate in patients treated with Ad2/CFTR- I (Figures 21 A-2 1 C) and in control patients (Figures 21 G-2 1I). These changes were probably due to local anesthesia and vasoconstrietion because when an additional patient was exposed to Ad2/CFTR- in a method which did not require the use of local anesthesia or vasoconstriction, there were no symptomns and the nasal inucosa appeared normal (Figures 2 1 D-21 figure 22 is a photomicrograph of a hernatoxylin and cosin stained biopsy of human nasal rnucosa obtained frm the third patient three days after Ad2/CFTR- I administration. This section shows a morphology consistent with CF, a thickened basement membrane and occasional morphonuclear cells in the submucosa, but no abnormalities that could be attributed to the adenovirus vector.

Figure 23 shows transepithelial voltage (Vt) across the nasal epithelium of a normal human subject. Arniloride (giM) and terbutaline (IM) were perfused onto the 3- 2-98; 11:58 /7 9/ 9mucosal surface beginning at the times indicated. Under basal conditions Vt was electrically negative, Perfiasion of amiloride onto the mucosal surface inhibited Vt by blocking apical Na7 channels.

Figures 24A and 24B show transepithelial voltage (Vt) across the nasal epithelium of normal human subjects (Figure 24A) and patients with CF (Figure 243).

Values were obtained under basal conditions, during perfusion with amiloride tM) and during perfusion of arniloride plus terbutaline (pM) onto the mucosal surface. Data are from seven normal subjects and nine patients with CF. In patients with CF, Vt was more~ electrically negative than in normal subjects (Figure 24B). Amiloride inhibited Vt in CF patients, as it did in normal subjects. However, Vt failed to hyperpolarize when terbutaline was perfuised onto the epithelium in the presence of amiloride. Instead, Vt either did not change or became less negative, a result very different from that observed in normal subjects.

Figures 25A and 25B show transepithelial voltage (Vt) across the nasal epithelium of a third patient before (Figure 25A) and after (Figure 25B) administration of approximately 25 MOI of Ad2/CFTR-l. Arniloride and terbutaline were perfused onto the mucosal surface beginning at the times indicated. Figure 25A shows an example from the third patient before treatment. Figure 25B shows that in contrast to the response before Ad2/CFTR- I w aipplied, after virus replication, in tht; piesence of wmiloride, terbutaline stimulated Vt.

3- 2-98;11:58 10/ 10 Figures 26A-26F show the time of course changes in transepithelial electrical properties before and after administration of Ad2/CFTR-1. Figures 26A and 26B are from the first patient who received approximately 1 MOI; Figures 26C and 26D are from the second patient who received approximately 3 MOI; and Figures 26E and 26F are from the third patient who received approximately 25 MOI. Figures 26A, 26C, and 26E show values of basal transepithelial voltage (Vt) and Figures 26B, 26D, and 26F show the change in transepithelial voltage (AVt) following perfusion ofterbutaline in the presence of amiloride. Day zero indicates the day of Ad2/CFTR-l administration.

Figures 26A, 26C, and 26E show the time course of changes in basal Vt for all three patients. The decrease in basal Vt suggests that application of Ad2/CFTR-1 corrected the CF electrolyte transport defect in nasal epithelium of all three patients. Additional evidence came from an examination of the response to terbutaline. Figures 26B, 26D, and 26F show the time course of the response. These data indicate that Ad2/CFTR-l corrected the CF defect in Cl" transport.

Figures 27A and 27B show the time course of changes in transepithelial electrical properties before and after administration of saline instead of Ad2/CFTR-1 to CF patients. Day zero indicates the time of mock administration. The top graph shows basal transepithelial voltage (Vt) and the bottom graph shows the change in transepithelial voltage following perfusion with terbutaline in the presence of amiloride (AVt). Closed symbols are data from two patients that received local anesthetic/vasoconstriction and placement of the applicator for thirty minutes. Open 3--2-98;11:58 11/ symbol is data from a patient that received local anesthetic/vasoconstriction, but not placement of the applicator. Symptomatic changes and physical findings were the same as those observed in CF patients treated with a similar administration procedure and Ad2/CFTR-1.

Figure 28 is a schematic of Ad2-QRF6IPGK-CFrR which differs from Ad2/CFTR-1 in that the latter utilized the endogenous Ela promoter, had no poly A addition signal directly downstream of CFTR and retained an intact E4 region.

Figure 29 shows short-circuit currents from CF nasal polyp epithelial cells infected with Ad2-ORF6'PGK-CFTR at mrultiplicities of 0.3, 3, and 50. At the indicated times: 10 pM arniloride, cAMP agonists (10 VM forskolin and 100 p.M IBMX, and 1 mM diphenylamine-7-carboxylate were added to the mucosal solution.

Figures 3QA-30C show summaries of the clinical signs (or lack thereof) of infection with Ad2-ORF6/?GK-CFTR.

Figures 3 1 A-31IC show a summary of blood counts, sedimentation rate, and clinical chemistries after infection with Ad2-ORF6/?GK-CFTR for monkeys C, D, and E. There was no evidence of a systemic inflarrinatory response or other abnormalities of the clinical chemistries.

Figures 32A-32C show summaries of white blood cells counts in monkeys C, D, and E after infection with Ad2-ORF6IPGK-CFTR. These data indicate that the administration of Ad2-ORF6/PGK-CFTR caused no change in the distribution and number of inflammatory cells at any of the time points following viral administration.

3- 2-98fl11!58 #2/7 12/

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Figures 33A-33C show antibody titers to adcnovirus prior to and aftcr the first and second administrations of Ad2-ORF6PGK-CFTR. Prior to administration of Ad2-OPF6/PGK- MFR, the monkeys had received instillations of Ad2ICFTR- 1. Antibody titers measured by ELISA rose within one week after the first and second administration of Ad2-QRF6IPGK-CFTFl Serum neutralizing antibodies also rose within a week after viral admninistration and peaked at day 24. No anti-adenoviral antibodies were detected by ELISA or neutralizing assay in nasal washings of any of the monkeys.

Detailed Descdption anqd Best Mode As used herein, the phrase "gene therapy" refers to the vrsfer of genetic material (e.g.,'DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition. The genetic material of intmrst encodes a product a protein polypeptide, peptide or functional RN4A) whose production in vivo is desired. For example, the genetic miaterial of interest can encode a hormone, receptor, enzyme or (poly) peptide of therapeutic value. Examples of genetic material of interest include DNA encoding: the cystic fibrosis transuaerbrane regulator (CFTR), Factor VII low de nsity lipoprotein receptor, betagalaciosidase, alpba-galacrosidease, bca-guocerebroidise~insuizi, OarathYroid hormone, and alpha-lI-mntitrypsin.

Although The potential for gene therapy to treat genetic diseases has been appreciated for marny years, it is only recently that such approaches have become practical with the ,teatment of two patients with adenosine dcanidase defiiency. The protocol consists of removing lymnphocytes from the patients, simrulating themn to'grow in tissue cultue, ifecting them with an appropriately engineered retrovirus followed by reintroduction of the cells into the patient (Kantoff, P. et al. (1987) J Exz. )ded 166:219). Iniial results, of treatment are very encoura.ging. With the approval of a rrirber of other human gene therapy protocols for limited clinuical use, and with the demnonstraition of the feasibility of complementing the CF defect by gene transfer, gene therapy for CF appears a very viable option.

The concept of gene replacement therapy for cystic fibrosis is very simple; a preparation of CFTR coding sequenc es in some suitable vector in a viral or other carrier delivered directly to the airways of CF patients-. Since disease of the pulmonary airways is the major cause of morbidity and is responsible for 95% of mortality, airway epitbelial cells are preferred target cells for CF gone therapy. The first gencration of CF gone terapy is likely to be transient and to require repeated delivery to the airways. Eventually, however, gene therapy may offer a cure for CF when the identity of the precursor or stemn cellI to air epithelia] cells becomes known. If DNA were incorporated into airway stern cells, all subsequent generations of such cells would make authentic CFTh from the integrated sequences and would correct the physiological defect almost irrespective of the biochemical basis of the action of CFTR.

00 0~ 12- Although simple in concept, scientific and clinical problems face approaches to gene therapy, not least of these being that CF requires an in vivo approach while all gene therapy treatments in humans to date have involved ex vivo treatment of cells taken from the patient followed by reintroduction.

One major obstacle to be overcome before gene therapy becomes a viable treatment approach for CF is the development of appropriate vectors to infect tissue manifesting the disease and deliver the therapeutic CFTR gene. Since viruses have evolved very efficient means to introduce their nucleic acid into cells, many approaches to gene therapy make use of engineered defective viruses. However, the use of viruses in vivo raises safety concerns.

Although potentially safer, the use of simple DNA plasmid constructs containing minimal additional DNA, on the other hand, is often very inefficient and can result in transient protein ~expression.

The integration of introduced DNA into the host chromosome has advantages in that such DNA will be passed to daughter cells. In some circumstances, integrated DNA may 15 also lead to high or more sustained expression. However, integration often, perhaps always, requires cellular DNA replication in order to occur. This is certainly the case with the present generation of retroviruses. This limits the use of such viruses to circumstances where cell division occurs in a high proportion of cells. For cells cultured in vitro, this is seldom a problem, however, the cells of the airway are reported to divide only infrequently (Kawanami, O. et al. (1979) An. Rev. Respir. Dis. 120:595). The use ofretroviruses in CF will probably require damaging the airways (by agents such as SO 2 or 03) to induce cell division. This may prove impracticable in CF patients.

Even if efficient DNA integration could be achieved using viruses, the human genome contains elements involved in the regulation of cellular growth only a small fraction of which 25 are presently identified. By integrating adjacent to an element such as a proto-oncogene or an anti-oncogene, activation or inactivation of that element could occur leading to uncontrolled growth of the altered cell. It is considered likely that several such activation/inactivation steps are usually required in any one cell to induce uncontrolled proliferation (R.A.Weinberg (1989) Cancer Research 49:3713 which may reduce somewhat the potential risk. On the other hand, insertional mutagenesis leading to tumor formation is certainly known in animals with some nondefective retroviruses Weinberg, supra; Payne, G.S. et al. (1982) Nature 295:209), and the large numbers of potential integrations occurring during the lifetime of a patient treated repeatedly in vivo with retroviruses must raise concerns on the safety of such a procedure.

In addition to the potential problems associated with viral DNA integration, a number of additional safety issues arise. Many patients may have preexisting antibodies to some of the viruses that are candidates for vectors, for example, adenoviruses. In addition, repeated use of such vectors might induce an immune response. The use of defective viral vectors 13may alleviate this problem somewhat, because the vec ors will not lead to productive viral life cycles generating infected cells, cell lysis or large numnbers of progeny viruses.

Other issues associated with the use of viruses are the possibility of recombination with related viruses naturally infecting the treated patient, complementation of the viral defects by simultaneous expression of wild type virus proteins and containment of aerosols of the engineered viruses.

Gene therapy approaches to CF will face many of the same clinical challenges at protein therapy. These include the inaccessibility of airway epithelium caused by mucus build-up and the hostile nature of the environment in CF airways which may inactivate viruses/vectors. Elements of the vector carriers may be immunogenic and introduction of the DNA may be inefficient. These problems, as with protein therapy, are exacerbated by the absence of a good animal model for the disease nor a simple clinical end point to measure the efficacy oftreatment.

15 CF Gene Therapy Vectors Possible Options Retroviruses Although defective retroviruses are the best characterized system and so far the only one approved for use in human gene therapy (Miller, A.D. (1990) Blood 76:271), the major issue in relation to CF is the requirement for dividing cells to achieve DNA integration and gene expression. Were conditions found to induce airway cell division, the in vivo application of retroviruses, especially if repeated over many years, would •necessitate assessment of the safety aspects of insertional mutagenesis in this context.

Adeno-Associated Virus (AAV) is a naturally occurring defective virus that requires 25 other viruses such as adenoviruses or herpes viruses as helper viruses(Muzyczka, N. (1992) in Current Topics in Microbiology and Immunology 158:97). It is also one of the few viruses that may integrate its DNA into non-dividing cells, although this is not yet certain. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. CFTR DNA may be towards the upper limit of packaging. Furthermore, the packaging process itself is presently inefficient and safety issues such as immunogenecity, complementation and containment will also apply to AAV.

Nevertheless, this system is sufficiently promising to warrant further study.

Plasmid DNA Naked plasmid can be introduced into muscle cells by injection into the tissue. Expression can extend over many months but the number of positive cells is low (Wolff, J. et al. (1989) Science 247:1465). Cationic lipids aid introduction of DNA into some cells in culture (Felgner, P. and Ringold, G.M. (1989) Nature 337:387). Injection of cationic lipid plasmid DNA complexes into the circulation of mice has been shown to result in expression of the DNA in lung (Brigham, K. et al. (1989) Am. J. Med. Sci. 298:278).

-14- Instillation of cationic lipid plasmid DNA into lung also leads to expression in epithelial cells but the efficiency of expression is relatively low and transient (Hazinski, T.A. et al. (1991) Am. J. Respir., Cell Mol. Biol. 4:206). One advantage of the use of plasmid DNA is that it can be introduced into non-replicating cells. However, the use of plasmid DNA in the CF airway environment, which already contains high concentrations of endogenous DNA may be problematic.

Receptor Mediated Entry In an effort to improve the efficiency ofplasmid DNA uptake, attempts have been made to utilize receptor-mediated endocytosis as an entry mechanisms and to protect DNA in complexes with polylysine (Wu, G. and Wu, C.H. (1988) J. Biol. Chem. 263:14621). One potential problem with this approach is that the incoming plasmid DNA enters the pathway leading from endosome to lysosome, where much incoming material is degraded. One solution to this problem is the use of transferrin DNA-polylysine complexes linked to adenovirus capsids (Curiel, D.T. et al. (1991) Proc. Natl. Acad. Sci. USA 15 88:8850). The latter enter efficiently but have the added advantage of naturally disrupting the "endosome thereby avoiding shuttling to the lysosome. This approach has promise but at present is relatively transient and suffers from the same potential problems of immunogenicity as other adenovirus based methods.

Adenovirus Defective adenoviruses at present appear to be a promising approach to CF gene therapy (Berkner, K.L. (1988) BioTechniques 6:616). Adenovirus can be manipulated such that it encodes and expresses the desired gene product, CFTR), and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle.

In addition, adenovirus has a natural tropism for airway epithelia. The viruses are able to 25 infect quiescent cells as are found in the airways, offering a major advantage over retroviruses. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis.

Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz, A.R. et al. (1974) Am. Rev. Respir. Dis. 109:233-238).

Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155).

Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Nail. Acad. Sci. USA 76:6606).

The following properties would be desirable in the design of an adenovirus vector to transfer the gene for CFTR to the airway cells of a CF patient. The vector should allow sufficient expression of the CFTR, while producing minimal viral gene expression. There should be minimal viral DNA replication and ideally no virus replication. Finally, .1VV recombination to produce new viral sequences and complementation to allow growth of the defective virus in the patient should be minimized. A first generation adenovirus vector encoding CFTR (Ad2/CFTR), made as described in the following Example 7, achieves most of these goals and was used in the human trials described in Example Figure 14 shows a map of Ad2/CFTR-1. As can be seen from the figure, this first generation virus includes viral DNA derived from the common relatively benign adenovirus 2 serotype. The Ela and Elb regions of the viral genome, which are involved in early stages of viral replication have been deleted. Their removal impairs viral gene expression and viral replication. The protein products of these genes also have immortalizing and transforming function in some non-permissive cells.

The CFTR coding sequence is inserted into the viral genome in place of the Ela/Elb region and transcription of the CFTR sequence is driven by the endogenous Ela promoter.

This is a moderately strong promoter that is functional in a variety of cells. In contrast to some adenovirus vectors (Rosenfeld, M. et al. (1992) Cell 68:143), this adenovirus retains 15 the E3 viral coding region. As a consequence of the inclusion of E3, the length of the adenovirus-CFTR DNA is greater than that of the wild-type adenovirus. The greater length of the recombinant viral DNA renders it more difficult to package. This means that the growth of the Ad2/CFTR virus is impaired even in permissive cells that provide the missing Ela and Elb functions.

The E3 region of the Ad2/CFTR-1 encodes a variety of proteins. One of these proteins, gpl9, is believed to interact with and prevent presentation of class I proteins of the major histocompatability complex (MHC) (Gooding, C.R. and Wold, W.S.M. (1990) Crit.

Rev. Immunol. 10:53). This property prevents recognition of the infected cells and thus may allow viral latency. The presence of E3 sequences, therefore, has two useful attributes; first, 25 the large size of the viral DNA renders it doubly defective for replication it lacks early functions and is packaged poorly) and second, the absence of MHC presentation could be useful in later applications of Ad2/CFTR-1 in gene therapy involving multiple administrations because it may avoid an immune response to recombinant virus containing cells.

Not only are there advantages associated with the presence of E3; there may be disadvantages associated with its absence. Studies of E3 deleted virus in animals have suggested that they result in a more severe pathology (Gingsberg, H.S. et al. (1989) Proc.

Natl. Acad. Sci. (USA) 86:3823). Furthermore, E3 deleted virus, such as might be obtained by recombination of an El plus E3 deleted virus with wild-type virus, is reported to outgrow wild-type in tissue culture (Barkner, K.L. and Sharp, P. (1983) Nucleic Acids Research 11:6003). By contrast, however, a recent report of an E3 replacement vector encoding hepatitis B surface antigen, suggests that when delivered as a live enteric vaccine, such a virus replicates poorly in human compared to wild-type.

-16- The adenovirus vector (Ad2/CFTR-1) and a related virus encoding the marker 0galactosidase (Ad2/p-gal) have been constructed and grown in human 293 cells. These cells contain the El region of adenovirus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. Because the size of its genome is greater than that of wild-type virus, Ad2/CFTR is relatively difficult to produce.

The Ad2/CFTR-I virus has been shown to encode CFTR by demonstrating the presence of the protein in 293 cells. The Ad2/p-gal virus was shown to produce its protein in a variety of cell lines grown in tissue culture including a monkey bronchiolar cell line (4MBR-5), primary hamster tracheal epithelial cells, human HeLa, human CF PAC cells (see Example 8) and airway epithelial cells from CF patients (Rich, O. et al. (1990) Nature 347:358).

Ad2/CFTR-I is constructed from adenovirus 2 (Ad2) DNA sequences. Other varieties of adenovirus Ad3, Ad5, and Ad7) may also prove useful as gene therapy 15 vectors. This may prove essential if immune response against a single serotype reduces the effectiveness of the therapy.

Second Generation Adenoviral Vectors Adenoviral vectors currently in use retain most 80%) of the parental viral genetic material leaving their safety untested and in doubt. Second-generation vector systems containing minimal adenoviral regulatory, packaging and replication sequences have therefore been developed.

Pseudo-Adenovirus Vectors (PAV)-PAVs contain adenovirus inverted terminal 25 repeats and the minimal adenovirus 5' sequences required for helper virus dependent replication and packaging of the vector. These vectors contain no potentially harmful viral genes, have a theoretical capacity for foreign material of nearly 36 kb, may be produced in reasonably high titers and maintain the tropism of the parent virus for dividing and nondividing human target cell types.

The PAV vector can be maintained as either a plasmid-bome construct or as an infectious viral particle. As a plasmid construct, PAV is composed of the minimal sequences from wild type adenovirus type 2 necessary for efficient replication and packaging of these sequences and any desired additional exogenous genetic material, by either a wild-type or defective helper virus.

Specifically, PAV contains adenovirus 2 (Ad2) sequences as shown in Figure 17, from nucleotide (nt) 0-356 forming the 5' end of the vector and the last 109 nt of Ad2 forming the 3' end of the construct. The sequences includes the Ad2 flanking inverted terminal repeats (5'ITR) and the 5' ITR adjoining sequences containing the known packaging signal and Ela enhancer. Various convenient restriction sites have been incorporated into the -17fragments, allowing the insertion ofpromoter/gene cassettes which can be packaged in the PAV virion and used for gene transfer for gene therapy). The construction and propagation of PAV is described in detail in the following Example 11. By not containing most native adenoviral DNA, the PAVs described herein are less likely to produce a patient immune reponse or to replicate in a host.

In addition, the PAV vectors can accomodate foreign DNA up to a maximum length of nearly 36 kb. The PAV vectors therefore, are especially useful for cloning larger genes CFTR (7.5 Factor VIII (8 kb); Factor IX (9 which, traditional vectors have difficulty accomodating. In addition, PAV vectors can be used to transfer more than one gene, or more than one copy of a particular gene. For example, for gene therapy of cystic fibrosis, PAVs can be used to deliver CFTR in conjunction with other genes such as anti proteases antiprotease alpha-i -antitrypsin) tissue inhibitor of metaloproteinase, antioxidants superoxide dismutase), enhancers of local host defense interferons), mucolytics DNase); and proteins which block inflammatory cytokines.

.15 Ad2-E4/ORF6 Adenovirus Vectors An adenoviral construct expressing only the open reading frame 6 (ORF6) of adenoviral early region 4 (E4) from the E4 promoter and which is deleted for all other known E4 open reading frames was constructed as described in detail in Example 12. Expression of E4 open reading frame 3 is also sufficient to provide E4 functions required for DNA replication and late protein synthesis. However, it provides these functions with reduced efficiency compared to expression of ORF6, which will likely result in lower levels of virus production. Therefore expressing ORF6, rather than ORF3, appears to be a better choice for producing recombinant adenovirus vectors.

25 The E4 region of adenovirus is suspected to have a role in viral DNA replication, late mRNA synthesis and host protein synthesis shut off, as well as in viral assembly (Falgout, B.

and G. Ketner (1987) J. Virol. 61:3759-3768). Adenovirus early region 4 is required for efficient virus particle assembly. Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. Halbert, D.N. et al.

(1985) J. Virol. 56:250-257.

The deletion of non-essential open reading frames of E4 increases the cloning capacity of recombinant adenovirus vectors by approximately 2 kb of insert DNA without significantly reducing the viability of the virus in cell culture. When placed in combination with deletions in the El and/or E3 regions of adenovirus vectors, the theoretical insert capacity of the resultant vectors is increased to 8-9 kb. An example of where this increased cloning capacity may prove useful is in the development of a gene therapy vector encoding CFTR. As described above, the first generation adenoviral vector approaches the maximum packaging capacity for viral DNA encapsidation. As a result, this virus grows poorly and may occassionaly give rise to defective progeny. Including an E4 deletion in the adenovirus S-18vector should alleviate these problems. In addition, it allows flexibility in the choice of promoters to drive CFTR expression from the virus. For example, strong promoters such as the adenovirus major late promoter, the cytomegalovirus immediate early promoter or a cellular promoter such as the CFTR promoter, which may be too large for first-generation adenovirus can be used to drive expression.

In addition, by expressing only ORF6 ofE4, these second generation adenoviral vectors may be safer for use in gene therapy. Although ORF6 expression is sufficient for viral DNA replication and late protein synthesis in immortalized cells, it has been suggested that ORF6/7 of E4 may also be required in non-dividing primary cells (Hemstrom, C. et al.

(1991) J. Virol. 65:1440-1449). The 19 kD protein produced from open reading frame 6 and 7 (ORF6/7) complexes with and activates cellular transcription fact6r E2F, which is required for maximal activation of early region 2. Early region 2 encodes proteins required for viral DNA replication. Activated transcription factor E2F is present in proliferating cells and is involved in the expression of genes required for cell proliferation DHFR, c-myc), whereas activated E2F is present in lower levels in non-proliferating cells. Therefore, the expression of only ORF6 of E4 should allow the virus to replicate normally in tissue culture cells 293 cells), but the absence of ORF6/7 would prevent the potential activation of i" transcription factor E2F in non-dividing primary cellls and thereby reduce the potential for viral DNA replication.

Target Tissue Because 95% of CF patients die of lung disease, the lung is a preferred target for gene therapy. The hallmark abnormality of the disease is defective electrolyte transport by the epithelial cells that line the airways. Numerous investigators (reviewed in Quinton, F. (1990) 25 FASEB J. 4:2709) have observed: a) a complete loss of cAMP-mediated transepithelial chloride secretion, and b) a two to three fold increase in the rate of Na+ absorption. cAMPstimulated chloride secretion requires a chloride channel in the apical membrane (Welsh, M.J.

(1987) Physiol Rev. 67:1143-1184). The discovery that CFTR is a phosphorylation-regulated chloride channel and that the properties of the CFTR chloride channel are the same as those of the chloride channels in the apical membrane, indicate that CFTR itself mediates transepithelial chloride secretion. This conclusion was supported by studies localizing CFTR in lung tissue: CFTR is located in the apical membrane of airway epithelial cells (Denning, G.M. et al. (1992) J. Cell Biol. 118:551) and has been reported to be present in the submucosal glands (Taussig et al., (1973) J. Clin. Invest. 89:339). As a consequence of loss of CFTR function, there is a loss of cAMP-regulated transepithelial chloride secretion. At this time it is uncertain how dysfunction of CFTR produces an increase in the rate of Naabsorption. However, it is thought that the defective chloride secretion and increased Na+ absorption lead to an alteration of the respiratory tract fluid and hence, to defective mucociliary clearance, a normal pulmonary defense mechanism. As a result, clearance of -19inhaled material from the lung is impaired and repeated infections ensue. Although the presumed abnormalities in respiratory tract fluid and mucociliary clearance provide a plausible explanation for the disease, a precise understanding of the pathogenesis is still lacking.

Correction of the genetic defect in the airway epithelial cells is likely to reverse the CF pulmonary phenotype. The identity of the specific cells in the airway epithelium that express CFTR cannot be accurately determined by immunocytochemical means, because of the low abundance of protein. However, functional studies suggest that the ciliated epithelial cells and perhaps nonciliated cells of the surface epithelium are among the main cell types involved in electrolyte transport. Thus, in practical terms, the present preferred target cell for gene therapy would appear to be the mature cells that line the pulmonary airways. These are S. not rapidly dividing cells; rather, most of them are nonproliferating and many may be terminally differentiated. The identification of the progenitor cells in the airway is uncertain.

Although CFTR may also be present in submucosal glands (Trezise, A.E. and Buchwald, M.

15 (1991) Nature 353:434; Englehardt, J.F. et al. (1992)J. Clin. Invest. 90:2598-2607), there is no data as to its function at that site; furthermore, such glands appear to be relatively inaccessible.

The airway epithelium provides two main advantages for gene therapy. First, access to the airway epithelium can be relatively noninvasive. This is a significant advantage in the development of delivery strategies and it will allow investigators to monitor the therapeutic Sresponse. Second, the epithelium forms a barrier between the airway lumen and the interstitium. Thus, application of the vector to the lumen will allow access to the target cell yet, at least to some extent, limit movement through the epithelial barrier to the interstitium and from there to the rest of the body.

Efficiency of Gene Delivery Required to Correct The Genetic Defect It is unlikely that any gene therapy protocol will correct 100% of the cells that normally express CFTR. However, several observations suggest that correction of a small percent of the involved cells or expression of a fraction of the normal amount of CFTR may be of therapeutic benefit.

a. CF is an autosomal recessive disease and heterozygotes have no lung disease.

Thus, 50% of wild-type CFTR would appear sufficient for normal function.

b. This issue was tested in mixing experiments using CF cells and recombinant CF cells expressing wild-type CFTR (Johnson, L.G. et al. (1992) Nature Gen. 2:21). The data obtained showed that when an epithelium is reconstituted with as few as 6-10% of corrected cells, chloride secretion is comparable to that observed with an epithelium containing 100% corrected cells. Although CFTR expression in the recombinant cells is probably higher than in normal cells, this result suggests that in vivo correction of all CF airway cells may not be required.

c. Recent observations show that CFTR containing some CF-associated mutations retains residual chloride channel activity (Sheppard, D.N. et al. (1992) Pediatr.

Pulmon Suppl. 8:250; Strong, T.V. et al. (1991) N. Eng. J. Med. 325:1630). These mutations are associated with mild lung disease. Thus, even a very low level of CFTR activity may at least partly ameliorate the electrolyte transport abnormalities.

d. As indicated in experiments described below in Example 8, complementation of CF epithelia, under conditions that probably would not cause expression of CFTR in every cell, restored cAMP stimulated chloride secretion.

Levels of CFTR in normal human airway epithelia are very low and are barely detectable. It has not been detected using routine biochemical techniques such as immunoprecipitation or immunoblotting and has been exceedingly difficult to detect with immunocytochemical techniques (Denning, G.M. et al. (1992) J. Cell Biol. 118:551).

Although CFTR has been detected in some cases using laser-scanning confocal microscopy, the signal is at the limits of detection and cannot be detected above background in every case.

Despite that minimal levels of CFTR, this small amount is sufficient to generate substantial cAMP-stimulated chloride secretion. The reason that a very small number of CFTR chloride channels can support a large chloride secretory rate is that a large number of ions can pass through a single channel (106-107 ions/sec) (Hille, B. (1984) Sinauer Assoc. Inc., Sunderland, MA 420-426).

f. Previous studies using quantitative PCR have reported that the airway epithelial cells contain at most one to two transcripts per cell (Trapnell, B.C. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6565).

Gene therapy for CF would appear to have a wide therapeutic index. Just as partial expression may be of therapeutic value, overexpression of wild-type CFTR appears unlikely to cause significant problems. This conclusion is based on both theoretical considerations and experimental results. Because CFTR is a regulated channel, and because it has a specific function in epithelia, it is unlikely that overexpression of CFTR will lead to uncontrolled chloride secretion. First, secretion would require activation of CFTR by cAMP-dependent phosphorylation. Activation of this kinase is a highly regulated process. Second, even if CFTR chloride channels open in the apical membrane, secretion will not ensue without regulation of the basolateral membrane transporters that are required for chloride to enter the cell from the interstitial space. At the basolateral membrane, the sodium-potassium-chloride -21 cotransporter and potassium channels serve as important regulators of transeptihelial secretion (Welsh, M.J. (1987) Physiol. Rev. 67:1143-1184).

Human CFTR has been expressed in transgenic mice under the control of the surfactant protein C(SPC) gene promoter (Whitesett, J.A. et al. (1992) Nature Gen. 2:13) and the casein promoter (Ditullio, P. et al (1992) Bio/Technology 10:74). In those mice, CFTR was overexpressed in bronchiolar and alveolar epithelial cells and in the mammary glands, respectively. Yet despite the massive overexpression in the transgenic animals, there were no observable morphologic or functional abnormalities. In addition, expression of CFTR in the lungs of cotton rats produced no reported abnormalities (Rosenfeld, M.A. et al. (1992) Cell 68:143-155).

The present invention is further illustrated by the following examples which in no way should be construed as being further limiting. The contents of all cited references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLES

Example 1 Generation of Full Length CFTR cDNAs Nearly all of the commonly used DNA cloning vectors are based on plasmids containing modified pMB I replication origins and are present at up to 500 to 700 copies per cell (Sambrook et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989). The partial CFTR cDNA clones isolated by Riordan et al. were maintained in such a plasmid. It was postulated that an alternative theory to intrinsic clone 25 instability to explain the apparent inability to recover clones encoding full length CFTR protein using high copy number plasmids, was that it was not possible to clone large segments of the CFTR cDNA at high gene dosage in E. coli. Expression of the CFTR or portions of the CFTR from regulatory sequences capable of directing transcription and/or translation in the bacterial host cell might result in inviability of the host cell due to toxicity of the transcript or of the full length CFTR protein or fragments thereof. This inadvertent gene expression could occur from either plasmid regulatory sequences or cryptic regulatory sequences within the recombinant CFTR plasmid which are capable of functioning in E. coli.

Toxic expression of the CFTR coding sequences would be greatly compounded if a large number of copies of the CFTR cDNA were present in cells because a high copy number plasmid was used. If the product was indeed toxic as postulated, the growth of cells containing full length and correct sequence would be actively disfavored. Based upon this novel hypothesis, the following procedures were undertaken. With reference to Figure 2, partial CFTR clone T16-4.5 was cleaved with restriction enzymes SPh and Psi I and the resulting 3.9 kb restriction fragment containing exons 11 through most of exon 24 (including -22an uncharacterized 119 bp insertion reported by Riordan et al. between nucleotides 1716 and 1717), was isolated by agarose gel purification and ligated between the Sph and PstlI sites of the pMBI based vector pkk223-3 (Brosius and Holy, (1984) Proc. Natl. Acad. Sci.

81:6929). It was hoped that the pMB I origin contained within this plasmid would allow it and plasmids constructed from it to replicate at 15-20 copies per host E. coli cell (Sambrook et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989). The resultant plasmid clone was called Partial CFTR clone T11 was cleaved with EO R1 and Hinc I and the 1.9 kb band encoding the first 1786 nucleotides of the CFTR cDNA plus an additional 100 bp of DNA at the 5' end was isolated by agarose gel purification. This restriction fragment was inserted between the Eco Rl site and Sma I restriction site of the plamid Bluescript Sk- (Stratagene, catalogue number 212206), such that the CFTR sequences were now flanked on the upstream side by a Sal 1 site from the cloning vector. This clone, designated T 11-R, was cleaved with Sal 1 and Sh 1 and the resultant 1.8 kb band isolated by agarose gel purification.

15 Plasmid pkk-4.5 was cleaved with Sal and Sph 1 and the large fragment was isolated by agarose gel purification. The purified T11-R fragment and pkk-4.5 fragments were ligated to construct pkk-CFTR1. pkk-CFTR1 contains exons 1 through 24 of the CFTR cDNA. It was discovered that this plasmid is stably maintained in E. coli cells and confers no measureably disadvantageous growth characteristics upon host cells.

pkk-CFTR1 contains, between nucleotides 1716 and 1717, the 119 bp insert DNA derived from partial cDNA clone T16-4.5 described above. In addition, subsequent sequence analysis of pkk-CFTR1 revealed unreported differences in the coding sequence between that portion of CFTRI derived from partial cDNA clone TI 1 and the published CFTR cDNA sequence. These undesired differences included a 1 base-pair deletion at position 995 and a 25 C to T transition at position 1507.

To complete construction of an intact correct CFTR coding sequence without mutations or insertions and with reference to the construction scheme shown in Figure 3, pkk-CFTR1 was cleaved with Xba 1 and Hp I, and dephosphorylated with calf intestinal alkaline phosphatase. In addition, to reduce the likelihood of recovering the original clone, the small unwanted Xba I/ipa I restriction fragment from pKK-CFTRI was digested with Sp-hl. T16-1 was cleaved with Xba I and AceI and the 1.15 kb fragment isolated by agarose gel purification. TI 6-4.5 was cleaved with Acc I and Hpa I and the 0.65 kb band was also isolated by agarose gel purification. The two agarose gel purified restriction fragments and the dephosphorylated pKK-CFTRI were ligated to produce pKK-CFTR2. Alternatively, pKK-CFTR2 could have been constructed using corresponding restriction fragments from the partial CFTR cDNA clone C1-1/5. pKK-CFTR2 contains the uninterrupted CFTR protein coding sequence and conferred slow growth upon E. coli host cells in which it was inserted, whereas pKK-CFTRI did not. The origin of replication of pKK-CFTR2 is derived from pMB 1 and confers a plasmid copy number of 15-20 copies per host cell.

23- Example 2 Improving Host Cell Viability An additional enhancement of host cell viability was accomplished by a further reduction in the copy number of CFTR cDNA per host cell. This was achieved by transferring the CFTR cDNA into the plasmid vector, pSC-3Z. pSC-3Z was constructed using the pSC 101 replication origin of the low copy number plasmid pLG338 (Stoker et al., Gene J8, 335 (1982)) and the ampicillin resistance gene and polylinker ofpGEM-3Z (available from Promega). pLG338 was cleaved with Sph I and Pvu II and the 2.8 kb fragment containing the replication origin isolated by agarose gel purification. pGEM-3Z was cleaved with Alw N, the resultant restriction fragment ends treated with T4 DNA polymerase and deoxynucleotide triphosphates, cleaved with Sph I and the 1.9 kb band containing the ampicillin resistance gene and the polylinker was isolated by agarose gel purification. The pLG338 and pGEM-3Z fragments were ligated together to produce the low copy number cloning vector pSC-3Z. pSC-3Z and other plasmids containing pSC101 origins of replication are maintained at approximately five copies per cell (Sambrook et aL supra).

With additional reference to Figure 4, pKK-CFTR2 was cleaved with Ec RV, PEs I and Sal I and then passed over a Sephacryl S400 spun column (available from Pharmacia) according to the manufacturer's procedure in order to remove the SalU to Eco RV restriction fragment which was retained within the column. pSC-3Z was digested with Sma I and EP I and also passed over a Sephacryl S400 spun column to remove the small Sma 1/PTs I restriction fragment which was retained within the column. The column eluted fractions from the pKK-CFTR2 digest and the pSC-3Z digest were mixed and ligated to produce pSC- CFTR2. A map of this plasmid is presented in Figure 5. Host cells containing CFTR cDNAs at this and similar gene dosages grow well and have stably maintained the recombinant 25 plasmid with the full length CFTR coding sequence. In addition, this plasmid contains a bacteriophage T7 RNA polymerase promoter adjacent to the CFTR coding sequence and is therefore convenient for in vitro transcription/translation of the CFTR protein. The nucleotide sequence of CFTR coding region from pSC-CFTR2 plasmid is presented in Sequence Listing 1 as SEQ ID NO:1. Significantly, this sequence differs from the previously published (Riordan, J.R. et al. (1989) Science 245:1066-1073) CFTR sequence at position 1990, where there is C in place of the reported A. See Gregory, R.3. et al. (1990) Nature 347:382-386. E. coli host cells containing pSC-CFTR2, internally identified with the number pSC-CFTR2/AG1, have been deposited at the American Type Culture Collection and given the accession number: ATCC 68244.

Example 3 Alternate Method for Improving Host Cell Viability A second method for enhancing host cell viability comprises disruption of the CFTR protein coding sequence. For this purpose, a synthetic intron was designed for insertion between nucleotides 1716 and 1717 of the CFTR cDNA. This intron is especially -24advantageous because of its easily manageable size. Furthermore, it is designed to be efficiently spliced from CFTR primary RNA transcripts when expressed in eukaryotic cells.

Four synthetic oligonucleotides were synthesized (1195RG, I 196RG, 1197RG and 1198RG) collectively extending from the Sph I cleavage site at position 1700 to the Hin II cleavage site at position 1785 and including the additional 83 nucleotides between 1716 and 1717 (see Figure These oligonucleotides were phosphorylated with T4 polynucleotide kinase as described by Sambrook et al., mixed together, heated to 95°C for 5 minutes in the same buffer used during phosphorylation, and allowed to cool to room temperature over several hours to allow annealing of the single stranded oligonucleotides. To insert the synthetic intron into the CFTR coding sequence and with reference to Figures 7A and 7B, a subclone ofplasmid Tl 1 was made by cleaving the Sal I site in the polylinker, repairing the recessed S. ends of the cleaved DNA with deoxynucleotide triphosphates and the large fragment of DNA Polymerase I and religating the DNA. This plasmid was then digested with EcoE RE and Nru I and religated. The resulting plasmid TI6-A5' extended from the Nru I site at position 490 of the CFTR cDNA to the 3' end of clone T16 and contained single sites for Sh I and Hinc 1 at positions corresponding to nucleotides 1700 and 1785 of the CFTR cDNA. T16-A5' plasmid was cleaved with Sph I and Hinc I and the large fragment was isolated by agarose gel purification. The annealed synthetic oligonucleotides were ligated into this vector fragment to generate TI6-intron.

T16-intron was then digested with EM El and Sma I and the large fragment was .isolated by agarose gel purification. TI6-4.5 was digested with Eco I and Sca I and the 790 bp fragment was also isolated by agarose gel purification. The purified T16-intron and TI 6fragments were ligated to produce T16-intron-2. T16-intron-2 contains CFTR cDNA sequences extending from the Nru I site at position 490 to the Sea I site at position 2818, and 25 includes the unique Hpa I site at position 2463 which is not present in T16-1 or T16-intron-1.

T-16-intron-2 was then cleaved with Xba I and Hpa I and the 1800 bp fragment was isolated by agarose gel purification. pKK-CFTR1 was digested with Xba I and Hpa I and the large fragment was also isolated by agarose gel purification and ligated with the fragment derived from T16-intron-2 to yield pKK-CFTR3, shown in Figure 8. The CFTR cDNA within pKK-CFTR3 is identical to that within pSC-CFTR2 and pKK-CFTR2 except for the insertion of the 83 bp intron between nucleotides 1716 and 1717. The insertion of this intron resulted in improved growth characteristics for cells harboring pKK-CFTR3 relative to cells containing the unmodified CFTR cDNA in pKK-CFTR2.

Example 4 In vitro Transcription/Translation In addition to sequence analysis, the integrity of the CFTR cDNA open reading frame was verified by in vitro transcription/translation. This method also provided the initial CFTR protein for identification purposes. 5 micrograms of pSC-CFTR2 plasmid DNA were linearized with Sal I and used to direct the synthesis of CFTR RNA transcripts with T7 RNA polymerase as described by the supplier (Stratagene). This transcript was extracted with phenol and chloroform and precipitated with ethanol. The transcript was resuspended in microliters of water and varying amounts were added to a reticulocyte lysate in vitro translation system (Promega). The reactions were performed as described by the supplier in the presence of canine pancreatic microsomal membranes (Promega), using 3 5 S-methionine to label newly synthesized proteins. In vitro translation products were analysed by discontinuous polyacrylamide gel electrophoresis in the presence of 0.1% SDS with 8% separating gels (Laemmii, U.K. (1970) Nature 227:680-685). Before electrophoresis, the in vitro translation reactions were denatured with 3% SDS, 8 M urea and 5% 2 -mercaptoethanol in 0.65 M Tris-HC1, pH 6.8. Following electrophoresis, the gels were fixed in methanol:acetic acid:water (30:10:60), rinsed with water and impregnated with 1 M sodium salicylate. 3 5 S labelled proteins were detected by fluorgraphy. A band of approximately 180 kD was detected, consistent with translation of the full length CFTR insert.

i Example 5 Elimination of Cryptic Regulatory Signals Analysis of the DNA sequence of the CFTR has revealed the presence of a potential E. coli RNA polymerase promoter between nucleotides 748 and 778 which conforms well to the derived consensus sequence for E. coli promoters (Reznikoff and McClure, Maximizing Gene Expression, 1, Butterworth Publishers, Stoneham, MA). If this sequence functions as a promoter functions in E. coli, it could direct synthesis of potentially toxic partial CFTR polypeptides. Thus, an additional advantageous procedure for maintaining plasmids containing CFTR cDNAs in E.coli would be to alter the sequence of this potential promoter such that it will not function in E. coli. This may be accomplished without altering the amino 25 acid sequence encoded by the CFTR cDNA. Specifically, plasmids containing complete or partial CFTR cDNA's would be altered by site-directed mutagenesis using synthetic olignucleotides (Zoller and Smith, (1983) Methods Enzymol. 100:468 More specifically, altering the nucleotide sequence at position 908 from a T to C and at position 774 from an A to a G effectively eliminates the activity of this promoter sequence without altering the amino acid coding potential of the CFTR open reading frame. Other potential regulatory signals within the CFTR cDNA for transcription and translation could also be advantageously altered and/or deleted by the same method.

Futher analysis has identified a sequence extending from nucleotide 908 to 936 which functions efficiently as a transcriptional promoter element in E. coli (Gregory, R.J. et al.

(1990) Nature 347:382-386). Mutation at position 936 is capable of inactivating this promoter and allowing the CFTR cDNA to be stably maintained as a plasmid in E. coli (Cheng, S.H. et al. (1990) Cell 63:827-834). Specifically position 936 has been altered from a C to a T residue without the amino acid sequence encoded by the cDNA being altered.

Other mutations within this regulatory element described in Gregory, R.J. et al. (1990) -26- Nature 347:382-386 could also be used to inactivate the transcriptional promoter activity.

Specifically, the sequence from 908 to 913 (TTGTGA) and from 931 to 936 (GAAAAT) could be altered by site directed mutagenesis without altering the amino acid sequence encoded by the cDNA.

Example 6 Cloning of CFTR in Alternate Host Systems Although the CFTR cDNA displays apparent toxicity in E. coli cells, other types of host cells may not be affected in this way. Alternative host systems in which the entire CFTR cDNA protein encoding region may be maintained and/or expressed include other bacterial species and yeast. It is not possible a priori to predict which cells might be resistant and which might not. Screening a number of different host/vector combinations is necessary to find a suitable host tolerant of expression of the full length protein or potentially toxic fragments thereof.

Example 7 Generation of Adenovirus Vector Encoding CFTR (Ad2/CFTR) 1. DNA preparation Construction of the recombinant Ad2/CFTR-I virus (the sequence of which is shown in Table II and as SEQ ID NO:3) was accomplished as follows: The :CFTR cDNA was excised from the plasmid pCMV-CFTR-936C using restriction enzymes Spel and EcII361. pCMV-CFTR-936C consists of a minimal CFTR cDNA encompassing nucleotides 123-4622 of the published CFTR sequence cloned into the multiple cloning site of pRC/CMV (Invitrogen Corp.) using synthetic linkers. The CFTR cDNA within this plasmid has been completely sequenced. The Spl/EIl361i restriction fragment contains 47 bp of 5' sequence derived from synthetic linkers and the multiple cloning site of the vector.

25 The CFTR cDNA (the sequence of which is shown as SEQ ID NO:1 and the amino acid sequence encoded by the CFTR cDNA is shown as SEQ ID NO:2) was inserted between the Nhel and SnaBI restriction sites of the adenovirus gene transfer vector pBR-Ad2-7. pBR- Ad2-7 is a pBR322 based plasmid containing an approximately 7 kb insert derived from the 10680 bp ofAd2 inserted between the Clal and BamHl sites of pBR322. From this Ad2 fragment, the sequences corresponding to Ad2 nucleotides 546-3497 were deleted and replaced with a 12 bp multiple cloning site containing an Nhel site, an Mlul site, and a SnaBI site. The construct also contains the 5' inverted terminal repeat and viral packaging signals, the Ela enhancer and promoter, the Elb 3' intron and the 3' untranslated region and polyadenylation sites. The resulting plasmid was called pBR-Ad2-7/CFTR. Its use to assemble virus is described below.

2. Virus Preparation from DNA To generate the recombinant Ad2/CFTR-l adenovirus, the vector pBR-Ad2-7/CFTR was cleaved with BstBl at the site corresponding to the unique BstB1 site at 10670 in Ad2. The cleaved plamid DNA was ligated to BstB1 restricted Ad2 -27- DNA. Following ligation, the reaction was used to transfect 293 cells by the calcium phosphate procedure. Approximately 7-8 days following transfection, a single plaque appeared and was used to reinfect a dish of 293 cells. Following development of cytopathic effect (CPE), the medium was removed and saved. Total DNA was prepared from the infected cells and analyzed by restriction analysis with multiple enzymes to verify the integrity of the construct. Viral supematant was then used to infect 293 cells and upon delvelopment of CPE, expression of CFTR was assayed by the protein kinase A (PKA) immunoprecipitation assay (Gregory, R.J. et al. (1990) Nature 347:382 Following these verification procedures, the virus was further purified by two rounds of plaque purification.

Plaque purified virus was grown into a small seed stock by inoculation at low multiplicities of infection onto 293 cells grown in monolayers in 925 medium supplemented with 10% bovine calf serum. Material at this stage was designated a Research Viral Seed Stock (RVSS) and was used in all preliminary experiments.

3. Virus Host Cell Ad2/CFTR-l is propagated in human 293 cells (ATCC CRL 1573).

These cells are a human embryonal kidney cell line which were immortalized with sheared fragments of human Ad5 DNA. The 293 cell line expresses adenovirus early region 1 gene products and in consequence, will support the growth of El deficient adenoviruses. By analogy with retroviruses, 293 cells could be considered a packaging cell line, but they differ from usual retrovirus lines in that they do not provide missing viral structural proteins, rather, they provide only some missing viral early functions.

Production lots of virus are propagated in 293 cells derived from the Working Cell Bank (WCB). The WCB is in turn derived from the Master Cell Bank (MCB) which was grown up from a fresh vial of cells obtained from ATCC. Because 293 cells are of human 25 origin, they are being tested extensively for the presence of biological agents. The MCB and WCB are being characterized for identity and the absence of adventitious agents by Microbiological Associates, Rockville, MD.

4. Growth of Production Lots of Virus Production lots of Ad2/CFTR-1 are produced by inoculation of approximately 5-10 x 107 pfu of MVSS onto approximately 1-2 x 10 7 Web 293 cells grown in a T175 flask containing 25 mls of 925 medium. Inoculation is achieved by direct addition of the virus (approximately 2-5 mis) to each flask. Batches of 50-60 flasks constitute a lot.

Following 40-48 hours incubation at 37"C, the cells are shaken loose from the flask and transferred with medium to a 250 ml centrifuge bottle and spun at 1000 xg. The cell pellet is resuspended in 4 ml phosphate buffered saline containing 0.1 g/l CaCI 2 and 0.1 g/l MgCI 2 and the cells subjected to cycles of freeze-thaw to release virus. Cellular debris is removed by centrifugation at 1000 xg for 15 min. The supernatant from this centrifugation is layered on top of the CsCl step gradient: 2 ml 1.4g/ml CsCI and 3 ml 1.25g/ml CsCI in -28mM Tris, 1 mM EDTA (TE) and spun for I hour at 35,000 rpm in a Beckman SW41 rotor.

Virus is then removed from the interface between the two CsCl layers, mixed with 1.35 g/ml CsCI in TE and then subjected to a 2.5 hour equilibrium centrifugation at 75,000 rpm in a TLN-100 rotor. Virus is removed by puncturing the side of the tube with a hypodermic needle and gently removing the banded virus. To reduce the CsCI concentration, the sample is dialyzed against 2 changes of 2 liters of phosphate buffered saline with 10% sucrose.

Following this procedure, dialyzed virus is stable at 4 0 C for several weeks or can be stored for longer periods at -80 0 C. Aliquots of material for human use will be tested and while awaiting the results of these tests, the remainder will be stored frozen. The tests to be performed are described below: 5. Structure and Purity of Virus SSDS polyacrylamide gel electrophoresis of purified virions reveals a number of polypeptides, many of which have been characterized. When preparations of virus were subjected to one or two additional rounds of CsCI centrifugation, the protein profile obtained was indistinguishable. This indicates that additional equilibrium centrifugation does not purify the virus further, and may suggest that even the less intense bands detected in the virus preparations represent minor virion components rather than contaminating proteins. The identity of the protein bands is presently being established by N-terminal sequence analysis.

Contaminating Materials The material to be administered to patients will be 2 x 106 pfu, 2 x 107 pfu and 5 x 107 pfu of purified Ad2/CFTR-1. Assuming a minimum particle to pfu ratio of 500, this corresponds to 1 x 109, 1 x 1010 and 2.5 x 1010 viral particles, these correspond to a dose by mass of 0.25 gg, 2.5pg and 6.25 pg assuming a moleuclar mass for 25 adenovirus of 150 x 106.

The origin of the materials from which a production lot of the purified Ad2/CFTR-l is derived was described in detail above and is illustrated as a flow diagram in Figure 6. All the starting materials from which the purified virus is made MCB, and WCB, and the MVSS) will be extensively tested. Further, the growth medium used will be tested and the serum will be from only approved suppliers who will provide test certificates. In this way, all the components used to generate a production lot will have been characterized. Following growth, the production lot virus will be purified by two rounds of CsCI centrifugation, dialyzed, and tested. A production lot should constitute 1-5 x 1010 pfu Ad2/CFTR-1.

As described above, to detect any contaminating material aliquots of the production lot will be analyzed by SDS gel electrophoresis and restriction enzyme mapping. However, these tests have limited sensitivity. Indeed, unlike the situation for purified single chain recombinant proteins, it is very difficult to quantitate the purity of the AD2/CFTR-1 using SDS polyacrylamide gel electrophoresis (or similar methods). An alternative is the immunological detection of contaminating proteins (IDCP). Such an assay utilizes antibodies -29raised against the proteins purified in a mock purification run. Development of such an assay has not yet been attempted for the CsCI purification scheme for Ad2/CFTR-l. However, initially an IDCP assay developed for the detection of contaminants in recombinant proteins produced in Chinese hamster ovary (CHO) cells will be used. In addition, to hamster proteins, these assays detect bovine serum albumin (BSA), transferrin and IgG heavy and light chain derived from the serum added to the growth medium. Tests using such reagents to examine research batches of Ad2/CFTR-l by both ELISA and Western blots are in progress.

Other proteins contaminating the virus preparation are likely to be from the 293 cells that is, of human origin. Human proteins contaminating therapeutic agents derived from human sources are usually not problematic. In this case, however, we plan to test the production lot for transforming factors. Such factors could be activities of contaminating human proteins or of the Ad2/CFTR-l vector or other contaminating agents. For the test, it is proposed that 10 dishes of Rat 1 cells containing 2 x 106 cells (the number of target cells in tthe patient) with 4 times the highest human dose of Ad2/CFTR-l (2 x 108 pfu) will be 15 infected. Following infection, the cells will be plated out in agar and examined for the o. appearance of transformed foci for 2 weeks. Wild type adenovirus will be used as a control.

Nucleic acids and proteins would be expected to be separated from purified virus preparations upon equilibrium density centrifugation. Furthermore, the 293 cells are not expected to contain VL30 sequences. Biologically active nucleic cells should be detected.

Example 8 Preliminary Experiments Testing the Ability of Ad2/Gal or Ad2/CFTR Virus to Enter Airway Epithelial Cells a. Hamster Studie 25 Initial studies involving the intratracheal instillation of the Ad-pGal viral vector into Syrian hamsters, which are reported to be permissive for human adenovirus are being performed. The first study, a time course assessment of the pulmonary and systemic acute inflammatory response to a single intratracheal administration of Ad-pGal viral vector, has been completed. In this study, a total of 24 animals distributed among three treatment groups, specifically, 8 vehicle control, 8 low dose virus (1 x 1011 particles; 3 x 108 pfu), and 8 high dose virus (1.7 x 1012 particles; 5 x 109 pfu), were used. Within each treatment group, 2 animals were analyzed at each of four time points after viral vector instillation: 6 hrs, 24 hrs, 48 hrs, and 7 days. At the time of sacrifice of each animal, lung lavage and blood samples were taken for analysis. The lungs were fixed and processed for normal light-level histology. Blood and lavage fluid were evaluated for total leukocyte count and leukocyte differential. As an additional measure of the inflammatory process, lavage fluid was also evaluated for total protein. Following embeddings, sectioning and hematoxylin/eosin staining, lung sections were evaluated for signs of inflammation and airway epithelial damage.

I r With the small sample size, the data from this preliminary study were not amenable to statistical analyses, however, some general trends could be ascertained. In the peripheral blood samples, total leukocyte counts showed no apparent dose- or time- dependent changes.

In the blood leukocyte differential counts, there may have been a minor dose-related elevation in percent neutrophil at 6 hours; however, data from all other time points showed no elevation in neutrophil percentages. Taken together, these data suggest little or nor systemic inflammatory response to the viral administration.

From the lung lavage, some elevation in total neutrophil counts were observed at the first three time points (6 hr, 24 hr, 48 hr). By seven days, both total and percent neutrophil values had returned to normal range. The trends in lung lavage protein levels were more difficult to assess due to inter-animal variability; however, no obvious dose- or timedependent effects were apparent. First, no damage to airway epithelium was observed at any time point or virus dose level. Second, a time- and dose- dependent mild inflammatory response was observed, being maximal at 48 hr in the high virus dose animals. By seven days, the inflammatory response had completely resolved, such that the lungs from animals in all treatment groups were indistinguishable.

In summary, a mild, transient, pulmonary inflammatory response appears to be associated with the intratracheal administration of the described doses of adenoviral vector in the Syrian Hamster.

A second, single intratracheal dose, hamster study has been initiated. This study is designed to assess the possibility of the spread of ineffective viral vectors to organs outside of the lung and the antibody response of the animals to the adenoviral vector. In this study, the three treatment groups (vehicle control, low dose virus, high dose virus) each contained 12 animals. Animals will be evaluated at three time points: 1 day, 7 days, and 1 month. In this SS 25 study, viral vector persistence and possible spread will be evaluated by the assessment of the presence of infective virions in numerous organs including lung, gut, heart, liver, spleen, kidney, brain and gonads. Changes in adenoviral antibody titer will be measured in peripheral blood and lung lavage. Additionally, lung lavage, peripheral blood and lung histology will be evaluated as in the previous study.

b. Primate studies.

Studies of recombinant adenovirus are also underway in primates. The goal of these studies is to assess the ability of recombinant adenoviral vectors to deliver genes to the respiratory epithelium in vivo and to assess the safety of the construct in primates. Initial studies in primates targeted nasal epithelia as the site of infection because of its similarity to lower airway epithelia, because of its accessibility, and because nasal epithelia was used for the first human studies. The Rhesus monkey (Macaca mulatta) has been chosen for studies, because it has a nasal epithelium similar to that of humans.

-31 How expression of CFTR affects the electrolyte transport properties of the nasal epithelium can be studied in patients with cystic fibrosis. But because the primates have normal CFTR function, instead the ability to transfer a reporter gene was assessed. Therefore the Ad-pGal virus was used. The epithelial cell density in the nasal cavity of the Rhesus monkey is estimated to be 2 x 106 cells/cm (based on an average nasal epithelial cell diameter of 7 and the surface near 25-50 cm 2 Thus, there are about 5 x 107 cells in the nasal epithelium of Rhesus monkey. To focus especially on safety, the higher viral doses (20-200 MOI) were used in vivo. Thus doses in the range of 109-1010 pfu were used.

In the first pilot study the right nostril of Monkey A was infected with Ad-p-Gal (-1 ml). This viral preparation was purified by CsCI gradient centrifugation and then by gel filtration chromatography one week later. Adenoviruses are typically stable in CsCI at 4 0

C

.for one to two weeks. However, this viral preparation was found to be defective it did *not produce detectable P-galactosidase activity in the permissive 293 cells). Thus, it was concluded that there was no live viral activity in the material. P-galactosidase activity in 15 nasal epithelial cells from Monkey A was also not detected. Therefore, in the next study, two different preparations of Ad-p-Gal virus: one that was purified on a CsCI gradient and then dialyzed against Tris-buffered saline to remove the CsCI, and a crude unpurified one was used. Titers of Ad-p-Gal viruses were -2 x 1010 pfu/ml and 1 x 1013 pfu/ll, respectively, and both preparations produced detectable P-galactosidase activity in 293 cells.

Monkeys were anesthetized by intramuscular injection of ketamine (15 mg/kg). One week before administration of virus, the nasal mucosa of each monkey was brushed to establish baseline cell differentials and levels of P-galactosidase. Blood was drawn for baseline determination of cell differentials, blood chemistries, adenovirus antibody titers, and viral cultures. Each monkey was also examined for weight, temperature, appetite, and 25 general health prior to infection.

The entire epithelium of one nasal cavity was used in each monkey. A foley catheter (size 10) was inserted through each nasal cavity into the pharynx, inflated with 2-3 ml of air, and then pulled anteriorly to obtain tight posterior occlusion at the posterior choana. Both nasal cavities were then irrigated with a solution ml) of 5 mM dithiothreitol plus 0.2 U/ml neuraminidase in phosphate-buffered saline (PBS) for five minutes. This solution was used to dissolve any residual mucus overlaying the epithelia. (It was subsequently found that such treatment is not required.) The washing procedure also allowed the determination of whether the balloons were effectively isolating the nasal cavity. The virus (Ad-p-Gal) was then slowly instilled into the right nostril with the posterior balloon inflated. The viral solution remained in contact with the nasal mucosa for 30 minutes. At the end of 30 minutes, the remaining viral solution was removed by suction. The balloons were deflated, the catheters removed, and the monkey allowed to recover from anesthesia. Monkey A received the CsCIpurified virus ml) and Monkey B received the crude virus ml). (note that this was the second exposure of Monkey A to the recombinant adenovirus).

-32- Both monkeys were followed daily for appearance of the nasal mucosa, conjunctivitis, appetite, activity, and stool consistency. Each monkey was subsequently anesthetized on days 1, 4, 7, 14, and 21 to obtain nasal, pharyngeal, and tracheal cell samples (either by swabs or brushes) as described below. Phlebotomy was performed over the same time course for hematology, ESR, general screen, antibody serology and viral cultures. Stools were collected every week to assess viral cultures.

To obtain nasal epithelial cells from an anesthetized monkey, the nasal mucosa was first impregnated with 5 drops of Afrin (0.05% oxymetazoline hydrochloride, Schering- Plough) and 1 ml of 2% Lidocaine for 5 min. A cytobrush (the kind typically used for Pap smears) was then used to gently rub the mucosa for about 10 seconds. For tracheal brushings, a flexible fiberoptic bronchoscope; a 3 mm cytology brush (Bard) was advanced through the bronchoscope into the trachea, and a small area was brushed for about 10 seconds. This procedure was repeated twice to obtain a total of ~10 6 cells/ml. Cells were then collected on slides (approximately 2 x 10 4 cells/slide using a Cytospin 3 (Shandon, PA)) for subsequent 15 staining (see below).

To determine viral efficacy, nasal, pharyngeal, and tracheal cells were stained for 1galactosidase using X-gal (5 bromo-4-chloro-3-indolyl-p-D-galactoside). Cleavage of X-gal by 1-galactosidase produces a blue color that can be seen with light microscopy. The Ad-pgal vector included a nuclear-localization signal (NLS) (from SV40 large T-antigen) at the amino-terminus of the 1-galactosidase sequence to direct expression of this protein to the nucleus. Thus, the number of blue nuclei after staining was determined.

RT-PCR (reverse transcriptase-polymerase chain reaction) was also used to determine viral efficacy. This assay indicates the presence of P-galactosidase mRNA in cells obtained S*I: by brushings or swabs. PCR primers were used in both the adenovirus sequence and the 25 LacZ sequence to distinguish virally-produced mRNA from endogenous mRNA. PCR was also used to detect the presence of the recombinant adenovirus DNA. Cytospin preparations was used to assess for the presence of virally produced P-galactosidase mRNA in the respiratory epithelial cells using in-situ hybridization. This technique has the advantage of being highly specific and will allow assessment which cells are producing the mRNA.

Whether there was any inflammatory response was assessed by visual inspection of the nasal epithelium and by cytological examination of Wright-stained cells (cytospin). The percentage ofneutrophils and lymphocytes were compared to that of the control nostril and to the normal values from four control monkeys. Systemic repsonses by white blood cell counts, sedimentation rate, and fever were also assessed.

Viral replication at each of the time points was assessed by testing for the presence of live virus in the supematant of the cell suspension from swabs or brushes. Each supematant was used to infect (at several dilutions) the virus-sensitive 293 cell line. Cytopathic changes in the 293 cells were monitored for 1 week and then the cells were fixed and stained for 3galactosidase. Cytopathic effects and blue-stained cells indicated the presence of live virus.

-33- Positive supernatants will also be subjected to analysis of nonintegrating DNA to identify (confirm) the contributing virus(es).

Antibody titers to type 2 adenovirus and to the recombinant adenovirus were determined by ELISA. Blood/serum analysis was performed using an automated chemistry analyzer Hitachi 737 and an automated hematology analyzer Technicom H6. The blood buffy coat was cultured in A549 cells for wild type adenovirus and was cultured in the permissive 293 cells.

Results: Both monkeys tolerated the procedure well. Daily examination revealed no evidence of coryza, conjunctivitis or diarrhea. For both monkeys, the nasal mucosa was mildly erythematous in both the infection side and the control side; this was interpreted as being due to the instrumentation. Appetites and weights were not affected by virus administrated in either monkey. Physical examination on days 1,4,7, 14 and 21 revealed no evidence of lymphadenopathy, tachypnea, or tachycardia. On day 21, monkey B had a temperature 39.1 C (normal for Rhesus monkey 38.8°C) but had no other abnormalities on 15 physical exam or in laboratory data. Monkey A had a slight leukocytosis on day 1 post infection which returned to normal by day 4; the WBC was 4,920 on the day of infection, 8,070 on day 1, and 5,200 on day 4. The ESR did not change after the infection. Electrolytes and transaminases were normal throughout.

Wright stains of cells from nasal brushing were performed on days 4, 7, 14, and 21.

They revealed less than 5% neutrophils and lymphocytes. There was no difference between the infected and the control side.

X-Gal stains of the pharyngeal swabs revealed blue-stained cells in both monkeys on days 4, 7, and 14; only a few of the cells had clear nuclear localization of the pigment and some pigment was seen in extracellular debris. On day 7 post infection, X-Gal stains from 25 the right nostril of monkey A, revealed a total of 135 ciliated cells with nuclear-localized blue stain. The control side had only 4 blue cells Monkey B had 2 blue cells from the infected nostril and none from the control side. Blue cells were not seen on day 7, 14, or 21.

RT-PCR on day 3 post infection revealed a band of the correct size that hybridized with a P-Gal probe, consistent with P-Gal mRNA in the samples from Monkey A control nostril and Monkey B infected nostril. On day 7 there was a positive band in the sample from the infected nostril of Monkey A, the.same specimen that revealed blue cells.

Fluid from each nostril, the pharynx, and trachea of both monkeys was placed on 293 cells to check for the presence of live virus by cytopathic effect and X-Gal stain. In Monkey A, live virus was detected in both nostrils on day 3 after infection; no live virus was detected at either one or two weeks post-infection. In Monkey B, live virus was detected in both nostrils, pharynx, and trachea on day 3, and only in the infected nostril on day 7 after infection. No live virus was detected 2 weeks after the infection.

-34c. Human Explant Studies In a second type of experiment, epithelial cells from a nasal polyp of a CF patient were cultured on permeable filter supports. These cells form an electrically tight epithelial monolayer after several days in culture. Eight days after seeding, the cells were exposed to the Ad2/CFTR virus for 6 hours. Three days later, the short-circuit current (Isc) across the monolayer was measured. cAMP agonists did not increase the Isc, indicating that there was no change in chloride secretion. However, this defect was corrected after infection with recombinant Ad2/CFTR. Cells infected with Ad2/CFTR (MOI=5; MOI refers to multiplicity of infection; 1 MOI indicates one pfu/cell) express functional CFTR; cAMP agonists stimulated Isc, indicating stimulation of Cl- secretion. Ad2/CFTR also corrected the CF chloride channel defect in CF tracheal epithelial cells. Additional studies indicated that Ad2/CFTR was able to correct the chloride secretory defect without altering the transepithelial electrical resistance; this result indicates that the integrity of the epithelial cells and the tight junctions was not disrupted by infection with Ad2/CFTR. Application of 1 MOI 15 of Ad2/CFTR was also found to be sufficient to correct the CF chloride secretory defect.

The experiments using primary cultures of human airway epithelial cells indicate that the Ad2/CFTR virus is able to enter CF airway epithelial cells and express sufficient CFTR to correct the defect in chloride transport.

Example 9 -In Vivo Delivery to and Expression of CFTR in Cotton Rat and Rhesus Monkey Epithelium MATERIALS AND METHODS Adenovirus vector 25 Ad2/CFTR-1 was prepared as described in Example 7. The DNA construct comprises a full length copy of the Ad2 genome of approximately 37.5 kb from which the early region 1 genes (nucleotides 546 to 3497) have been replaced by cDNA for CFTR (nucleotides 123 to 4622 of the published CFTR sequence with 53 additional linker nucleotides). The viral Ela promoter was used for CFTR cDNA. Termination/polyadenylation occurs at the site normally used by the Elband protein IX transcripts. The recombinant virus E3 region was conserved. The size of the Ad2-CFTR-1 vector is approximately 104.5% that of wild-type adenovirus. The recombinant virus was grown in 293 cells that complement the El early viral promoters. The cells were frozen and thawed three times to release the virus and the preparation was purified on a CsC1 gradient, then dialyzed against Tris-buffered saline (TBS) to remove the CsCI, as described.

Animals Rats. Twenty two cotton rats (6-8 weeks old, weighing between 80-100 g) were used for this study. Rats were anesthetized by inhaled methoxyflurane (Pitman Moore, Inc., Mundelen, Ill). Virus was applied to the lungs by nasal instillation during inspiration.

Two cotton rat studies were performed. In the first study, seven rats were assigned to a one time pulmonary infection with 100 pl solution containing 4.1 x 109 plaque forming units (pfu) of the Ad2/CFTR-1 virus and 3 rats served as controls. One control rat and either two or three experimental rats were sacrificed with methoxyflurane and studies at each of three time points: 4, 11, or 15 days after infection.

The second group of rats was used to test the effect of repeat administration of the recombinant virus. All 12 rats received 2.1 x 108 pfu of the Ad2/CFTR-l virus on day 0 and 9 of the rats received a second dose of 3.2 x 108 pfu of Ad2/CFTR-1 14 days later. Groups of one control rat and three experimental rats were sacrificed at 3, 7, or 14 days after the second administration of virus. Before necropsy, the trachea was cannulated and 15 brochoaveolar lavage (BAL) was performed with 3 ml aliquots of phosphate-buffered saline.

A median stemotomy was performed and the right ventricle cannulated for blood collection.

The right lung and trachea were fixed in 4% formaldehyde and the left lung was frozen in liquid nitrogen and kept at -70"C for evaluation by immunochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and viral culture. Other organs were removed and quickly frozen in liquid nitrogen for evaluation by polymerase chain reaction (PCR).

Monkeys. Three female Rhesus monkeys were used for this study; a fourth female monkey was kept in the same room, and was used as control. For application of the virus, the monkeys were anesthetized by intramuscular injection of ketamine (15 mg/kg). The entire epithelium of one nasal cavity in each monkey was used for virus application. A foley 25 catheter (size 10) was inserted through each nasal cavity into the pharynx, the balloon was inflated with 2-3 ml of air, and then pulled anteriorly to obtain a tight occlusion at the posterior choana. The Ad2/CFTR-1 virus was then instilled slowly in the right nostril with the posterior balloon inflated. The viral solution remained in contact with the nasal mucosa for 30 min. The balloons were deflated, the catheters were removed, and the monkeys were allowed to recover from anesthesia. A similar procedure was performed on the left nostril, except that TBS solution was instilled as a control. The monkeys received a total of three doses of the virus over a period of 5 months. The total dose given was 2.5 x 109 pfu the first time, 2.3 x 109 pfu the second time, and 2.8 x 109 pfu the third time. It was estimated that the cell density of the nasal epithelia to be 2 x 106 cells/cm 2 and a surface area of 25 to cm 2 This corresponds to a multiplicity of infection (MOI) of approximately The animals were evaluated 1 week before the first administration of virus, on the day of administration, and on days 1, 3, 6, 13, 21, 27, and 42 days after infection. The second administration of virus occurred on day 55. The monkeys were evaluated on day 55 and then on days 56, 59, 62, 69, 76, 83, 89, 96, 103, and 111. For the third administration, on day 134.

-36only the left nostril was cannulated and exposed to the virus. The control monkey received instillations of PBS instead of virus. Biopsies of the left medial turbinate were carried out on day 135 in one of the infected monkeys, on day 138 on the second infected monkey, and on day 142 on the third infected monkey and on the control monkey.

For evaluations, monkeys were anesthetized by intramuscular injection ofketamine mg/kg). To obtain nasal epithelial cells, the nasal mucosa was first impregnated with drops of Afrin (0.05% oxymetazoline hydrochloride, Schering-Plough) and 1 ml of 2% Lidocaine for 5 minutes. A cytobrush was then used to gently rub the mucosa for about 3 sec. To obtain pharyngeal epithelial swabs, a cotton-tipped applicator was rubbed over the back of the pharynx 2-3 times. The resulting cells were dislodged from brushes or applicators into 2 ml of sterile PBS. Biopsies of the medial turbinate were performed using cupped forceps under direct endoscopic control.

Animals were evaluated daily for evidence of abnormal behavior of physical signs. A record of food and fluid intake was used to assess appetite and general health. Stool 15 consistency was also recorded to check for the possibility of diarrhea. At each of the evaluation time points, rectal temperature, respiratory rate, and heart rate were measured.

The nasal mucosa, conjunctivas, and pharynx were visually inspected. The monkeys were also examined for lymphadenopathy.

Venous blood from the monkeys was collected by standard venipuncture technique.

Blood/serum analysis was performed in the clinical laboratory of the University of Iowa Hospitals and Clinics using a Hitachi 737 automated chemistry analyzer and a Technicom H6 automated hematology analyzer.

0 Serolog 25 Sera were obtained and anti-adenoviral antibody titers were measured by an enzymelinked immunoadsorbant assay (ELISA). For the ELISA, 50 ng/well of filled adenovirus (Lee Biomolecular Research Laboratories, San Diego, Ca) in 0.1M NaHCO 3 were coated on 96 well plates at 4 0 C overnight. The test samples at appropriate dilutions were added, starting at a dilution of 1/50. The samples were incubated for 1 hour, the plates washed, and a goat anti-human IgG HRP conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) was added and incubated for 1 hour. The plates were washed and O-Phenylenediamine (Sigma Chemical Co., St. Louis, MO) was added for 30 min. at room temperature. The assay was stopped with 4.5 M H 2 S0 4 and read at 490 nm on a Molecular Devices microplate reader. The titer was calculated as the product of the reciprocal of the initial dilution and the reciprocal of the dilution in the last well with an OD>0.100.

Neutralizing antibodies measure the ability of the monkey serum to prevent infection of 293 cells by adenovirus. Monkey serum (1:25 dilution) [or nasal washings (1:2 dilutions)] was added in two-fold serial dilutions to a 96 well plate. Adenovirus (2.5 x 105 pfu) was added and incubated for 1 hour at 37°C. The 293 cells were then added to all wells and the -37plates were incubated until the serum-free control wells exhibited >95% cytopathic effect.

The titer was calculated as the product of the reciprocal of the initial dilution times the reciprocal of the dilution in the last well showing >95% cytopathic effect.

Bronchoalveolar lavage and nasal brushings for cytologv Bronchoalveolar lavage (BAL) was performed by cannulating the trachea with a silastic catheter and injecting 5 ml of PBS. Gentle suction was applied to recover the fluid.

The BAL sample was spun at 5000 rpm for 5 min. and cells were resuspended in 293 media at a concentration of 106 cells/ml. Cells were obtained from the monkey's nasal epithelium by gently rubbing the nasal mucosa for about 3 sec. with a cytobrush. The resulting cells were dislodged from the brushes into 2 ml of PBS. Forty microliters of the cell suspension were cytocentrifuged onto slides and stained with Wright's stain. Samples were examined by light microscopy.

Histology of lung sections and nasal biopsies S* The right lung of each cotton rat was removed, inflated with 4% formaldehyde, and embedded in paraffin for sectioning. Nasal biopsies from the monkeys were also fixed with 4% formaldehyde. Histologic sections were stained with hematoxylin and eosin Sections were reviewed by at least one of the study personnel and by a pathologist who was "unaware of the treatment each rat received.

Immunocvtochemistrv Pieces of lung and trachea of the cotton rats and nasal biopsies were frozen in liquid 25 nitrogen on O.C.T. compound. Cryosections and paraffin sections of the specimens were used for immunofluorescence microscopy. Cytospin slides of nasal brushings were prepared on gelatin coated slides and fixed with paraformaldehyde. The tissue was permeabilized with Triton X-100, then a pool of monoclonal antibodies to CFTR (M13-1, M1-4) (Denning, G.M.

et al. (1992) J. Clin. Invest. 89:339-349) was added and incubated for 12 hours. The primary antibody was removed and an anti-mouse biotinylated antibody (Biomeda, Foster City, CA) was added. After removal of the secondary antibody, streptavidin FITC (Biomeda, Foster City, Ca) was added and the slides were observed under a laser scanning confocal microscope. Both control animal samples and non-immune IgG stained samples were used as controls.

PCR was performed on pieces of small bowel, brain, heart, kidney, liver, ovaries, and spleen from cotton rats. Approximately 1 g of the rat organs was mechanically ground and mixed with 50 gl sterile water, boiled for 5 min., and centrifuged. A 5 ul aliquot of the -38supernatant was removed for further analysis. Monkey nasal brushings suspensions were also used for PCR.

Nested PCR primer sets were designed to selectively amplify Ad2/CFTR- DNA over endogenous CFTR by placing one primer from each set in the adenovirus sequence and the other primer in the CFTR sequence. The first primer set amplifies a 723 bp fragment and is shown below: Ad2 5' ACT CTT GAG TGC CAG CGA GTA GAG TTT TCT CCT CCG 3' (SEQ ID NO:4) CFTR 5' GCA AAG GAG CGA TCC ACA CGA AAT GTG CC 3' (SEQ ID The nested primer set amplifies a 506 bp fragment and is shown below: Ad2 5' CTC CTC CGA GCC GCT CCG AGC TAG 3' (SEQ ID NO:6) CFTR 5' CCA AAA ATG GCT GGG TGT AGG AGC AGT GTC C 3' (SEQ ID NO:7) A PCR reaction mix containing 10mM Tris-Cl (pH 50mM KCI, 1.5 mM MgCl 2 0.001% gelatin, 400 gM each dNTP, 0.6 1M each primer (first set), and 2.5 units 15 AmpliTaq (Perkin Elmer) was aliquoted into separate tubes. A 5 gl aliquot of each sample prep was then added and the mixture was overlaid with 50 pl of light mineral oil. The samples were processed on a Barnstead/Thermolyne (Dubuque, IA) thermal cycler programmed for 1 min. at 94°C, 1 min. at 65*C, and 2 min. at 72*C for 40 cycles. Post-run dwell was for 7 min. at 72*C. A 5 pl aliquot was removed and added to a second PCR reaction using the nested set of primers and cycled as above. A 10 pI aliquot of the fmal amplification reaction was analyzed on a 1% agarose gel and visualized with ethidium bromide.

To determine the sensitivity of this procedure, a PCR mix containing control rat liver supernatant was aliquoted into several tubes and spiked with dilutions of Ad2/CFTR-1.

Following the amplification protocols described above, it was determined that the nested PCR procedure could detect as little as 50 pfu of viral DNA.

RT-PCR

RT-PCR was used to detect vector-generated mRNA in cotton rat lung tissue and samples from nasal brushings from monkeys. A 200 gl aliquot of guanidine isothiocyanate solution (4 M guanidine isothiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarcosyl, and 0.1 M P-mercaptoethanol) was added to a frozen section of each lung and pellet from nasal brushings and the tissue was mechanically ground. Total RNA was isolated utilizing a single-step method (Chomczynski, P. and Sacchi, N. et al. (1987) Analytical Biochemistry 162:156-159; Hanson, C.A. et al. (1990) Am. J. Pathol. 137:1-6). The RNA was incubated with 1 unit RQ1 RNase-free DNase (Promega Corp., Madison WI)) at 37°C for 20 min., denatured at 99°C for 5 min., precipitated with ammonium acetate and ethanol, and redissolved in 4 pl diethylpyrocarbonate treated water containing 20 units RNase Block 1 (Stratagene, La Jolla CA). A 2 pl aliquot of the purified RNA was reverse transcribed using -39the GeneAmp RNA PCR kit (Perkin Elmer Cetus) and the downstream primer from the first primer set described in the previous section. Reverse transcriptase was omitted from the reaction with the remaining 2 pl of the purified RNA prep, as a control in which preparations (both RT) were then amplified using nested primer sets and the PCR protocols described above. A 10 .l aliquot of the final amplification reaction was analyzed on a 1% agarose gel and visualized with ethidium bromide.

Southern analysis.

To verify the identity of the PCR products, Southern analysis was performed. The DNA was transferred to a nylon membrane as described (Sambrook el al., supra). A fragment ofCFTR cDNA (amino acids #1-525) was labeled with 32 P]-dCTP (ICN Biomedicals, Inc. Irvine CA) using an oligolabeling kit (Pharmacia, Piscataway, NJ) and purified over a NICK column (Pharmacia Piscataway, NJ) for use as a hybridization probe.

The labeled probe was denatured, cooled, and incubated with the prehybridized filter for 15 hours at 42 0 C. The hybridized filter was then exposed to film (Kodak XAR-5) for 10 min.

Culture of Ad2CFTR- Viral cultures were performed on the permissive 293 cell line. For culture of virus from lung tissue, 1 g of lung was frozen/thawed 3-6 times and then mechanically disrupted in 200 pl of 293 media. For culture of BAL and monkey nasal brushings, the cell suspension was spun for 5 min and the supernatant was collected. Fifty pl of the supernatant was added in duplicate to 293 cells grown in 96 well plates at 50% confluence. The 293 cells were 25 incubated for 72 hr at 37*C, then fixed with a mixture of equal parts of methanol and acetone for 10 min. and incubated with FITC-labeled anti-adenovirus monoclonal antibodies (Chemicon, Light Diagnostics, Temecuca, CA) for 30 min. Positive nuclear immunofluorescence was interpreted as positive culture. The sensitivity of the assay was evaluated by adding dilutions of Ad2/CFTR-l to 50 pi of the lung homogenate from one of the control rats. Viral replication was detected when as little as 1 pfu was added.

RESULTS

Efficacy of Ad2/CFTR-1 in the lungs of cotton rats.

To test the ability of Ad2/CFTR-l to transfer CFTR cDNA to the intrapulmonary airway epithelium, several studies were performed. 4 x 10 pfu IU of Ad2/CFTR-l in 100 pl was adminstered to seven cotton rats; three control rats received 100 pl of TBS (the vehicle for the virus). The rats were sacrificed 4, 10 or 14 days later. To detect viral transcripts encoding CFTR, reverse transcriptase was used to prepare cDNA from lung homogenates.

The cDNA was amplified with PCR using primers that span adenovirus and CFTR-encoded sequences. Thus, the procdure did rnoi detect endogenous rat CFTR.

The lungs of animals which received Ad2ICFTR-l were posifive for virally-encoded CFTR rnRNA. The lungs of all control rats were inegative.

To detect the protein, lung sections were irnunostained with antibodies specific to CFTL_ CFTR was detected at the apical membrane of bronchial epithelium from all rats exposed to Ad2/CFTR-l1, but not from control rats. The location of recombinant CFTR at the apical membrane is consistent with the locatiop of endogenous CFTR in human airway epithelium. Recombinant CFTR was detected above backgrouind levels because endogenous levels of CFTR in airway epithelia are very low and thus, difficult to detect by imrnunocytochemnistry (Trapnell, B_ et al. (1991) Proc. NaIL. Acad Sei. USA 88:6565-6569; Detning, G.M. et al. (1992) J1 Cell hil. i 18:51-59).

These results show that Ad2ICFTR-l directs the expression of CFTR mRNA in The lung of the cotton rut and CFTR protein in the intrapulmnonary airways.

Safety of Ad2/CFTR-l in cotton rats.

Because the ElI region of Ad.2 is deleted in the Ad2/CFTR-lI virus, thce v ecto r was expected to be replication-impaired (Berkner, KL. (1988) BioTechiqudes 6:6-16--629) anid that it would be unable to shut off host cell protein synthesis (Basuss, L.E. 'et al- (1 989) J -Viral 50:202-212)- Previous in -vitro studies have suggested that this is the cast in a variety of cells* including primary cultures of human airway epithelial cells (Rich. D.P. et al. (.1993) Humant Gene Therapy 4:461-476). However, it is imnportant to confirm this in vivo in-the cottoni rat,: which is the most permissive animal model for human 4denovinis infec-tioin (Ginsberg, H.S.

et al. (1989) Proc Mod. Acad. Sd. USA 96:3823-3827; Prtintce, G.A. et al. (1993) J Viral 67; 101 -111). Although dose of viu of 4.1 x 10 10 pfus per kg was used, noneC of -the rats died. More importantly, extracts from lung homogenates from each of the cotton rats were cultured in the permissive 293 cell line. With this assay I pfu of recombinant virus was detected in lung homogenate. However, virus was not detected by culture in the lungs of any of the treated animnals. Thus, the virus did not appear -to replicate ina vivo.

ht is also possible that administration of Ad2/CFTR-1 could cause an inflammnatory response, either due to a direct effect of The virus or as a result of admiaistration of viral particles. Several studies were performed to test this possibility. None of the rats had a change in the total or differential white blood cell count, suggesting that there was no major systemic inflammatory response. To assess the pulmonary inflammatory response more dircctly, bronchoalveolar lavage was performed on each of the rats Figure I IA shows that there was no change in the total number of cells recovered from the lavage or in the differential cell count.

Sections of the lung stained by H&E were also prepared. T'here was no evidence of viral izKlusions or any other changes charactcristic of adenoviral infection (Prince, G.A. el al.

(1993) J Virol. 67:101 -111). When coded lung sections were evaluated by a skilled reader 3- 2-98;11!~58 4/7 14/ -41 who was unaware of which sections were treated, she was unable to distinguish between sections from the treated and untreated lungs.

It seemed possible that the recombinant adenovirus could escape from the lung into other tissues. To test for this possibility, other organs from the rats were evaluated using nested PCR to detect viral DNA. All organs tested from infected rats were negative, with the exception of small bowel which was positive in 3 of?7 rats.

The presence of viral DNA in the small bowel suggests that the rats may have swallowed some of the virus at the time of instillation or, alternatively, the normal airway clearance mechanisms may have resulted in deposition of viral DNA in the gastrointestinal tract Despite the presence of viral DNA in homogenates of small intestine, none of the rats developed diarrhea. This result suggests that if the virus expressed CEIR in the intestinal epitheliumr, there was nio obvious adverse consequence.

Repeat drninistratio of Ad2/CFTR-1 to coTton rats.

Because adenovirus DNA int~egration into chromosomal DNA'is not necessary for gene expression and only occurs at very low fequency, expression following any given treatment, was anticipated to be fluite and that reputed -administration of recombinant adenqvirus would be required for treatment of CF airway disease. Therefore, the: effect of repeated administration of Ad2/CFTR- 1 cotton rats Was examnined. Twelve cotton rats received 50 pl of Ad2JCFTR-J Two weeks later. 9 of the rats received a second dose of 50 p 1 of Ad2ICFT"R-1 and 3 rats received 50 0i of TBS. Rats were sacrificed on day 3, 7, or 14 after virus administration. At the time of the second vector adroinisifation all cotton rats had an increased antibody titer to adenoviras.

After the second intrapulmonary administration of virus. none of the rats died- Moreover, the results of studies assessing safety and efficacy were similar to results obtained in arimals receiving adenovirus for the first time. Viral culturs of rat lung homogenates on 293 cells were negative at all time points, suggesting that there was no virus replication.

There was no difference between treated and control rats in the total or differntial white bWood count at any of the ti 'me points. The lungs were evaluated by histologic sections stained with H&E; and -found no observable differences between the control and treaed raic when sections UWeeread'by ws or by a blinded skilled reader, When organs were examined for viral DNA using PCR. viral DNA was found only in the smfall intestine of 2 rats, Despite seropositivity of the rats at the time of the second administration, expression of CFTR (as assessed by RT-PCR and by inurunocytochemistry of sections stained with MFR antibodies) similar to thai seen in animals that received a single admninismrtion was observed.

I~RA//,

4 /VT O S1 04-02-98 14:27 (3 4 E 631 #2 Davies Collison Cave 42 Thcsc rcsults suggest that prior administnation of Ad2/CFTR- I and the development of an antibody response did not cause an inflamnmatory response in the rats nor did it prevent virus-dependent production of CFTR.

Evidcrice that Ad2/CFTR- I expresSes CFTR in priMat aiyiheliuin The cells lining the respiratory tract and the immune system of primates are similar to those of hiunans. To test the ability of Ad2JCFTR-l to transfer CFTR to the respiratory epithelium of primates, Ad2/CFTR was applied on three occasions as described in the methods to the nasal epithelium of three Rhesus monkeys. To obtain cells from the respiratory epithelium, the epithelium was brushed using a procedure similar to that used to sample the airway epitheliumn of humans during fiberoptic bronchoscopy.

To assess gene transfer, RT-PCR was used as described above for the cotton rats. RT PCR was positive on cells brushed from the right nostril of all three monkeys, although it was only detectable for 18 days after virus administration. An example of the results are shown in Figure 19A. The presence of a positive reaction in cells from the left nostil most likely represents some virus movement to the left side due to drainage, or possibly from the monkey moving The virus from one nostri to the other with its fingers after it recovered from anesthesia.I The specificity of the RT-PCR is shown in Figure 19B. A Southern blot with a probe to CFTR hybridized with the RT-PCR product from the monkey infected with Ad2JCFTR- 1.

As a control, one monkey received a different virus (Ad2/I3Gal-l) which encodes J3galactosidase. When different primers were used to reverse transcribe the P-galactosidase niRNA and amplify the cDNA, the appropriate PCR product was detected. However, the PCR product did not hybridize to the CFTR probe on Southern blot. This result shows the specificity of the reaction for amnpiffication of the adenovirus-directed CFTR transcript.

The failure to detect evidence of adenovirus-eneaded CFTR toRNA at 18 days or beyond suggests that the sensitivity of the RT-PCR may be low becuse of limited efficacy of the reverse u-anscriptase or because RNAses may have degraded RNA after cell acquisition.

viral DNA, however, was detected by PCR in brushings from the nasal epithelium for seventy days after application of the virus. This result indicates that although rnRNA was not detected after 2 weeks, viral DNA waas present for a prolonged period and may have been trancriptionally active.

To assess the presence of CFTR proteins directly, cells obtained by brushing were plated onto slides by cvrospin arnd stained with antibodies to CFTR.

A positive reaction was clearly ev-idcrit in cells exposed 10 Ad2JCFTR- 1. 'The cells were scored as positive by imrnusiocytochemristry when evaluated by a reader uninformed to the identity of the samples.

Immunocytochemistry remained positive for five io six weeks for the three monkeys, even A. after the second administration ofT Ad2/CFTR- 1. On occasion, a few positive staining cells 3- 2-98; 11!58 5/7 15/ Were observed from the conu'alateral nostril of the monkeys. However, This was of short duration, lasting at most one week.

Sections of nasal turbinate biopsies obtained within a week after the third infection were also examined. In sections from the control monkey, little if any irmunofluorescencc from the surface epithelium was observed, but the subrnucosal glands showed signficant staining of CFTR These observations are consistent with results of previous studies (Engelhardt, and Wilson, J.M. (1992) Nature Gen. 2:240-248.) In contrast, sections from monkeys that received Ad2ICFTR-l revealed increased imntunofluoreseence at the apicaf membrane of the surface epithelium. Tle submizcosal glands did not appear to have greater iznmunostraining than was observed under control conditions, These results indicate that Ad2ICFTR-1 can tr-ansfer the CFTR cDNA to the airway epitheliumr of Rhesus monkeys, even in seropositive animals (see below).

Saa--fAVE -Iadiitrdt aky Figure 20A shows that all three treated monkeys developed antibodies against adenoviru. Antibody titers measured by ELISA rose within two weeks after the :first infection. With subsequent infections the titer rose within days. The sentinel monkey had low antibody titers throughout the experiment. Tests for the presence of neutral izing antibodies were also pefornd. After the first administration, neutralizing antibodies were not obsczved, but they were detected after the second administration and during the third viral administration (Fig. To detect virus, supernatants from nasal brushngs,.and swabs were cultured on 293 cells. All monkeys had positive cultures on day I and on day 3 or 4 frcm. the infected nostril.

Cultures remained positive in one of te monkeys at seven days after administration, but cultures were never positive beyond 7 days. Live virus was occasionally detected in swabs frorn the contra lateal nostril during the first 4 days after infection. The rapid loss of detectable virus suggests that there was not viral replication. Stools were routinely cultured, but virus was never detected in stools from any of the monkeys.

None of the monkeys developed any clinical signs of viral infection or inflammration.

Visual inspection of the nasal epithelium revealed slight crytherna in all three monkeys in both nostrils on the first day after infection; but similar crythemna was observed in the control monkey and likely resulted from the instrwnentarion. There was no visible abnormalities at days 3 or 4, or on weekly inspection thereafter. Physical examnination revealed no fever, lymphadenopathy, conjunctivitis, tachypnea, or tacbycardia at ay of The june points. No abnormalities were found in a complete blood count or sedimenation rate, nor were abnormualities observed in serurn electrolytes. transarninases. or blood urea nitrogen and creatinine.

Examination of Wright-stained cells from thde nasal brushings showed that neurrophils and lymphocytes accounted for less than 5% of tota cells in all Wh~ee monkeNys.

I' RAi,, T SEC i4 u /VT O 3- 2-98:11!58 16/ -44- Administration of the Ad2/CFTR-) caused no change in the distribution or number of infflamrmatory cells at any of the time points following virus administration. H&E stains of the nasal turbinate biopsies specimens from the control monkey could not be differenitiated from that of the expermental monkey when the specimens were reviewed by an independent pathologist.

These results dernonstrate the ability of a recombinant adenovirus encoding CFTR (Ad2/CFTR-1) to express CFTR cDNA in the airway epithelium of cotton rats and monkeys during repeated administration. They also indicate that application of the virus involves little if any risk. Thus, they suggest that -such a vector may be of value in expressing CFTR in the airway epithelium of humans with cystic fibrosis.

Two methods were: used to show that AdYCFTR- I expresses CFTR in The airway epithelium of cotton rats and primates: CFTR mnPNA was detected using RT-PCR and protein was detected by irnmnunocytochemistry. Duration of expression as assessed immxunocytocbemically was five to six weeks. Because very little protei is required to gene maxe Cl- secretion (Welsh, M.I. (1997) PhysoL. Rev. 67:114341184; Trapnell, B.C. et al.

(1991.) Proc.-Nati. A cad. Sci, USA 88:6565-6569; Denning, G.M. et al. (1992) .1 Cell Mt 118:551-559), it is likely -that functional expression of CIFTR persists substantially longer than the period of time during which CPTR was detected by immunocytochernisiry- Support for this evidence copies fromt two consderauions: -first it is very difficult to detect CFTR immuncytochcrnically in the airway epithielium, yet the* expression of an apical membrane CI permeability due to the presence of CFTR C i channels is readily detected. The ability of a minimal amount of CFTR to have important functional effects is likely a result of the *fact that a single ion channel conducts a very large numWe of ions (106 107 ions/sec).

Thus, ion channels are not usually abundant proteins in epithlini. Second, previous work suggests that the defective electrolyte transport of CF epithelia can be corrected when only 6of cells in a CF airway epitheliurm overexprcss wild-type CFTR (Johnson. LG_ et al.

(1992) Nature Gem~ 2:21-25). Thus, correction of the biologic defect in CF patients may be possible when only a small percent of the cells express CFTR. This is also consistent with our previous studies in vitro showing that Ad2/CFTR- I at relatively low multiplicities of infection generated a cAIMP-stimulated CI- secrctory response in CF epithelia (Rich, D.P. et al. (1993) Hwrnan Gene Therapy 4:461-476).

This study also provides the firs comprehensive data on the safety of adenovirus vectors for gene rnsfer to airway epithtelium. Several aspects of the stu~dies are encouraging. There was no evidence of viral replication, rather infectious viral particles were rapidly clcared from both cotton rats and primates. These data, together with our previous in vitro studies, suggest that replication of recombinant virus in humans will likely not be a problem. The other major consideration for safety of an adenovinis vector in the treatment of CIF is the possibility of an inflammatory response. The data indicate that the virus generated an antibody response in both cotton rats and monkeys. Despite this, -no evidence of a

SE

NT 0o( systemic or local inflammatory response was observed. The cells obtained by bronchoalveolar lavage and by brushing and swabs were not altered by virus application.

Moreover, the histology of epithelia treated with adenovirus was indistinguishable from that of control epithelia. These data suggest that at least three sequential exposures of airway epithelium to adenovirus does not cause a detrimental inflammatory response.

These data suggest that Ad2/CFTR-I can effectively transfer CFTR cDNA to airway epithelium and direct the expression of CFTR. They also suggest that transfer is relatively safe in animals. Thus, they suggest that Ad2/CFTR- may be a good vector for treating patients with CF. This was confirmed in the following example.

Example 10 CFTR Gene Therapy in Nasal Epithelia from Human CF Subjects EXPERIMENTAL PROCEDURES 15 Adenovirus vector The recombinant adenovirus Ad2/CFTR- I was used to deliver CFTR cDNA. The construction and preparation of Ad2/CFTR-l, and its use in vitro and in vivo in animals, has been previously described (Rich, D.P. et al. (1993) Human Gene Therapy 4:461-476; Zabner, J. et al. (1993) Nature Gen. (in press)). The DNA construct comprises a full length copy of the Ad2 genome from which the early region 1 genes (nucleotides 546 to 3497) have been replaced by cDNA for CFTR. The viral Ela promoter was used for CFTR cDNA; this is a low to moderate strength promoter. Termination/polyadenylation occurs at the site normally used by Elb and protein IX transcripts. The E3 region of the virus was conserved.

Patients Three patients with CF were studied. Genotype was determined by IG Labs (Framingham, MA). All three patients had mild CF as defined by an NIH score (Taussig, L.M. et al. (1973) J. Pediatr. 82:380-390), a normal weight for height ratio, a forced expiratory volume in one second (FEVI) greater than 50% of predicted and an arterial

PO

2 greater than 72. All patients were seropositive for type 2 adenovirus, and had no recent viral illnesses. Pretreatment cultures of nasal swabs, pharyngeal swabs, sputum, urine, stool, and blood leukocytes were negative for adenovirus. PCR of pretreatment nasal brushings using primers for the adenovirus El region were negative. Patients were evaluated at least twice by FEV I, cytology of nasal mucosa, visual inspection, and measurement of Vt before treatment. Prior to treatment, a coronal computed tomographic scan of the paranasal sinuses and a chest X-ray were obtained.

The first patient was a 21 year old woman who was diagnosed at 3 months after birth.

She had pancreatic insufficiency, a positive sweat chloride test (101 mEq/l), and is homozygous for the AF508 mutation. Her NIH score was 90 and her FEV1 was 83% Dav ies l 9 1ison Cave 04-02-98 14:28 [63] #4 9411649PCTUS93fl 1667 -46predicted. The second patient was a 36 year old man -who was diagnosed at the age of IS when be prescnted Aith symptoms of pancreatic insufficiency. A sweat chloride test revealed a chloride- concentration of 70 m.Eq/1- He is a heterozygote with The AF508 and G5S1D mutations. His NIH score was 88 and Is FEVI was 66% predicted. The third patient was a 50 ear od womn, dignosed at the age of 9 with a positive sweat chloride test (104 mEq/l) She has pancreatic insufficiency and insulin dependent diabetes mellitus. She is hornozvgous for the AF508 mutation. Her NIH score was 73 and her FEV I was 65% predicted.

Tra n &ep ithclal~o totag e The transepithelial electric potential difference across the nasal cpithelium was measured using techniques similar to those previously described (Alton, E.W.F.W. et a] (1987) Thurazx 42:815-817; Knowles, M. et al. (1981) N Eng. .1 Med. 305:1489-1495), A 23 gauge subcutaneous needle connected with sterile normal saline solution to a silver/silver chloride pellet Wright, Giuiford, CT) was used as a reference electrode. The exploring electrode was a size 8 rubber catheter (modified ArgyleR Foley catheter, St. Louis, MO) With one side hole at the tip. The catheter was filled -with Ringer's solution containing (in mM), 135 N~aCI, 2.4 KH 2

PO

2

K

2 H{P0 4 I 2CaCL 2 1.2 MgCl 2 and 10 Hepes (titrated to pH 7,4 with NaOH) and was connected to a silver/silver chloride pellet. Voltage was measured with a voltmeter (Keithley Instruments Inc., Cleveland, Offi) connected to a strip chart recorder (Servocorder, Watanabe Instruments, Japan). Prior to the measurements, the silver/silver chloridc pellets were connected in series with the Ringer's solution; the pellets were changed if the recorded Vt was g~reater than ±4 mV. The rubber catheter was introduced into the nostril under telescopic guidance (Hopkins Telescope, Karl Storz, Turtlingen West G~ermnany) and the side hole of the catheter was placed next to the study area in the medical aspect of the inferior nasal turbinaie. The distance from the anterior tip of the inferior turbinaie and the spatial relationship Aith the medial turbinaie, the maxillary sinus ostiuxn, and in one patient a srnall polyp, were used to loc-ate the area of Ad2/CFTR- I administration for measurements.

Photographs and video recorder images were also used. Basal Vt was recorded until no changes in Vt were observed after slow intermittent 100 p1l/min infusion of the Ringer's solution. Once a stable baseline was achieved, 200 p1l of a Ringer's solution containing 100 p1 M amtiloride (Mcrck and Co. Inc., West Point PA) was instilled through the catheter and thanees in V, were recorded until no further change were obser-ved after intermittent instillations. Finally, 200 jil Ringer's solution containing 100 liM amiloride plus 10 IAM terbutalile (Geigy Pharmnaceuticals, Ardslev, NY) was instilled and the changes in \7 Were recorded.

Mcasuremenis of basal Vt were reproducible over time: in the three treated pat ients, the coefficients of variation before admrinaion of Ad2/CFTR-1 w,%ere 3.6%7 12%, and 12%. The changes induced by terbutaline were also reproducible. In 30 measurements in 9 CF patients, ilhe ierbutaline-induced changes in Vt ranged from 0 mV to +4 m\1; 3- 2-98;11!58 1/7 .17/ 7 hyperpolarization of V, was never observed, In contrast, in '7 normal subjects &Vt ranged from -I mV to -5 mV: byperpolarization was always observed.

Ad2/CFTR-1 "pplicatiou and cell apQuiin The patients were taken to the operiting room and monitoring was commenced using continuous EKG and pulse oxirnetry recording as well as amrnatic intermittent blood pressure measurement After mild sedation, the nasal snucosa was anesahetized by atomizing MI Of 5% Cocaine. The iucosa in the ama of the inferior turbinate wams then packed with cotton pledgets previously soaked in a mixtue of 2 nml of 0. 1% adrenaline and 9 ml of I1% tetracaine. The pledgms remained in place for 10-40 mmi- Using endoseopic: visualization with a television monitoring system, the applicator was introduced through the nostril and tnositioned on the media] aspect of the inferior turbinate, at least three centimeters from its anterior tip (Figures 2 1A-211). The viral suspension was infused into the applicator through coxnecting catheters. The position of the applicator was monitored endoscopically to ensure that it did not move and that enough pressure was applied to prevent leakage. After the virus was in contact with the nansal epithelium for thirt minutes, the viral suspension was removed, and The applicator was withdrawn. In the third patient's right nasal cavity, the virus was applied using the modified Foley catheter used for Vt measurements. The catheter was introduced without anesthetic under endoseopic guidance until The side bole of the catheter was in contact with the area of interest in the inferior-turbinaie. The viral solution was infused slowly until a drop of solution was seen w4ith the telescope. The catheter- was left in place for thirty minutes and then removed, Cells were obtained from The area of virus admiinisration approximately 2 weeks before treatment and then at weekly intervals after tratment. The inferior turbinaie %-as packed for 10 minutes with cotton pledgets previously soaked in I =1 of 5% cocaine. Under endoscopic control, the area of administration was gently bnushed for 5 seconds. The brushed cells were dislodged in PBS. Swabs of the nasal epithbelia were collected using cotton tipped applicators without anesthesia. Cy'ospi slides were prepared and stained wvith Wrighs stain. Light microscopy was used to assess the respiratory epithelial cells and inflammatory cells. For biopsies, sedatives/anesthesia was administered as described for the application procedure. After endoscopic inspetion, and identification of the Site to be biopsied, the subucosa was injected with 1% xylocaine, with 1/100,000 epinephrine. The area of virus application on the inferior rubinate was removed. The specimen was fixed in 4% formaldehyde and stained.

RESULTS

On day one after Ad2/CFTR-lI administration anid at all subsequent time points, Ad2/CFTR-1 from the nasal epithelium.i pharynx, blood, urine, or stool could rnot be cultured, As a control for the sensitivity of the culture assay, samples were: routinely spiked -AiTh 3- 2-98;11!58 18/ -48 and 100 IU Ad2/CFTR-1. In every case, the spiked samples were positive, indicating that, at a minimum, 10 IU of Ad2/CFTR should have been detected- No evidence of a systemic response as'assessed by history, physical examination, serum chemistries or cell counts, chest and sinus X-rays, pulmonary function tests, or mrterial blood gases performed before and after Ad2/CFT.R-1 administration. An increase in antibodies to adeniovirus was not delectable by ELISA or by neutralization for 35 days after txeatinent.

Three to four hours after Ad2ICFTR-lI administration, at the time that local anesthesia arnd localized vasoconstriction abated, all patients began to complain of nasal congestion and in onec case, mild rhinorrhca. These were isolated symptoms that diminished by 18 hours and resolved by 28 to 42 hours. Inspection of the nasal mucosa showed mild to moderate crythernia, edemna, and exudate (Figures 21A-21C). These physical findings followed a time course similar to the symptoms. The physical findings were nol limited to the site of virus application, even though preliminary st~udies using the applicator showed that marker methylene blue was limited to the area of application. In two additional patients with CF, the 1S identical anesthesia and application procedure were used, but saline was applied instead of virus, yet the same symptoms and physical findings Were observed in these patients (Figures 21G-211)- Moreover, the local anesthesia and vasoconstrictioh generated similar changes even when the applicator was not used, suggestizg that the anesThesia/vasoconstriction caused some, if not all the injury- Twenty-four hours after the application procedure, analysis of cells removed from nasal swabs revealed an equivalent increase in the p=rcnt neutrophils; in patients treated with Ad2/CFTR- I or mith W~ine. One wielt after application, the racutrophilia had resolved in both groups. Respiratory epithieiiid cells obtained, by nasal brushing appeared normal at one week and at subsequent time points, and showed no evidence of inclusion bodies. To further evaluate the mucosa, the epithelium was biopsied on day three in the first patient and day one in the second patient- Independent evaluation by two pathologists not otherwise associated -with the study suggested changes consistent with mild trauma and possibic ischemnia (probably secondary wo the anestheficlvasoconstrictors used before virus administration), but there were no abnormalities suggestive of virusmediated damage.

Because tiac application procedure produced some mild injury in the first two patients, the method of administration was altered in the third -patient. The method used did not require the use of local anesthesia oi- vasoconastriction and which was thus less likely to cause injury, but which was also less certain irn its ability to constrain Ad2ICFT-l in a precisely defined arem On the right side, Ad2/CFTPR-l was administered as in the first two paticnts, anid on the left side, -the %rirus was administered without anesthesia or the applicator, instead using a small Foley catheter to apply and rnainiuin Ad2ICFTR- 1 in a relatively defined area by surface tension (Figure 2 1E)_ On the right side, the symptoms and physical findings were the same as those observed in the firs Two patients. By contrast, or, the left side there were no symptoms and on inspection the nasal mnucosa appeared normal (Figures 2 1D-2 iF). Nasal SRAk/

EV

SE4 7lOCLL .IX "I //Air 0- 3- 2-38;1158 5/7 19/ -49swabs obtaned from the right side showed neutrophilia similar to that observed in the first two patients. In contrast, the left side which had no anesthesia and minimal manipulation, did not develop neutrophilia. Biopsy of the left side on day 3 after administration (Figure 22), showed morphology consistent with CF- a thickened basement membrane and ocasonal polytuorphonuclear cells in the subajucosa- but no abnormalitics that coulid be attributed to the adenovirus vector.

.The first patient developed symptoms of a sore throat and increased cough that began three weeks after treatuient and persisted for two days. Six weeks after neatzment she developed an exacerbation of her bronchitislbronchiectasjs and hemoptysis that required hospitalization. The second patient had a transient episode of rninimal hemoptysis three weeks after treatent; it was not accompanied by any other symptoms before or after the episode.. The third patient has an exacerbation of bronchitis three weeks after treatment for which she was given oral antibiotics. Based on each patient's pretrealmenit clinical history, evaluation of the episodes, and viral cultures, no evidence could be discerned that linked these episodes to admirnistration of Ad2/CFTR-1. Rather the episodes appeared consistent with the normal course of disease in each individual..

The loss of CFTR Cl- channel function causes abnormal ion transport across aff~ected epithelia, which in turn contributes to mime pathogencsis of CF-associated airway disease (Boat, T-F. et ira The Metabolic Basis of Inherited'Diseases (Scriver, C.R. et al- eds., McGraw-Hill, NewYork 0I 989): Quinton, P.M. (1990) FASE.Bj. 4:2709-2717). In airway epithelia, ion transport is dominated by two electrically conductive processes: amidlondesensitive absorption of Na+ from the mucosal to thme subanucosal surface and cAMP'stimulated C1- secretion in the opposite direction. (Quinaton. P.M. (1990) FASEB J. 4:2709- 2717; Welsh, M-1 (1987) PhysioL Rev. 67:1143-1194). These two transport processes can be assessed noninvasively by measuring the voltage across the nasal epithelium (VeJ in vivo (Knowles, M. et a] (198 1) X. Eng. J Med 305:1499-1495; Alton, E.W.F.W. et al.(1 987) Thorax! 42:815-817). Figure 23 shows an example from a normnal subject. Under basil conditions, Vt was electrically negative (lumen referenced to thme subetucosal surface).

Perfusion of amiloride (100 p~M) onto the mucosal surface inhibited Vt by blocking apical MNa+ channels (Knowles, M. et al (198 1) X. Eng- J. Mod. 3 05:1489-149:5, Quinton, P.M.

(1990) FASEB J. 4:2709-271 7; Welsh. M.1- (1992) Neuron 8:8$21-829). Subsequent perfasion of terbutaline (10 psM) a P-adrenergic agonist, hyperpolarized Vt by increasing cellular levels of cAMP, opening CFTR. C1- channels, and stimulating chloride secretion (Ouinton. P.M. (1990) FASEB J. 4:2709-2717; Welsh, M.J. et al. (1992) Neuro 'n 3:821-829).

Figure 24A shows results from seven normal subjects: basal Vt was-1-I 1 .Onv, and in the presence of arrailoride, terbutaline hyperpolarized Vt by -2.3 4t In patients with CF, Vt was more electrically negative than in normal subjects (Figure 24B), as bas been previously reported (Knowles, M- et al. (198 1) Y. £ng J Med 305:14 89- 1495). Basal Vt was -37.01~2-4 mV. much more negative than values in normal subjects (P< AA Z 4, A iuue A16 LU V4 41C rLY 1111'4J1 IIIL. Ira PIALM 01 IOC OCICICO Sequecs, a LJ.JNA Davies Collison Cave0-2-8 1:9(3 04-02-98 14:29 631 #5 0.00 (Note the difference in scale in Figure 24A and Figure 24B). Amliloride inhibitcd V,' as it did in normal subjects. However, V 1 failed to hyperpolarize when terbu~alirae was perfused onto the epitheliumn in the presence of ainilaride. Instead, V 1 citber did not change or became less negative: orn average V, depolarized by +1.8 0.6 mV, a result very different from that observed in normal subjects. (P<0O.00 1).

After Ad2VCFTh- I was applied, basal Vt became less negative in allI three CF patients: Figure 25A shows an example from the third patient before (Figure 25A) and after (Figurc 253) treatment and Figures 26A, 26C, and 26F show the time course of changes in basal V, fur all three patients- The decrease in basal Y1 suggests that application of Ad2/CF'TR- I corrccted the CF electrolyte transport defect in nasal epithclium of all three patients. Additional evide.ri"~ fame from an examination of the response to terbutaline. Figure 2511 shows that in contra-,t LL) the riuspunse before Ad2/CFTR- I was applied, after virus replication, in the presence of anailoridc, ierbuialine stimulated Figures 2613, 26D, and 26F show the time course of the responise. These data indicate that Ad2/CFTR- I corrected the CF defect in C1 transport.

Correction of the C1- defect cannot be attributed to the anesthesia/appl icat ion procedure because it did not occur in patients trcatud with saline instead of Ad2/CFTR- I (Figure 29). Moreover, the effects of the anesthesia were generali7vd on the nasal mucosa, but basal Vt decreased only in the area of virus admninistration. Finally, similar changes were observed in the left nasal mucosa of the third patient (Figures 26E and 261F), which had no symptomatic or physical response after the modified application procedure.

Unsuccessfu aftempts were made to detect CFIR uranscripts by reverse trartsciptasc- PCR and by immnunocytochemistry irn cells from nasal -:rushings and biopsies. Although similar studies in artimals have been successfta (Zabrier, I, et al. (1993) ANarure Gen. (in press)), those studies used much higher doses of Ad2ICFTrR- I1. The lack of success in the present case iy reflects the small amount of available tissue, the low MOI, the fact that only a fraction of cells may have been corrected, anid the fact ihat Ad21CFTR- 1 contains a low to moderate strength promoter (Ela) Wuich produces much less rnPJIA and. protein than comparable constructs using a much stronger CMV promoter (unpublished observation). The Ela promoter was chosen because CFTR normally expressed at very low levels in airway epithelial cells (Trapnell, B.C. et al. (1991) Proc. Nail, Acad ScO. USA 88:6565-6569). It is also difficult to detect CMTR protein arnd raRNA in normal human airway epithelia, although function is readily detected because a single ion channel can conduct a vecry large number of ions per second and thus efficiently support Cl1- transport.

With time, the electrical changes that indicate correction of the CF defect reverred toward pretreatment values. However. the basal V, appeared to revert more slowly than did the change in Vt produced by terbutaline. The significance of this difference is uanknovom but it may reflect the relative sensitivity of the two measurements to expressIon of normal CFTR.

In any case, this study was not designed to test the duration of correction because the treated -51area was removed by biopsy on one side and the nasal.mucosa on the other side was brushed to obtain cells for analysis at 7 to 10 days after virus administration, and then at approximately weekly intervals. Brushing the mucosa removes cells, disrupts the epithelium, and reduces basal Vt to zero for at least two days afterwards, thus preventing an accurate assessment of duration of the effect of Ad2/CFTR-1.

Efficacy ofadenovirus-mediated gene transfer.

The major conclusion of this study is that in vivo application of a recombinant adenovirus encoding CFTR can correct the defect in airway epithelial C1- transport that is characteristic of CF epithelia.

Complementation of the C1- channel defect in human nasal epithelium could be measured as a change in basal voltage and as a change in the response to cAMP agonists.

Although the protocol was not designed to establish duration, changes in these parameters were detected for at least three weeks. These results represent the first report that 15 administration of a recombinant adenovirus to humans can correct a genetic lesion as measured by a functional assay. This study contrasts with most earlier attempts at gene transfer to humans, in that a recombinant viral vector was administered directly to humans, rather than using a in vitro protocol involving removal of cells from the patient, transduction of the cells in culture, followed by reintroduction of the cells into the patient.

Evidence that the CF Cl" transport defect was corrected at all three doses of virus.

*....'corresponding to 1, 3, and 25 MOI, was obtained. This result is consistent with earlier studies showing that similar MOIs reversed the CF fluid and electrolyte transport defects in primary cultures of CF airway cells grown as epithelia on permeable filter supports (Rich, D.P. et al. (1993) Human Gene Therapy 4:461-476 and Zabner et al. submitted for 25 publication): at an MOI of less than 1, cAMP-stimulated C1- secretion was partially restored, and after treatment with 1 MOI Ad2/CFTR-1 cAMP agonists stimulated fluid secretion that was within the range observed in epithelia from normal subjects. At an MOI of 1, a related adenovirus vector produced P-galactosidase activity in 20% of infected epithelial cells as assessed by fluorescence-activated cell analysis (Zabner et al. submitted for publication).

Such data would imply that pharmacologic dose of adenovirus in CF airways might correspond to an MOI of one. If it is estimated that there are 2xl 06 cells/cm 2 in the airway (Mariassy, A.T. in Comparative Biology of the Normal Lung (CRC Press, Boca Raton 1992), and that the airways from the trachea to the respiratory bronchioles have a surface area of 1400 cm 2 (Weibel, E.R. Morphometry of the Human Lung (Springer Verlag, Heidelberg, 1963) then there would be approximately 3x109 potential target cells. Assuming a particle to IU ratio of 100, this would correspond to approximately 3x1011 particles of adenovirus with a mass of approximately 75 pg. While obviously only a crude estimate, such information is useful in designing animal experiments to establish the likely safety profile of a human dose.

-52- It is possible that an efficacious MOI of recombinant adenovirus could be less than the lowest MOI tested here. Some evidence suggests that not all cells in an epithelial monolayer need to express CFTR to correct the CF electrolyte transport defects. Mixing experiments showed that when perhaps 5-10% of cells overexpress CFTR, the monolayer exhibits wild-type electrical properties (Johnson, L.G. et al. (1992) Nature Gen. 2:21-25).

Studies using liposomes to express CFTR in mice bearing a disrupted CFTR gene also suggest that only a small proportion of cells need to be corrected (Hyde, S.C. et al. (1993) Nature 362:250-255). The results referred to above using airway epithelial monolayers and multiplicities of Ad2/CFTR-1 as low as 0.1 showed measurable changes in Cl- secretion (Rich, D.P. et al. (1993) Human Gene Therapy 4:461-476 and Zabner et al. submitted for publication).

Given the very high sensitivity of electrolyte transport assays (which result because a single C1" channel is capable of transporting large numbers of ions/sec) and the low activity of the Ela promoter used to transcribe CFTR, the inability to detect CFTR protein and CFTR 15 mRNA are perhaps not surprising. Although CFTR mRNA could not be detected by reverse .transcriptase-PCR, Ad2/CFTR-I DNA could be detected in the samples by standard PCR, demonstrating the presence of input DNA and suggesting that the reverse transcriptase reaction may have been suboptimal. This could have occurred because of factors in the tissue that inhibit the reverse transcriptase. Although there is little doubt that the changes in electrolyte transport measured here result from expression of CFTR, it remains to be seen whether this will lead to measurable clinical changes in lung functioi.

Safety considerations.

Application of the adenovirus vector to the nasal epithelium in these three patients 25 was well-tolerated. Although mild inflammation was observed in the nasal epithelium of all three patients following administration of Ad2/CFTR-l, similar changes were observed in two volunteers who underwent a sham procedure using saline rather than the viral vector.

Clearly a combination of anesthetic- and procedure-related trauma resulted in the changes in the nasal mucosa. There is insufficient evidence to conclude that no inflammation results from virus administration. However, using a modified administration of the highest MOI of virus tested (25 MOI) in one patient, no inflammation was observed under conditions that resulted in evidence of biophysical efficacy that lasted until the area was removed by biopsy at three days.

There was no evidence of replication of Ad2/CFTR-l. Earlier studies had established that replication of Ad2/CFTR-1 in tissue culture and experimental animals is severely impaired (Rich, D.P. et al. (1993) Human Gene Therapy 4:461-476; Zabner, J. et al. (1993) Nature Gen. (in press)). Replication only occurs in cells that supply the missing early proteins of the El region of adenovirus, such as 293 cells, or under conditions where the El region is provided by coinfection with or recombination with an El-containing adenovirus Davies Gollison Cave040-8430 6 04-02-98 14:30 631 #6 (Graham, F.L. and Prevec, L. Vaccines: New Approaches to Irnmunologeical Problems (R.

Ellis, ed., Boston, Burtemwoh-Heinermnn, 1992); Berkner, K.L. (1988) Blot echtiiques 6:636-629), The patients studied here were seropositive for adenovirus types 2 and 5 prior 1o the study were negative for adenovirus upon culture of nasal swabs prior to adminisiraTion of Ad2JCFTR-1, and were showvn by PCR methods to lack endogenous El DNA sequences such as have been reported in somei human subjects (Matsuse T. et al. (1992) Am. Rev. Respir. Dis- 146:177-184).

Exam~le I1I Construction.tand Packaging of Escud~o Adenoviral vector (EAV) With reference to Figure 16, the PAV construct was made by inserting the Ad2 packaging signal and El enhancer region (0-358 nr) in Bluescript 11 SK- (Strataiaree, LaJolla, CA). A variation of this vector, known as PAV IT was constructed similarly, except the Ad27 packaging signal and ElI enhancer region contained 0-3 80 r. The addition of nucleotides aT the 5' end results in larger PAVs, wl~tich may be rno'e efficiently packaged, yet would inciudo mot adenovirual sequences and therefore could potentially be more immunogenic or more capab!c of replicating.

To allow ease of manipulation for CiteT the insertion of gene coding regions Or cori.picic excision and use in transfections for the purpose of generating infiectiouS paril~es, coni'l1cmentazy plasmid was also built in pBluescript SKII-. This cornplemnentary plasmid contains the Ad2 major late promoter (MLP) and *aipartite leader (TPL) DNA and an T-antigcn isudcq localization signal (NLS) and polyadentylation signal (SVpA). As can be scen inFigure 16, Whs plasmid contains a convenient -restriction site for the insertion of genes of interst between The MLP/TPL and SV40 poly A. This construct is engineered such that the entire cassette may be excised and in~serted into the former ?AV I or PAy 11 construct.

Generation of PAV infectious particles was performed by excision of PAV from the plasroid with the AM I and Sag 11 restriction endonucleases and co-transfection into 293 cells (an Ela/Elb expressing cell line) (Grahamn, F.L. ei al, (1977) J. Gen Virol 36:59-74) wvith cither wild-type Ad.2, or packaging/replic-ation deficient helper virus. Purification of ?AV from helper can be accompanied by CsCI gradient isolation as PAy viral particles will be of a lower density and will band at a higher position in the gradient.

For gene therapy, it is desirable to generate significant quantities of PAV v'irion free from contaminating helper virus. The primary adv'antage of PAV over standard adenoviral 'vectors is the ability to package large. DNA inserts into virion (up to about 36 kb). Howev'er.

PAV requires a helper virus for replication and packaging and this helper virus will be the predominant species in any PAV preparation. To increase the propontion of PAV in viral preparation scveral approaches can be employed. For example, one can use a helper virus which is partially defective for packaging ino virons (either by virtue of mutations in the packaging sequences (Grable. M. and Hearing P. (1992) J. Virol. 66: 723-73 or by virrue of its size -viruses with Senome sizes greater than approximately 37.5 kb package Davies Collison Cave Davis Cflisn Cve4-02-98 14:32 (64] #42 -54 inefficiently. In mixed infections Aith packaging defective virus, PAY would be expected to be represenited at higher levecls in the virus mixture than would occur w;ith non-packaging defective helper viruses.

Anothcr approach is to make the helper irus dependent upon PAV for its ownm S replication. This may most easily be accomplished by deleting an essential gene from the helper virus IX or a terminal protein) and placing that gene in the PAV vector. In this way neither PAy nor the helper virus is capable of independent replication ?AV and the helper virus are therefore co-dependent, This should result in Whighe PAY representation in the resulting virus preparation.

I1D A third approach is to develop a novel packaging cell line, which is capable of generating significant quantities of PAV viriori free from coinarninatiig helper virus. A novel protein IX, (pIX) packaging system has been developed. Ths system exploits several documented features of adenovirus molecular biology. The first is that adenoviral defective particles are known to comprise up to 30% or m~ore of standard wild-type adenoviral preparations. These defective or incomplete particles are stable and contain 13-95% of the adenoviraJ genome, typically 15-30%. Packaging of a PAV genome (15-30% of wild-Type genonie) shou.ld package comparably. Secondly, stable packaging of full-length Ad geno me but not genomnes <95% required the presence of the aderiovirual gene designated pIX.

The novel packaging system is based on t~he generation of an Ad protein pIX cxpressing 293 cell linei In addition, an adenoviraJ helper virus engineered such that the El region is deleted but, enough exogenous material is inserted to equal or slightly exceed the full length 36 kb size. Both of these twvo constructs would be introduced into the 293/pIX cell line as purified DNA. In the presence of pIX, yields of both predicted progeny viruses as seen in current PAV/Ad2 production experiments can be obtained. Virus containing lysates from these cells can then be titered independently (for the marker gene activity specific to either vectorj and used to infect standard 293 (lacking pMX at a multiplicity of infection of I relative to PAV. Since resmach wit ths line as well as from incomplete lur defective particl e research inicates that full length gepornes, have a comrpetitive packaging advantage, it is expected that infection with an MOI of I relative to PAV will necessarily equate to an effec-tive MOI for helper of greater than All cells will presumably contain both ?AV (at least 1) and helper (greater than Replication and viral capsid production in this cell shouild occur normally but only PAV genomes should be packaged. Harvesting these 293IpDC cultures is expected to yield essentially helper-free PAV.

Example 12 Construction of Ad2-E4/DRF6 Ad2-E4/ORF6 (Figure 17 shows the plasmid construction of Ad2-E41'ORF6) which is an Adenoevirus 2 based vector deleted for all Ad2 sequences between nucleotides 32815 and 35577. This deletion removes all open reading frames of E4 but leaves the E4 promoter and first 32-37 nucleotides of the E4 mRJIA intact. In place of the deleted sequences, a DNA Davies Collison Cave Davis Cllion ave04-02-98 14:32 [64] #43 fragment encoding 0RF6 (Ad2 nucleolides 34082-33] 78) which was derived by polyrnerase chain reaction of Ad2 DNA Aith ORF6 specific DNA primers (Genzyrne oligo. 2371 CGGATCC1TTATTATAGGGGAAGTCCACGCCTAC

(SEQ.

ID NO:8) and oligo. #2372 CGGGATCCATCGATGAAATATGACTACGTCCG

(SEQ.

DD NQ:9) were inserted). Additional sequences supplied by the oligonucleotides included a cloning site at the 5'and 3'ends of the 1'CR fragment (CWi and BarnHl respectively) and a Dolyadenylation sequence at the 3Tend to ensure correct polyadenylaion of the ORF6 niRNA. As illustrated in Figure 7, the PCR fragivicnt was first ligated to a DNA fragme~nt including the inverted terminal repeat (ITR) and E4 promoter region of Ad2 (Ad2 nucleolides 35937-35577) and cloned in the bacterial plasinid pBluescript (Stratagene) to create piasmid ORF6. After sequencing to verify the integrity of the ORF6 reading frame, the fragment encompassing the ITR and ORF6 w.as subcloned into a second plasmid, pAd A E4, which contains the 3' end of Ad2 from a Sac I site to the 3' ITR (Ad2 nucleotides 29 562-35937) and is deleted for all E4 sequences (promoter to poly A site Ad2 positions 3 2815-3 564 1) using flanking restriction sites. Ina this second plasmid, virus expressing only E4 ORF6, pAdORF6 was cut with restriction enzyme EW~ and ligated to Ad2 DNA digested with EW. This Enr1 site corresponds to Ad2 nuclcotidc 28612. 293 cells were irarasfected with the ligaiion and the resulting virus was subjected to restriction analysis to verify that the Ad2 E4 region had been substituted with the corresponding region of pAdORF6 and that. the only remaining E4 open reading frame was ORM6 A cell line could in theory be establisbed that would fully complement E4 functions deleted from a recombinant virus. The problem %kith This approach is that E4 functions in the regulation of host cell protein synthesis and is therefore toxic to cells. The present recombinant adenoviruses are deleted for the ElI region and must be grown in 293 cells which complement ElI functions. The E4 promoter is activated by the Ela gene product, and therefore to prevent inadv'ertent toxic expression of E4 transcription of E4 must be tightly regulated. The requirements of suchi a promoter or transactivating system is that in The uninduced siate expression must be low enough to avoid toxicity to the host cell, but in the induced state must be sufficiently ac tivated to make enough E4 gene product to complement the E4 deleted virus during virus produciion.

Exmnot-fl An adenoviral vector is prepared as described in Example 7 while substituting the phosphoglycerate kinase (PGK) promoter for the Ela promoter.

An adenoviral vector is prepared as described in Example 11I while substituting the PGK promoter for the Ad2 major laic promoter (MLP).

~SEC

04 /VT 0~ Davies Collison Cave Davis Cllion ave04-02-98 14:33 1641 44 56- Exanmple 15- Gelperafion of Ad)-ORF6/PQK-CFTR This protocol uses a second generation adenovirus vector named Ad2-ORF6PCGK- CFTR. This virus lacks ElI and in its place contains a modificd transcription unit with the P0K promoter and a poly A addition site flanking the CFTR cDNA. The PGK promoter is of only moderate strength but is long lasting and not subject to shut off. The E4 region of the vector has also been modified in that the whole coding sequence has been removed and replaced by ORF6, the only E4 gene essential for growth of Ad in tissu~e culture. This has the effect of generating a genome of 101 the size of wild type Ad2.

The DNA construct comprises a full length copy of the Ad.2 genome from which the ealy region 1 (El) genes (present at the 5'end of the viral genoine) have been deleted and replaced by an expression cassette encoding CFTR. The expression cassette includes the promoter for phosphoglycerate kinase (PGK) and a polyadenylation (poly A) addition signal from the bovine growth hormone gene (BGH). In addition, The E4 region of Ad.2 has been deleted and replaced with only open reading frame 6 (ORF6) of the Ad2 E4 region. The adenovixus vector is referred to as AD2-ORF6/PGK-CFTR and is illustrated schematically in Figur 28- 7he entire wild-type Ad2 genome has bee previously sequenced (Roberts, R.J., (1986) In Adenovirus DNA, W. Oberfier, editor, Matinus Nihoff Publishing, Boston) and the existing numbering system has been adopted here when referring to the wild type genorrne.

Ad2 genomic regions flanking El and E4 deletions, and insertions into the genome are being completely sequenced- Thie Ad2-ORF6fPGK-CFTIR. construt diff~ers from the one used in our earlier protocol (Ad2CFTR- 1) in that the loner utilized the endogenous El a promoter, had no poly A addition signal directly downstream of CFTR and retaned an intact E4 region. The properties of Ad2ICFR- I in tissue culture and in aniimal studies have been reported (Rich et al., (1993) Humanw Gene Therapy 4:461-467; and Zabner el aW. (1993) Nature Generics (in Press).

At ille V' end of the genorne, nucleotides 357 to 3328 of Ad2 have been decleted and replaced with (in order 5' to 3')22 nucleotides of linker, 534 nucleotides of the PGK promoter, 86 nucleotides of linker, nucleotides 123-4622 of ihe published CFTR sequence (Rtiordan et al. (1989) Scienzce 245:1066-1073), 21 nucleorides of linker, and a 32 nucleolide synthetic EGH poly A addition signal followed by a final I1I nucleoudes of linker. The topology of the 5' end of the recombinant molecule is illustrated in Figure 28.

At the end of the'genorne of Ad2-ORF6/PGK-CFTR, A42 sequences berween nucleotides 32315 and 35577 have been deleted to remove all open reading firmes of E4 but retain the E4 promoter, the E4 cap sites and first 32-37 nucleofides of E4 m.RNA. 'The deleted sequences were replaced with a fragment derived by PCR which contains open reading frame 6 of Ad2 (nucleotides 34082-33178) and a synthejic poly A addition sirnafl.

The topology of the 3'enid of the molecule is shown in Figure 28. The sequence of this segment of the molecule will be confirmed. The reaider of the Ad2 viral DNA sequence is 04 t -r O Davies Collison Cave Daves oilisol Cve04-02-98 14'35 (641 #48 57 published in Roberts, R.J. in Adenovirus DNA. Oberfler, Mahinus Nihoff Publshng.

Boston, 1986 The overall size of the Ad2-0R76/PGK-CFTR vector is 36,336 bp which is 101.3% of full length Ad2.

The CFTR transcript is predicted to initiate at one of three closely spaced transcripiional stat Sites in the cloned P0K promoter (Singer-San et al. (1984) Gene 32:409- 417) al nucleouides 828, 829 and 937 of the recombinant vector (Singer-Sam et al. (1984) Gene 32:409-417). A hybrid 5' untranslated region is comprised of 72, 80 or 8 1 nucleotides of PGK promoter region, 86 nucleotide of linker sequence, and 10 nucleolides deived from the CFTR insert. Transcriptional termination is expected to be directed by The BGH poly A addition signal at recombinant vector nuclootide 5530 yielding an approximately 4.7 kb transcript. The CFTR coding region comprises nucleotides 1010-5454 of the recombinant virus and nucleotides 182, 181 or 173 to 4624, 4623, or 4615 of the PGK-CFTR-BGE mRnNA respectively, depending on which transcriptional initiation site is used. "Within the CM~ cDNA there are two differences from the published (Riordan et al, cited supra) cDNA sequence. An A to C change at position 1990 of the CFTR cDNA (published CFTR cDNA coordinates) which was an error in the original published sequence, and a T to C change introduced at position 936. The change at position 936 is translationally silent but increases the stability of the cDNA when propagated in bacterial plasmids (Gregory et al. (1990) Nature 347:392-386; and Cheng et al. (1990) Cell 63.827-934). The 3'untransated region of the predicted CFTR transcnpi comprises 21 nucleotides of linker sequence and approximately 10 nucleotides of synthetic BGH poly A additional signal.

Although the activity of CFTR can be measured by electrophysiological methods, it is relatively difficult to detect biochemically or irnnunocyiochemically, particularly at low levels of expression (Gregory et al., cited supra; and Denning et al. (1992) J Cell Biol.

118:551-559). A high expression level reporter gene encoding the E. call 0 galactosidase protein fused to a nuclear localization signal derived from the SV40 T-antigen was xhereforr constructed. Reporter gene transcription is driven by the po-werft CNvV early gene constitutive promoter, Specifically, The El region of wild type Ad2 between nucleotides 357- 3498 has been deleted and replaced it with a 515 bp fragment containing the CMV promoier and a 3 252 bp fragment encoding the j3 galactosidase gene.

Re&Wggtry Characteriqmics of the Elements of the AI2-OR.F6/PGK-CFTR In general terms, the v'ector is similar to several earlier aderiovirus vectors encoding CFTR but it differs in three specific ways from the Ad2/CFTR-1 construct.

Traniscription of CFTR is fromi the PGK promoter. This is a promoter of only moderate strength but because it is a so-called house keeping promoter We considered it more likely to be capable of long term albeit perhaps low level expression. I may also be less C I RA4, ~104 o~ a L.JA Ij.3,J AUUWI S- 58likely to be subject to "shut-down" than some of the very strong promoters used in other studies especially with retroviruses. Since CFTR is not an abundant protein longevity of expression is probably more critical than high level expression. Expression from the PGK promoter in a retrovirus vector has been shown to be long lasting (Apperley et al. (1991) Blood78:310-317).

Polvadenylation Signal Ad2-ORG6/PGK-CFTR contains an exogenous poly A addition signal after the CFTR coding region and prior to the protein IX coding sequence of the Ad2 El region. Since protein is believed to be involved in packaging of virions, this coding region was retained.

Furthermore, since protein IX is synthesized from a separate transcript with its own promoter, S: to prevent possible promoter occlusion at the protein IX promoter, the BGH poly A addition signal was inserted. There is indirect evidence that promoter occlusion can be problematic in that Ad2/CMV pGal grows to lower viral titers on 293 cells than does Ad2/pgal-1. These 15 constructs are identical except for the promoter used for P galactosidase expression. Since the CMV promoter is much stronger than the Ela promoter it is probable that abundant transcription from the CMV promoter through the P galactosidase DNA into the protein IX coding region reduces expression of protein IX from its own promoter by promoter occlusion and that this is responsible for the lower titer ofAd2/CMV-pgal obtained.

Alterations of the E4 Region A large portion of the E4 region of the Ad2 genome has been deleted for two reasons.

The first reason is to decrease the size of the vector used or expression of CFTR. Adenovirus vectors with genomes much larger than wild type are packaged less efficiently and are therefore difficult to grow to high titer. The combination of the deletions in the El and E4 regions in Ad2-ORF6/PGK-CFTR reduce the genome size to 101% of wild type. In practice it is straightforward to prepare high titer lots of this virus.

The second reason to remove E4 sequences relates to the safety of adenovirus vectors.

A goal of these studies is to remove as many viral genes as possible to inactive the Ad2 virus backbone in as many ways as possible. The OF 6/7 gene of the E4 region encodes a protein that is involved in activation of the cellular transcription factor E2-F which is in turn implicated in the activation of the E2 region of adenovirus (Hemstrom et al. (1991) J. Virol.

65:1440-1449). Therefore removal of ORF6/7 from adenovirus vectors may provide a further margin of safety at least when grown in non-proliferating cells. The removal of the El region already renders such vectors disabled, in part because El a, if present, is able to displace E2-F from the retinoblastoma gene product, thereby also contributing to the stimulation of E2 transcription. The ORF6 reading frame of Ad2 was added back to the El-E4 backbone of the Ad2-ORF6/PGK-CFTR vector because ORF6 function is essential for production of the recombinant virus in 293 cells. ORF6 is believed to be involved in DNA replication, host WO 94/12649 Jr U I -7jl -59cell shut off and late mRNA accumulation in the normal adenovirus life cycle. The E1-E4- ORF6+ backbone Ad2 vector does replicate in 293 cells.

The promoter/enhancer use to drive transcription of ORF6 of E4 is the endogenous E4 promoter. This promoter requires Ela for activation and contains Ela core enhancer elements and SP1 transcription factor binding sites (reviewed in Berk, A.J. (1986) Ann. Rev.

Genet. 20:75-79).

Replication Origin The only replication origins present in Ad2-ORF6/PGK-CFTR are those present in the Ad2 parent genome. Replication of Ad2-ORF6/PGK-CFTR sequences has not been detected except when complemented with wild type El activity.

Steps Used to Derive the DNA Construct Construction of the recombinant Ad2-ORF6/PGK-CFTR virus was accomplished by S* 15 in vivo recombination of Ad2-ORF6 DNA and a plasmid containing the 5' 10.7 kb of adenovirus engineered to have an expression cassette encoding the human CFTR cDNA driven by the PGK promoter and a BGH poly A signal in place of the El coding region.

The generation of the plasmid, pBRAd2/PGK-CFTR is described here. The starting plasmid contains an approximately 7.5 kb insert cloned into the Clal and BamHI sites of 20 pBR322 and comprises the first 10,680 nucleotides of Ad2 with a deletion of the Ad2 sequences between nucleotides 356 and 3328. This plasmid contains a CMV promoter inserted into the Clal and Spel sites at the region of the El deletion and is designated pBRAd2/CMV. The plasmid also contains the Ad2 5' ITR, packaging and replication sequences and El enhancer. The El promoter, Ela and most of Elb coding region has been deleted. The 3' terminal portion of the Elb coding region coincides with the pIX promoter which was retained. The CMV promoter was removed and replaced with the PGK promoter as a £Cla and Spel fragment from the plasmid PGK-GCR. The resulting plasmid, pBRAd2/PGK, was digested with Avrll and BstBI and the excised fragment replaced with the Spel to BstBI fragment from the plasmid construct pAd2Ela/CFTR. This transferred a fragment containing the CFTR cDNA, BGH poly A signal and the Ad2 genomic sequences from 3327 to 10,670. The resulting plasmid is designated pBRAd2/PGK-CFTR. The CFTR cDNA fragment was originally derived from the plasmid pCMV-CFTR-936C using restriction enzymes Spel and Ecl13611. pCMV-CFTR-936C consists of a minimal CFTR cDNA encompassing nucleotides 123-4622 of the published CFTR sequence cloned into the multiple cloning site of pRC/CMV (Invitrogen Corp.) using synthetic linkers. The CFTR cDNA within this plasmid has been completely sequenced.

The Ad2 backbone virus with the E4 region that expresses only open reading frame 6 was constructed as follows. A DNA fragment encoding ORF6 (Ad2 nucleotides 34082- 33178) was derived by PCR with ORF6 specific DNA primers. Additional sequences Davies Collison Cave -298 4:4 [415 04-02-98 14:34 641 supplied by the oligonucleotides include cloning sites at the 5' and 3' ends of the PCR fragment. (QIn1 and DamHI respectively) and a pvIy A addition sequence AATAAA at the 3 end to ensure correct polyadenylation of ORF6 rnRNA. The ?CK fragment was cloned into pBhsesczipy (Stratagene) along with an Ad2 fragment (nucleotides 35937-35577) containing the inverted terminal repeat, E4 promoter, E4 mnRNA cap sites and first 3 2-37 nucleotides of E4 niRNA to create pORYF6. A &UailrnH1 fragment encompassing the ITR and ORF6 was used to replace thc Snl1-T~amMi fragment encomipassing -the ITR and E4 deletion in pAdAE4 contains the 3'end of Ad2 from a 5=1 site to the 3'ITR (nucleotides 27123-35937) azd is deleted for all E4 sequences including the promoter and poly A signal (nucleotides 328 3564 The resulting construct, pAdE4ORr6 was cut with k~sI and ligated to Ad2 DNA digested with EjNI nucleodde 28612). 293 cells were tranisfected with the ligation reaction to generate virus containing only open reading frame 6 from the E4 region.

In Vitrr Snudies with Ad2-R6PKCT The ability of Ad2-OR.F6IGK-CFTR to express CFTR in several cell lines, including human HeLa cells, humnan 293 cells, and primary cultures of normal and CFhurnan airwayepithelia was tested. As an example, the results from the human 293 cells is related here.

When human 293 cells were grown on culture dishes, the vector was able to transfer CFTR eDNA and express CFTR as assessed by inimunoprecipitation and by functional assays of halide efflux. Gregory, R.J. et al. (1990) Nature 347:382-386; Cheng, S.H. et al. (1990) Cell 63:827-434. More specifically, procedures for preparing cell lysates, immunoprecipitation ofproteins using anti-CFTR antibodies, one-dimensional peptide analysis and SDSpolyacrylarnide gel electrophoresis were as described by Cheng et al. Cheng, S.H. et al.

(1990) Cell 63:827-834. Halide efflux assays were performed as described by Cheng, S.H. et al. (1991) Cell 66:1027-1036. cAMP-stimulatedUCTR chloride chaxuiel activit~y was measured using thte halide sensitive fluorophore SPQ in 293 cells treated 'with 500 lU/cell Ad2-OKF6IPGK-CFTR- Stimulation of the infected cells with forskolin (20 jtM) and IBMX (100 jun) increased SPQ fluorescence indicating the presence of functional chloride channels; produced by the vector.

Additional studies using primary cultures of human airway (nasal polyp) epithelia] cells (from CF patients) infected with Ad2-ORF6JPGK-CFTR demonstrated that Ad2- ORF6/PGK-CFTR infection of the nasal polyp epithelial cells resulted in the expression of cAMP dependent CY channels. Figure 29 is an ex~ample of the results obtained fron such studies. Primary cultres of CF nasal polyp epithielial cells were infected wAith Ad2- ORF6/PGK-CFTR at multiplicities of 0.3, 5, and S0. Three days post infecion, monlay'ers were mounted in Ussing chambers and short-circuit current was measured. At the indicated times: 10 jtM arniloride, cAMP agonists (10 liM forskolin and 100 WiM 1MX). and 1 mnM diphenylainine-2-Car'boxylate wcre added lo the inucosal solution.

iV -1 &.Vll- I I -61- In Vivo Studies with Ad2-ORF6/PGK-CFTR Virus preparation Two preparations of Ad2-ORF6/PGK-CFTR virus were used in this study. Both were prepared at Genzyme Corporation, in a Research Laboratory. The preparations were purified on a CsC1 gradient and then dialyzed against tris-buffered saline to remove the CsCI. The preparation for the first administration (lot had a titer of 2 x 1010 IU/ml. The preparation for the second administration (lot had a titer of 4 x 1010 IU/ml.

Animals Three female Rhesus monkeys, Maacca mulatta, were used for this study. Monkey C (#20046) weighed 6.4 kg. Monkey D (#20047) weighed 6.25 kg. Monkey E (#20048) weighed 10 kg. The monkeys were housed in the University of Iowa at least 360 days before the start of the study. The animals were maintained with free access to food and water throughout the study. The animals were part of a safety study and efficacy study for a different viral vector (Ad2/CFTR-1) and they were exposed to 3 nasal viral instillation throughout the year. The previous instillation of Ad2/CFTR-1 was performed 116 days prior to the initiation of this study. All three Rhesus monkeys had an anti-adenoviral antibody response as detected by ELISA after each viral instillation. There are no known contaminants 20 that are expected to interfere with the outcome of this study. Fluorescent lighting was controlled to automatically provide alternate light/dark cycles of approximately 12 hours each. The monkeys were housed in an isolation room in separate cages. Strict respiratory and body fluid isolation precautions were taken.

25 Virus administration For application of the virus, the monkeys were anesthetized by intramuscular injection of ketamine (15 mg/kg). The entire epithelium of one nasal cavity in each monkey was used for this study. A foley catheter (size 10) was inserted through each nasal cavity into the pharynx, the balloon was inflated with a 2-3 ml of air, and then pulled anteriorly to obtain a tight occlusion at the posterior choana. The Ad2-ORF6/PGK-CFTR virus was then instilled slowly into the right nostril with the posterior balloon inflated. The viral solution remained in contact with the nasal mucosa for 30 min. The balloons were deflated, the catheters were removed, and the monkeys were allowed to recover from anesthesia.

On the first administration, the viral preparation had a titer of 2 x 1010 IU/ml and each monkey received approximately 0.3 ml. Thus the total dose applied to each monkey was approximately 6.5 x 109 IU. This total dose is approximately half the highest dose proposed for the human study. When considered on a IU/kg basis, a 6 kg monkey received a dose approximately 3 times greater that the highest proposed dose for a 60 kg human.

-62- Timing of evaluations.

The animals were evaluated on the day of administration, and on days 3, 7, 24, 38, and 44 days after infection. The second administration of virus occurred on day 44. The monkeys were evaluated on day 48 and then on days 55, 62, and 129.

For evaluations, monkeys were anesthetized by intramuscular injection of ketamine mg/kg). To obtain nasal epithelial cells after the first viral administration, the nasal mucosa was first impregnated with 5 drops of Afrin (0.05% oxymetazoline hydrochloride, Schering-Plough) and 1 ml of 2% Lidocaine for 5 minutes. A cytobrush was then used to gently rub the mucosa for about 3 sec. To obtain pharyngeal epithelial swabs, a cotton-tipped applicator was rubbed over the back of the pharynx 2-3 times. The resulting cells were dislodged from brushes or applicators into 2 ml of sterile PBS. After the second administration of Ad2-ORF6/PGK-CFTR, the monkeys were followed clinically for 3 weeks, and mucosal biopsies were obtained from the monkeys medial turbinate at days 4, 11 and 18.

15 Animal evaluation.

Animals were evaluated daily for evidence of abnormal behavior of physical signs. A record of food and fluid intake was used to assess appetite and general health.. Stool consistency was also recorded to check for the possibility of diarrhea. At each of the evaluation time points, rectal temperature, respiratory rate, and heart rate were measured.

20 The nasal mucosa, conjuctivas and pharynx were visually inspected. The monkeys were also examined for lymphadenopathy.

Hematology and serum chemistry Venous blood from the monkeys was collected by standard venipuncture technique.

Blood/serum analysis was performed in the clinical laboratory of the University of Iowa Hospitals and Clinics using a Hitatchi 737 automated chemistry analyzer and a Technicom H6 automated hematology analyzer.

Serology Sera from the monkeys were obtained and anti-adenoviral antibody titers were measured by ELISA. For the ELISA, 50 ng/well of killed adenovirus (Lee Biomolecular Research Laboratories, San Diego, Ca) was coated in 0.1 M NaHCO3 at 4* C overnight on 96 well plates. The test samples at appropriate dilutions were added, starting at a dilution of 1/50. The samples were incubated for 1 hour, the plates washed, and a goat anti-human IgG HRP conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) was added for 1 hour. The plates were washed and O-Phenylenediamine (OPD) (Sigma Chemical Co., St.

Louis, MO) was added for 30 min. at room temperature. The assay was stopped with 4.5 M H2SO4 and read at 490 nm on a Molecular Devises microplate reader. The titer was calculated as the product of the reciprocal of the initial dilution and the reciprocal of the *r -63dilution in the last well with an OD>0.100. Nasal washings from the monkeys were obtained and anti-adenoviral antibody titers were measured by ELISA, starting at a dilution of 1/4.

Nasal Washings.

Nasal washings were obtained to test for the possibility of secretory antibodies that could act as neutralizing antibodies. Three ml of sterile PBS was slowly instilled into the nasal cavity of the monkeys, the fluid was collected by gravity. The washings were centrifuged at 1000 RPM for 5 minutes and the supematant was used for anti-adenoviral, and neutralizing antibody measurement.

Cvtology Cells were obtained from the monkey's nasal epithelium by gently rubbing the nasal mucosa for about 3 seconds with a cytobrush. The resulting cells were dislodged from the brushes into 2 ml ofPBS. The cell suspension was spun at 5000 rpm for 5 min. and resuspended in 293 media at a concentration of 106 cells/ml. Forty pl of the cell suspension was placed on slides using a Cytospin. Cytospin slides were stained with Wright's stain and analyzed for cell differential using light microscopy.

Culture for Ad2-ORF6/PFK-CFTR To assess for the presence of infectious viral particles, the supernatant from the nasal brushings and pharyngeal swabs of the monkeys were used. Twenty-five pl of the supernatant was added in duplicate to 293 cells. 293 cells were used at 50% confluence and were seeded in 96 well plates. 293 cells were incubated for 72 hours at 37 0 C, then fixed with a mixture of equal parts of methanol and acetone for 10 min and incubated with an FITC label anti-adenovirus monoclonal antibodies (Chemicon, Light Diagnostics, Temecuca, Ca) for 30 min. Positive nuclear immunofluorescence was interpreted as positive culture.

Immunocvtochemistrv for the detection of CFTR.

Cells were obtained by brushing. Eighty pl of cell suspension were spun onto gelatincoated slides. The slides were allowed to air dry, and then fixed with 4% paraformaldehyde.

The cells were permeabilized with 0.2 Triton-X (Pierce, Rockford, Il) and then blocked for minutes with 5% goat serum (Sigma, Mo). A pool of monoclonal antibodies (M1 3-1, M1-4, and M6-4) (Gregory et al., (1990) Nature 347:382-386); Denning et al., (1992) J. Cell Biol.

118:(3) 551-559); Denning et al., (1992) Nature 358:761-764) were added and incubated for 12 hours. The primary antibody was washed off and an antimouse biotinylated antibody (Biomeda, Foster City, Ca) was added. After washing, the secondary antibody, streptavidin FITC (Biomeda, Foster City, Ca) was added and the slides were observed with a laser scanning confocal microscope.

Davies Collison Cave Davis Cllion ave04-02-98 14:34 [64] #6 -64- Biosie To assess for histologic evidence of safety, nasal media] ttzrbinate biopsies wvere obtaned on day 4, 11 and 18 after the second viral administration as described before (Zabner et al (1993) Human Gene Therapy, in press). Nasal biopsies were fixed in 4% formaldehyde and M&E stained sections were reviewed.

RESULTS

Studies of efficacv.

To directly assess the presence of CFTR, cells obtained by brushing were plated onto slides by cytospin and stained with antibodies to CFTR. A positive reaction is clearly evident in cells exposed to Ad2-ORF6IPGK-CFTR. The cells were scored as positive by imunocyochemnistry when evaluated by a reader blinded to the identity of the samples.

Cells obtained prior to infection and from other untreated monkeys were used as negative controls.

None of the monkeys developed any clinical signs of viral infections or inflammation.

There were no visible abnormalities at days 3. 4,7 or on weekly inspection thereafter.

Physical examination revealed no fever, lyraphadenopathy, conjunctivitis, coryza, tachypnea, or tachycardia at any of the time points. There was no cough, sneezig or diarrhea. The monkeys had no fever. Appetites and weights were not affected by virus administration int either monkey. The data are summarized in Figures 30A-30C.

The presence of live virus %%as tested in the supernatant of cell suspensions from swabs and brushes from each nostril and the pharynx. Each supernatant was used to infect the virus-sensitive 293 cell line. Live virus was never detected at any of tlc 'Lime points. The rapid loss of live virus suggests that there was no viral replication, The results of complete blood counts, sedimnentation rate. and clirical chemistries are shown in Figure 31A-3 IC- There %%as no evidence of a systemic jnflammuatory response or other abnormalities of The clinical chemistries.

Epithelial inflammation. was assessed by cytological examination of Wright-stained cells (cytospin) obtained from brushings of the nasal epitheliumn. The pcrcnragc of neutrophils arnd lymphocytes from the infected nostrils were compared to those of the control nostrils and values from four control monkeys. Wright stains of cells from nasal brushing were performed on each of the evaluation days. Ncutrophils and lymphocytes accounted for less than S% of total cells at all time points. The data am~ shown in Figures 32A-32C. The data indicate that administration of Ad2-ORF6/PGK-cFTR caused no change in the distribution 4RA4/ or number of inflammatory cells at any of the time points following virus administration.

E j 04C u' T14~ -t ?o P:\OPERUMS\43655-97 chn.doc-21/8/(K)X even during a second administration of the virus. The biopsy slides obtained after the second Ad2-ORF6/PGK-CFTR administration were reviewed by an independent pathologist, who found no evidence of inflammation or any other cytopathic effects.

Figures 33A-33C show that all three monkeys had developed antibody titers to adenovirus prior to the first infection with Ad2-ORF6/PGK-CFTR (Zabner et al. (1993) Human Gene Therapy (in press)). Antibody titers measured by ELISA rose within one week after the first and second administration and peaked at day 24. No anti-adenoviral antibodies were detected by ELISA or neutralizing assay in nasal washings of any of the monkeys.

These results combined with demonstrate the ability of a recombinant adenovirus encoding CFTR (Ad2-ORF6/PGK-CFTR) to express CFTR cDNA in the airway epithelium of monkeys. These monkeys have been followed clinically for 12 months after the first viral administration and no complications have been observed.

The results of the safety studies are encouraging. No evidence of viral replication was found; infectious viral particles were rapidly cleared. The other major consideration for safety of an adenovirus vector in the treatment of'CF is the possibility of an inflammatory response. The data indicate that the virus generated an antibody response, but despite this, no evidence of a systemic or local inflammatory response was observed. The cells obtained by brushings and swabs were not altered by virus application. Since these Monkeys had been S 20 previously exposed three times to Ad2/CFTR-I, these data suggest that at least five sequential exposures of airway epithelium to adenovirus does not cause a detrimental inflammatory response.

These data indicate that Ad2-ORF6/PGK-CFTR can effectively transfer CFTR cDNA to airway epithelium and direct the expression of CFTR. They also indicate that transfer and 25 expression is safe in primates.

SEquival.ents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention 30 described herein. Such equivalents are intended to be encompassed by the following claims.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or 0 R4, "comprising", will be understood to imply the inclusion of a stated integer or step or group S" of integers or steps but not the exclusion of any other integer or step or group of integers or 3- 2-98:11!58 #2/7 2 0/ 7 TABLE I -6 gyFo h C wTi1L DB 13)4w T 7 T)g K464M N 9 =EI AlSO Y 10 NBDI gm 10 IQ 3D NMI MR N 30 NRD1 3S491 y it NRD1 aSSID 'Y 11 JISDI NoWomQ N 15 sEo Kl2S3b N 20 NRD2 Thwi1 ii 22 -I-bm TABLE ii Sao. ID.NO: 2030 40 50 cATchZcAAT A^TAT^CCTT AlToATT GAOcCAATA TGOATGTA93 owG ar GTA~rAGTTA TTATATGGAA TAAAACCTAA CT!COOTAT ACTATTACTC CCCC-ACCTCA ____RVERTUD TERMIAL KEPETETON-ORXOIN OP REPLIC ATIOlJ so 0 090 1m 110 13D TTOA3CGIO 0cCOOOOCO TOAACMG OCOG=ArO TAGrAGTrGYO acG0OaArGr AACACrOCAC CGC13CCCG ACCCTTWCCC COCCCACTGC ATCATCAC.AC CGCCTrCAc-A INVRTD TDINAL RBPTITI ON- RIGN OF R 130 140 1.W 160 110 160 GATITOCAA GTGGCGOA ACACA=GA; GCOCCOGATO TOOTAAAAaT OAC~rITTG CTACAAWIT CACACCOCCT TOMGACATr CGCO0CCTAC ACCATITCA CTIMAA"Ar 19D 2 210 230 230 240 GGrGGCG GrGrTACOO OGTGACAA T=XKrGCO GrTAGGCG GAXTT@?AO cACACOCOOC CACATATGCC CTTCA=TF AAAAWGCGC CAAA&TCCOGC CTACAACXI'C 2.10 ?w0 7m 29029 300 ,hAA&TTTOOO carAACCAAr. TAATOrTTOO CCATTrTc~c owAAAACrO AATAAOGA Aa-rTAA^CCC oCAT-1GTTC ATTACAAACC GGrAAAOC CCCrZrGALC rTATTrCCT 0 Gb EA ENHANCER AND VIAL PACKAGING DOMAIN 0-b 110 310 330 330 340 so034 AUrOAAAXCT QAATAATTCT GMGTACMIA IA3JCOOAA, TATrrTCrA OOCCCG TCAC=TAGA CTrATrAAO& CACAATGAMT AICQCOCAfl ATAAACAOAT CCCOOCOCCC 120b bIA IANCEi AND VIRAL PACKAGIG IDOMA0h 170 310 34 4W0 410 2 QwhCTTrGACC GMTAC(MW~ AGArCVOCC ALITMrTZ CTCAGUIGIT TTCC0ZTC CTOAAACTOO CMAATOC&CC TCTW.OCG00 TCCACAAAA& OAOFCCAC,#A AAWGAWG 2__1A ENHANCER -0 10 BIAPIOMOTISR RGIO?40 430 440 4= 470 CGCAAA0 i-rOOoTT ATrArrATArw TCA0=QTACO COCAUQt&T TrAICCCOO GCAGrTTC AACCOAAA& TAATAhAAC A0TCOA,C GCeICACATA ^&TATOOC R__IAPROM'OTBUGION c 100 490 510 520 530 s T*A~wrcTc AAQoAmicC& TCTrmAn-m cAaomU1?AO Aarx-rcTcc TmcoADccGC ACTCAAGGAG TrCTCCOOTO AOA.ACTCACO GrCOCICATC TCAAAAGAGO AOCTCOOCO IIflMOB HY13RMDBA-*e"rR.]D MSSAGE d E1AMRNA5'UNTkANSLATED d

'TSEC

-04 wO c) 'ANr 0 3- 2-98;11!58 1/7 2 1 7 67 TABLE fl-oaad SEQ NO- 3 SSO 50 5rO 58 590 6 TCCGA0CTAG TAACOGCGC CAOr1'OCrG CAGATATICAA AMGICACGGT ACCCOAGADa AOCTCGATC ATTGCCGCG GCrrACACOAC GrCTA.TA0TT 'TCA6WTGCCA TOOGCTTCT h JWD SDIA42PTR-ELD MESSAGE 1 SY3!flWTCLIK" SEQUENCES _40 6 610 620 650 60 650 665 ccATocAG GTCGccTcT* *AAAAaaccA GCGcrrrrcTC CAAAcTT; TTCAOCTO0A QGTACGrCTC CADCOAGAC C'r1TrCGa CGCAACAGAG GrTTTGAAAAA AACMQACCT 1AQ R LPL 3 KA S vY E KL F FS 6 W -cysTicPiURosisTIAISMHBRAXr!CON!DUC'TANCB REGULATOR; COD )CYZRlDEIA-CFTR-ElB N2SSA02 h> -140i 12__I3 T0622KUMAN CPTXCDNA 18__ob m 65 70 710 71 CCAOACCAAT TTTGAflGAAA GOATACADAC AOCGCCTGGA. ATTGTCAGAC ATATACCAA WTCTOGITA AAACTCCTrT CCTATGYCTG TCGG&CCrT AACA~rCTO TATATGGTM Ta P I--L*.RK GYM Q KL A L SD 3) YQ -CYSTICFIBROSIS TRANISMSMANE CONDUCTANCE REGULATOR: CODON HYBID 9 LA-CPTR-E13 MESSAGE rn -12TO 4627,OF uumArc rTR CDIIA '4 __226& ISO 74D 750 WD 770 1 TCCCrrCT0T TOATTCTUCT GACAAerAT CTGAAAATT GGAAAGAOAA TOoGh~Ac3AG AOOAGACA ACTAA~acGA CTOTTAGATA GACITTZAA CCrrrCTCTT ACCTArCTC! I p a V IDS A D N L E 3 WL 2 U. I WD ft, 10813 TRANS BIORAIE COND1JCTA1ICM mP.OULATOR:,.CODON XYRID 31A.CPT-E131NaSSAGE lax_ 123 TO46I22OF HUMAN CPTU. CDNA% 3 0 ___31C-b 82 m3 so AGCTooCTTC AA^GAh.AATA CCTAAACTCA flTAATG=C TCOCGATQT TIMTOO& TCG&CCAA TTTCTTTA GATTTIMIF AATr4CQWA AGCLAXTACA AAAAACY E L AS3 K N P KL 1 NAL. I itC PFP 'W> -CYSTIC FIBOSIS TRANSIONDRANS CONDUCTANCE RPOULATOR; CODQN HYBMX I IA-CPTI-E13 MESSAGE M -23 To462OP HUMAN CPTR CDNA 37W No0 360 W70 99O 9 CiAT-rrATM; CT*XG0"TC TFTrrATATT TADGOC3&AGr CACCAAAOCA GTACAOCCTC CTAAATAr-AA OATACCT!TAD AAAAAXAXAA ATCCCCTTCA 0IS~FFCT CAT=QIGAG U. p K F Y 0 I P LY L GEHV T KA ',VQ P -CYSTXCPMIR0EIB TkANSMEMEEANE CONDUCTANCE REULATEOft; CODOR HYBRID E1A-CPTIR-B15 MESSAGE mm 1;3To 4 622 OF HUMA CTRCD NAZA TCTrAarGOG AAGAATCATA GCTICCTATO ACCCGOIATAAI CmaAGAA COTCTATCo .AGAATACCC TICTTAISrA2 COAAOOATA% TOGOCCT~rr GftCCTCCTT GCOAOTAGC L L L G R. I I A S Y D F D N X R E R 9 1> -CYSTICFIB105$ TRAl 3)flNEANE CONDUCTANCE RGULATOR; COD ON -k RYIBRXD RIA-CPTR-2131 MESSAGE 123 TO 4622OFHUMAN CPT& CD)A___83 gm w0g 10 1010tw C"ArCT ACMAXAMC TT&TOCCTTC TCILKATT0T11 OAGOACACTO C'T=ACACC GCTAAATAOA TCCGZAX=CG AAXACOAA.O AOAAATAACA6 CTCCTIGAC: GAM&TGTG A I YL GI1G L CL L F I V R T L L Llb CYSTIC FIROsXS TRANUIRMBRANE CONDUTCTANCE REOULATOOI; CODON b HYBRID B1A.CPTI..II MSSAGE o_ 123 TO 4622 OP HUMAN CFT CIDNA _aXj

~SEC

/'VT 0~ 3- 2-98;11!.58 2/7 22/ TABL~E fl-countie SBQ. ID. Not~ 3 1030 I040 low 1060 1I0=(1 cAOcCATflT TGoccTTCALT C^CATTOGA& TOCAWAGAG AATAOCTAW TTTAGTITGA GrC~aFAMA ACOAAMlA efGrAACCTT AC~rCTACTC TACGArAC ATACA&ACT P A F LEH HI 0 X Q MR I A M PS L> CysTIC P111031 TRAN3MEMBRANE CONDUCTANCE REGULATOR; COPON 123 TO 46230?I HUMAN C FTR CVXA 6m L(O11i 1110 112D I13 1140 =rATAOA GACMFAMDG cTG1CAAGcc GTGITcrArzA TAAAATAACr A6TTOGACAAC AAATATTCTr CTGAAATTTC GACMITTcG CACAAOATCT ATFFFATrCA TAACCTGrrG I Y K TL K L 5 8 R VL D K I S 1 0 Qj CYSTICFIRROSIS TXAJISNMWMRAJIR CONDUCTANCE REGULATOR; CODOI( HYEJ.XD R1A-CFTR-BIS MESESAGE 123 T4622 OF VMAN CPTRCDWA1. 6, ILU 116 70 1I3 LB130

Z)

1-rarrAOTCT CrrrrCCA.C AACCrGAACK AAMl~r(IA AoGAcTTOCAL Tro~acAAATr AACAATCAOA 05AAIAGQrTO TTGGACTTGr TTAAACiACT TCCTGA.ACGr ACCGrrA.

L V S L L S N N L N4 K PD E 3L A V A CYST[CPIBIOSIS TRAN33LBUERANP- CONDUCTANCE REGUL&TOR; CODON R YBRWD ELA-CIR-E1R MESSA013 M1 ___13TOLd220PMATCPTRC7I ____73Cb I110 Wo 1330 124a x1D 260 TCGr=rGA7 cCTrCCFFIT CAAGTGWCAC TCCTCATOG GTAATCT00GAMT(qrAr~C ACCACACCrA GOOAOGA.AAC GMTACW0 ADGA0ACCC CG&TAACC crAACATG, F I A P L Q V A L L MO L L L TRANSMUG.ANE CONDUCTANE EGULATOR; CODON h HYBDE ZLA.(VT-X II MESSAGE 9i _123T046220FjEUMAXCPTR CDKA 177 1380 IZO 131.0 I AGGCGI==O CTTCTGYOOA CTTGOflCC TGA2TAOr1C TaGCccrrrrr C*~oaCT0 TCCCAMACO 0AhACAT OAACCAAAUG ACTATCAGA ACGG".AAA GrCGACC Q AS5 A P CO0 L G P LI V L A L F Q A G CYSTlCPIRROSIS TR-ANSME30X.A3I CONDUCTANE REGULATOR;. CODON h XTBRtD ElA-CFTR-E IS MESAGEh> SZ 33T0.6220PffUMANCPTIRC3A 1330 lam0 L 360 1370 LIM3 TAOKGWAAAT G&TCULTGAAG TALC~fAATC AGA09McOG OAAD&TCA~r G*AAGAMOT ATCCcTcTITA CTACTACrTC ATO1CCTrAa TCTCTCOACC C7'rcTAOTCA c-rMrT13AC L. GR MId MK Y R v Q R AG a KI S B R I> _CYSTICFURO81S TR"SURMRANE CONDUCTANCE REGULATOR; CODON h HYBRID 81A-CPTR411b M5A.GR 8_M_123TOA622OPKVXEANCFTRCDNAL 4Wi- 91 1390 1400 1410 1420 1430 LUD 0 TOAXTAMTC AGAAATGAT TACAG"C AATCTOITAA OOCATWTOC TGOGAADAW ACTAA.TooAO !CTTPTAA ATOrCTc[Ao ITAOACAATI CCGI ArGACG ACCCTTCTTC V I TB S M 1 7 R D Q 5Y V A Y C WE 2 CY8TZCPhDROSI TAtSEBRN CONDUTANE REGULA&TOR. CODON RYRRIDA-CPTR-IMZSSAGZ 9M 02 1221TO A622 OP HUAN CFTh CNA Ol 1450 1440 1470 1480 19 m CAATAA AATGArTAA ATAAOAC MAACAGAACT GAAACTGACT CGQAAGMAL CrTTAccrrrr TrAcTAAccT TTGAAMTCTio TTr~rCTTroA cTrTOACTaA OcrcCGr A MBK MISB N LR QTEL K L T R K A> _CYSTICP3R0S1S TRA24SME)MRANE CONDUCTAwTC REGULATOR: CODON__ h EMBRID 9 A-c~r& is IMSSAOR 123 TO 4622 OF AUlAAN C PTR CD A, luau>

~SEC

4 L T

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0~ 3- 2-98;11!58 3/7 23/ 69 TAALE UMcoawzutcd1 1510 LUG13 1~ 1550 CCrAGrGA0 ATACTTCAA.T A=CACCT TCTTCT7CTC AGIGrTTT OTOIrOrrTr GGATACACTC TATa&A~aTA TCGAGTCOOA AOAAGAAA TCCCAADAAA CACCACAAA A Y V R Y P N S A P F PS3 ap vP VP> -CYSTICFMIkOSIS TRANSEMBRMIB CONDUCTANCE REGULATOR; CODON h HBDEA-CMrIB31.CESAGZ 123 TO 482201HUMAN CPTI CDWA 1080 a TATCTOTrOCT TCCCrTOCA CTAATCAAAO OMTCkTCCT CCOOAATA. TrCACCACCA ATAGACACQ&AM3ATACOT GITrAGMflC CrA0TAOA GGCCFTIAT AAGTO~rOGT L 6 V L PTYA L I K 0 1 1L Rt KI P TT> -CYSTIC FIflOSIS TRANIISM5MRANrE CONDUCrANCE REGULATOR,. COD WI -h HYDRID 31A-CPTR-E 1Uh5SSAG, h 111 123 T0462 OPEHUMAN C3TILCD)(A 090 1630 16W 1650 190 1670 1680 TCTCA7TCTG CAIrOTTCTG~ CGCAT"OO TcACrCGOCA ATTCCCI0G 43CTGTACAAA AGiAGrAAGAC C'rA&CAAOAC OCOTACCOCC AeOTAWC~Or TA*AGQCC CG&CATOTT 1 3 P c t V L Rt K A V I I Q P P W A V Qo CYSTICPIBROSKS TRANSMAMBRANE CONDUCTANCE REGULATOR; CODONd HIyRIDEIA-CPTR-BIJMIS9AGB IW 1123 TO 4622 OF HUMAN CPTICIDNA 2005 11.

17W 1710 17;M 1730 1740 CATOWATIGA CTCTCTT43GA OCAATAIJ.CA A AAAAOA TTTCTTACA AAIGCAAG".T OTACCATACT GAGAGA.ACCT CGITATTGT TTrAITCCr AAAGA&TOF TM=L-rTCIA T V Y D L 0 A 1 19 K I Q D .F L Q K Q B> -CYSTIC FIROSIS TRAJISME)QRANfE CONDUCTAKcE IEGULATO)ft; CODON -h EryID B1A-CPT3.J15lMESSAGE 1J 12S T04622O0V1HUMAN CFTVCDNIA-__12Wi5 12 1750 1760 1770 178D 1790 low.

ATAAGACATT GOATXTAAC TTAACGACTA CAGAAGTAI GATGOALGAAW GrAACAGCCT TArrCTOar" CCrrArATFrG AATOCTGA6T arCTTCArCA CTACCICITA CATTOCOGA t ICT L Y N L TT TxJV v MEIN V T A TRANSIIGIGRANE CONDVCTANC33 REGULATOR; CODO >3 H1!3IZD B1AC1'TR41D SSAOR h LNX iSTO4622OFNEUMANCPTRCDONA ___13201i 133 1750 1820 IA30 1840 1350 1160 TCTGWAOOA G13ATTCOG GLAArATTTO AGAAAWCAA ACAAAACAAT A&CAATAOGAA AOkACCCrCtT CCTAAACC CrrAAXAiAC TCTrTC TGA t 11 ILIA TTGTTAXCTT p WEE G P G 8 LF P E AKX Q N N N Nm -CYSTICPIBROSIS TRAIIS3EMBRANP CONDVCTANCE REGULATOR; CODON h H"YBIA-CPTR-BXD MSSAGE -o -t40 2B__Iro 16220 OF UMAN CYTR CIDA I_ A.AACTI'CTAA T0GIGATO&C AOCCTC ITCT TCA~rAiTIT CTCACTCTr GACWCrO TTTOAA92&TT ACCACrACrO TCGGAAhA AGrCATTAAA GAGrGAA" cCA7UAGA3C K T 8 N4 GD D S L P P 3 N P 6 L L 0 T P> -CYSTIcPIDROSIS TRAWSME3CDIANE CONDUCTANCZ RELATOR; CODOW h KY2RD5LA-CMr-XI2MRSSAaE b Id 123 T046223OF HUMAN CFTRCDNA__ h___1 1930 iO m9~ 1960 1970 199 TCCTGAAAGA TATTAAMTC AAGAXTOAAA GAOGKCAGTT GorQOoeaar GCrGOATCCA AnGAcTCT ATAATIAAAG TTCTAXCrTT CTCTOTCAA CAAC=CCM cGA=cAiOGT V LKD I NP V C 9 B R 0Q L L, A V A -CYSTICFIBROSIS TRANSM3Ia lANZ CONDUCTANCE REOUUATOR; CODON__ h HYBRED IA-CFT-Ell MESSAGE 1460 1 23 TO46221OF HUMAN C FTR CD14A

SO

04

~SC)

/$vr 0~ 3- 2-98;11:~58 4/7 24/ 70 TASLK fl-cninir SBQ. ID. Ift 3 CTCGAGCAOO CAA=ATTCA, C'rrCTMTGA TOATrATc1 AGACTOGO CCTTCAGAMO GAMTCGTCC GrrC'rGMAr GLAV1ACT ACTAATACCC TCrTGACCrC GOAAWrCTCC T0A G X -rS IL M M I Id 0 9L PS 9 -CYST[CPI1OS1S TRANSMEMD1LANE CONDUCTANCE REGULATOR; COD)0N I h HYAR1DEIA.CVfl.VB DI1SAOR h 13zx 123 TO4622 OF flUrAN PTRCDNOA 155a r^TTAhA GcACAGTGA AOAATTTCAT TCTGFTC1=C GrTTrcC=00 ATTArG~CTG CATIAATT cGrawcAcT TCTrAAM3rA AGAA(3LT CAAAAWGACC OG4AATCTCC 0R K I xS G KI 1C 8 5c Q P 3w P S CYVSTIC FIROSIS TRANSME)MRAM4E CONDUCTANCE REGUJLATOR; CODON HYDRED BXA.C1rTK-1B MESSAGE b> 1Ii I23TO462IOFELUMANCPTRLCDNA _iwm 2110 212W 2130 2140 2110 216) GCACCATTAA AGAAAATATC AXCTFGGrO 1-1rCCTATGA TGA.ATATArrA TACACLAAWOC CQTWTrAA-WT TCTrTrA0 TAOAAACCAC AAAOOATACT ACTTAXCT AXGLCTICGC OTrI K a NI I p 0 va 0 D Y R Y I S -CYSTlCPIBROSXS TgANSIMBUIBRANE CONDUCTANCE REGULATOR; CO DON b ii HDRM32A-CFR-H1BMESS&GZ -123, To 4622 OF' HUMAX C FTR CDlA!~ -1 210 2113 2m~ 2 2210 2220 TCATCAAAW ATOCCAACTA GAGOGACA TCTCcmmGF TOCAGAGAA& GACAAT.&TAG AGTAUTTTCO TACCUTTOAX CTTCTCCT~r A&OQOFCAA AWOICTCTTT CTGTTAXATC VE IXA C QL a 3 D IS a F A BK DN b eYSTXCm=ROSZ.TWAISWZW3A1'B CONDUCTANCE REGULATOR. CODON__ HYBRID 21 A.CFrI-B 13 MESSAGE 17S1 ___123T4620FEUM.ANCTRCDNA 17m wr1it.

ZO2240 ma 220 aB rrVrrGGAGA. AOGT0OAA"C ACACTGAGIC 0A~TCAACG AOCAMOATE TCTTTxGCAA AAG&ACCTCT TCCACCTTAG TUrOA=TAc CT0CAOTTOC TCGrICTrM. AflAAATCGT V LO0B 0G I TiL G 0 Q R A R 1 S L A> CY11CIOSIS TRAN82MGR.AM' CONDUCTANCE REGULATOR:. CODON bI EEItm EIAcFth-R1isRMESAGE 176)1 12 TO0463207 HUMANCP Th CDNA ___1810).

GA3CAGTATA CAAAGATGCT CIATGrATr TA~rAOAr TCCrrrZTGG&k TACCrAGATG cTcarcATATr aTIcrAco eTAAAcATA& AXAA.TcT.G AoGAfAccT, ATOO*XCrTAC RIA V T K D A D L Y Li L, S P F G Y L Ib -CYSTICF[BIOSIS TIAJUMMRANE CONDUCTANCE RECULATOR: CODON h KY ID E1A4-O'TI-E131BfSSA*B __k i~i 122 TO'463301' HUMAN IR F~CIDNA -18101 I 1S7 TT TTAACA~k& AAAfiAG YA TAAAWOT GMUT1rAA ACTC&T(MOT AACAAAATA AAAI2TQ= TTITCTTAT AACTrrCQA CACAnhrCAT? TGACTACG& TTX~rrWTGA V L T Z KB 3 1 1E C VV C L M A .N KT> __cys=icIBROSIB TkA.NS NAMIE CoNbUCTANCS R=aULTOI-. CODON h NYXID3L ZA-O'T-ER1XRBACE M z8O ZITO-62ZZOFIU38AWCFThCIDNA,, -q LOU Ala 2420 2410 2440 2450 WO6 Oa,.rTaCM CACTTCTAAA AXOG&ACATT TAAAOAAAM TGACAAA T-rAA.TrTOC CCTAAAACCA 0GAAOA.rr TACCTTGTA- MTrTCITTCG AC~rmTT AKITAAAACG R I L V TSR h ME L 9K A D K L I 1> _CYSTXCPIOSIS TRANSMEMBRANR CONDUCTANCE EGULATOk- CODON h RYBD B lA-CPTR-EiB boSSAGE 19s01 1Z3TO4622OPHU]MANCPTRCDNA

SSEC

104 c)v o 3- 2-98;11!:58 5/7 2 5/ 7 71 TABLE II-DArnlinn SHLQ,. NotI0 3 2470 24I0 2490 2.50 2510 2W CAaCATTTT TAX00OACAX TCAOAACT CCMAAATCTA CAOCCAGACT TACCCATc G2TC~hAAAA ATAcccTaTA A.AJ4TCT7OA. GoTTTAGA OTCGM=GTA H E0o 3 y YP Y G 1 FS E L Q N L Q P D CYSTICPBhROS!S TZANSU!MNRANN CONWCTA?(Q REGULATOR; CODON k H"hRIDEIA-CFfR-l-U1EMBAG2 12__3 TO T 6 22 OF VMAJIC TICD NA __20di -250 23m 2540 2530 2m6 2570 2510 TrAA3CrCAAA. ACTCAIM"O TOrGATCTT TCWACAArr TAOTrCAA AUAAC3AAATr AATCOAO!TT TGAGIACCCT ACACTAAGAA A=QTAA ATCA.COTCTr TCTrCTIAA FS S K L XO C DS P VQ P S A B ZR. IN __CY9=tICPIOSIS TRW3EM6RAXrE CONDUCTAZI REGULATOR; CODO >2 hb I RJM RE IA.CPrR-Z I B MSSAGE -0 12 3 T04462 2 0F HUMAN C PT CD NA -=2MIi 202W2610 20D 2640 cAATCCTAAc ToAaArcCTTA cA~cGrTYcT cArrA*AAOO AGA&TCCTCCT GTCTCCTOM& OTTAGGATrO ACTCTOOkAAr GFOOCAAAGA ar"TCrrCC TCTACGA0OA CAOAOOACCT 9 1L T T L 9R F S L B- DA P V s __CrSTICPIBIO SIG rhAMSMEMBRNE C03RDUCTANCE REGULATOR, CODON ETRIDEBIA-CV1FR-E IBMESSAGE 2125i, 13 TO62O HMAN CPTRCDNA __21W0 17 2660 2670 2m V0m CAOAAACAD: AAACAATCT TITAAMCAG& CrGGA0A~fT TGOOAAA AaIAAM&ATT GrCrrrCMTT TlrrerrGA AAATrrOrCT GM=CrCAA ACCrflTTI TCCTr~mAA T X T X K Q S' F 1 9 T 0 3 V 0. RI I K w6 CYSTTPEUIOSIS TRA)ISMEMRAZN CONDUCTANCHREGULATOR; CODON 21501 123. TO 4622 OF HUhAN C FTR CID EA Z= -220:1 2710 2720 2740 2750 ?Tdo CTATrCTCAA TCCAATCAAC: =CTrACGAA AATFTCCAT TarCV-XAAO ACTCCCTrAC G&T,.AGAOTT ATAOTT AaA.TATOCTr TrAA.AAQGI7A ACACCITFFI T0Aa0GAXZO 3 1IL N1 pII Sif sN I I VQ X T PL> -CYSTXPIDZO SIB IMANr3hn3RANB CONDUCTACE REGULATOR; CODON -b XYIIDE1A.CTll~l13)8AGZ -2=i -1)23 TO4622OF HUMAN CPTRCDNA 22___20 2270 211 2m 20 2810 A AAATGAATOG CATCGAAraW GATTCTGAXO AO3CCTTTAOA GAOA6A0OCTO TCCrrACVLAC TrTACTTACC GrAPCICTC C1AAflACTAC TCGGA.AACT CTCTYCCGAkt AOCA&TCATO Q M P 0 x a D 3D R P L Z Rt RL a L Wh -CY3rIC7IMR6SS TIANSIIEMS?-AiI CONDUCTA14CE ItIOULA FOI; CODON> Ii YRRID 3 A-CFTR-E15hM:BSA0E '230M 13 TO 4622 OF UMAN CPTR CDNA __2MMD -235M- 200 2840 I=1 -w wm2 CAGAITCTOA GCMAU0GAGA.0 OCGATACT= CTCOWACAG C01GAXCA= A~rGOCCCC.A GtCTAAGACT cCCCTCTC COCTArOACo GAOQCGrA~rC GCACTAOCG TOA=CowI p 1S s Q a P AlIL p R 1 v VI T aP>m CYTCPIROSIS TRAN6WMhJJII CONEDUCTANCE MEULATOR: CODON zm 123 T046220PHUbMANCPTR CDfA umZ4iZ41 21 DIm 23m0 COCTTCWW ACGAAMGO CAGTrT0TCC TOAJ.CCTGAI GACAeACTCA, OrAACCJAO cicAAarcco rOCTlCTCrC orC~a~c~oo ACrroOACTA CrGrGaGo CAATrGrrc TLiQ A KKRI Q SV L EL1 M2H S V 11Q -cYSTICPRO9ls ThtAN9 GNRAKE CONDU3CTANCE REGULATOR: CODOIC xui RBIE1AMCT1.4IR)LBSAGEI ___2dQi 123TO 4L22 OF UMAY cvTR CDWA __2401 IRAk/ ~SE c 104W

C,

3- 2-98;11!:58 5/7 26/ 7 2 TABLE Uomdna1d SBQ. ID. NO-~ 3 200D 2960 v0 m %D3 GOAGAACAT TCALCOAAAG ACAACAGCAX CCACACOM6A AGTGTCACTG OCCCCrCA13; cAGTCTicTA ArOCTC TGTrraAr ooMrCi'rrT TCAOTOAC CauWAorCC o Q wI X R T T A i9T kI K S L AP p -cysr[CFIRROsISTRA)(3lE3~0AX3 CON~DUCTANCE REGUJLATOR; CODON h 1 1YDRID E1A-CPTR-EB MESAGE ___2401 123 TO 4 622or xumAN cITR CPNA 23 3010 3=3q0 300 "05) sO CAAACflGAC TGAArTGGAT ATATATTCA& GAACTATC TCAA0AAAC; CIGTTr3AAA GTTTOAACTG ATrrACL-rA TATATAM~rr CrIVCCAATAO AOITctTrG& CCOAACCIT A X( L T B L D IfV S R R L S Q E T G L lb -CYTMCFIBROSSTRANSMBRANE CONDUCTANCE P.JOUL&TOR; CODON ___HY33RM ID1ACPTR.ElMSSAGE L3TO4622OPHLMA4CFTRCDNA 2440i ___31b 3070 30 3100 3110 3120 TAAMG&&A AATTAACOAA CAAGACrlA& A000MMICT TTTTOAiVAT ATOGAGACCA A1TCACTTCT TTAATTGCTT CrrCTA&TT TCCTCACQa& AAAACTACTA TALCTCICGr 3 E B I NI a DL KBE C L P D D X E Si' CYSTICFIRROSIS TRANSMX3(BRANE CONDUCTANCE EGUL&TOR; CODON b HYBKE 1A-CFTR-B 10 MESSAGE Mi 1Z23 TO4622 OFHUMAN CFTI CVtA 3130 3m4 3130 391070 3180 TACCAOCA~r GACTACATOG AACACATACC TECC3AIATAI TACT'rTCCAC AAg3AQCFEAA ATGGTCGTCA CTl3&TG~tAtC TTOT8TAIGG AA(3CTATAXA ATOACAGOTrO TrCTCGAATr I P A V tTTW N TY LR Y I T V KSL 1 CYSTICYB131031 T3A3ESMh3B DANE CONDUCTANCE XBOULATOR; CODON 7"O 123TOA622OPHUMdANCFTRC DA -7M00 271W, 3030 3100 5210 3= 323 3210 TITT a TOxsT AAIrrCT0C TTAGTAATT TZCTIAGz& GG3TOOCTGLC TCTITGGIT AAAPJ.CACGA TTAAACCACG AATCATrAA.A AA0ACCT CCACCCOAOOA AGAAACICAAC I F V L I W C L VI F L A 2 V A A 9 L W~ CYSTZ0CFIROSIS TRAXMEMD ANE, CONDUCTACS REGUTLATOX; CODON 3rYB IDEIA-CPTR-EI11M58.A0H 2720i 123 TO4622 OrHUMAN CFTL CPNA 70 2770> 3= 32d 3770 3W 320 TGCTOTOCT CCTGOAAAC ACTCCTCTC AAGACAAAOO GA.AIAGTACT CATAGrAOAA ACOACACCOA GGOAJCcITO TG.A;C,40AAG 7rCTO1'ITCC CTTAXCATO& 47ATCATCTr V L W L L GiN T P L Q D 9 N 3 T HB s p CYSTICYFI3ROSIS TRANSMEAMEJAI(CONDUCTAN.E REGULATOR; CODON HYBRED LA-C~r%.El9 MSSAGE 27b0,23TO 46220F HUMAN CFT CDNA 3310 332D 3330 3423350 33w ATAACA3CTA TGCAGTOAXT ATCACCAOCA CCAGICTA TIATGrorTT TACATITACO TAT1rGTCGAT ACGTCA='AA TAGTGGrCar OG1CAMXAT AATACACAAA ATcjrAATM N N S Y A V I I TB3 T S 8 Y I v 7 VI I> CYSTICFZDROWI TXAil5~M3 lAN CONrDUCTANCZ REGULATOR; CODON> 3370 SSW0 3390 9i 410 sm~ TwawrAac cGAcAcTITG cTlGCTATOO GaXTCMTAO Ao=rTACCA CTGcGrOAT& ACCCTCAXCO OCTGTGAAAC GAACGATACC CTAAa"OTC TCCAGATGQT GAccAC~rAT V 0V A D TL L A~ M FF P R GL F L vV> PA __CYSTIC 71510 51 T3RANSI1EMB3RANK CONDUCTANCE REGULATOR: CODONI__ HYBRDD1A-CPTR-P.IB MIESSAGE 0 123 TO4622 0#HUMAN CPTR CDNA__ 0 Z:Ec: ~0040w T~N 0<- 3- 2-98;11!58 7/7 27/ -73 TABLE IE-cnfiue SIQ. ADX NO: 3 3i3 340 3~ 46 3670 um~ CTCTAATC&C AOrrCOAAA ATIrrACACC ACAAAATOTT d,~ATWCTGT CTrCAAWC GMrAACTO TCACAWTrr TA&ATGTcOG T~rTTTACAA T0TrAAGACAA GMAGTTCOTO T L I T vS sK I L X NKK M L R 8 V L Q A CYSTIC MIROSIS TRANIdMR 9 ANI CONDUC TANCE RIGULATO COD ON I_ RYU RD E A.CM -I B ME SS AGE 2M___LZ3 TO 46 2 2 F UM AN C PTRCD NA 010;0 360 3500 3510 31W 3530 3540 CTATQTCAAC CCtCAACAC0 TTGAAAGCAG GTO0GATTCT TAATAGAITC TCCAAAGATA oATAc~aGrG aaAafferac ~cTitcGIc cAicTA4GA ATTCTAAG AarTAT PM X 7 L N T L K A O G I L. N K p 6 K 112 -CYSUI)XOCSIS TRANSMEMBRANE CONDUCTANCE REGULATOR; COI3ON HYB RID Z1A-CFFR-N B M319MAOR 123 TO 6622 OF HUMAN~ CIPTR CDNA __306M =307C 3550 3560 3570 3%0330

W

TAGCAATTKT GGATGACCTT CTQCCFTTA. CCATA.TrrGA CTTCATCCAO TTGIrTAITAA ATCGTTAAAA CCrACTOGAA OAC00AG.ATX GOTATA.AT GAAGTACICTC AACAATAA'IT I AKI L D DL L P L T I P D P 1 Q LL I> TRANISKQE[BAE CONDUCTANCE REGULATOR; CODON ErYEDWIA.CPTR.!1b MESSAGE 123 TO 4622 OF RJMAN CFTR CDNA 3I- __3-00b 3610 3627 3630 3w1 3650 3660 TrcroATTOG AGCTATAOCA OTTOCOCA13 TTTTA.CACc cmAAcrrr GorcAAcAG AAcAc:TAACC TCGaT1GT CAACAOrGC AAAAITFFGGO GTMAGAA CAACOTTrC Zvi 0 A''vYA vL Q3? T I p V AT> CYSTICpojXOSIS TRANSMEMBRANE CONDUCTANC2 REGULATOR; CODON I_ ZYB5LDE1jACPT-Z MESSAGE 3140a 123TO4622OPEUMANCPTKCDNA __310i 31 3M7 317000 3710 3m2 TOCCA~IGAT AaG=ITTr ArTATofGax atACaXFI ccrccAAA~c TcAcAacAAc AC0=CACrA TCACAAAA TAAXACAACT CTC~YATAAA GGADOTTO AGTTC01rTo VF pVI A P I M L R AY P L aT SQ lp' __CYb-rlCFS51 TW(SMENDRAII CONDUCTrANCE REGULATOR. CODON HYBRID ZIA-CPTlK-BIB hMSSAGE _____Lz3TO462ZOPKM^KC]TRCDNA a_ 32m).

1740 3750 37W 3770 3740 TCAAACAACT GGAATCT0AA& OAGGAO1C chTTT~cAc TcAiTroIGT1 AcAA--crAA AazrGz-rQA. CCrrAACrr CCQTCCTCAG GITAAA~rO AGrAGAACAA TMflCOATT L 9 Q L ES R a5 R F T IfL v T S __CYSTICIV lOUIS TRANSU RANE CONDUCTANCE REUlaATORI CODON h HTJDfLMI IA-MPR-Wil MESSAGIE "0 -L23 TO 4622 OF HUMAN CUCDNA ___331m.

379 33 !10 38W3 30O 3540 AAOACTATO GACACTTCOT GCCTICCXIAC QW0CCrrA C'ITrQ.AACT CTGMTCCACA TrCCTGAXIAC C=GIAAQCA COAAGCCTO CCGTCOQAAT GAACTrIOA GACAAorar I OL T L AF 0 1QPY YEF T LF X o _CVSTICPIOSZS TRANSMEM RANH CONDUCTANCE REGULA&TOR; COD ON HYBRII) IA-PTRIS ESSAGE U21M 123 To46220OP UAN CPT CNA i~ 3~ 37M- 3M3860 3970 3540 lUm IUI AAQCTCTGAA TI-[ACATACT QCCAACTOQY TCTr=ACCr GrCAACAC TG CCTOcrrCC flv.OAGACTr AAArGFATIGA CGeITTACCA A0AACA*GA CAGFTGTGAC GOACCAA0G WAL 31 LET AWW PLYL S TL R W F -CYSTICFIhROSIS TXANSMEME RANE, CONDUCTANCE UGULATOR-, CODON h HYDRED IIIA.CPTR-I1R MSSACE I R 12Z3 TO'4622 OF HUMAN CFTR.CDNA __m=23 343b 3- 2-98;11!58 8/7 28/ 74 TABLE H1-coatinued =BQ. ID. No-; 3 3910 19M 30 3 3m 3ME MAATOA.OAT AaAATT TTTorCATCT TCrCAa7OC TGTTAcCTT ATr TCCATT mrACTCTrA TCTrTACIMA AAACAMIlA ACAA0TAACG ACAATOOAA TAMOOIAA& Qm RI M1I F vI FF I A V TF IS s CYSrICYIBRO SIS TRANSMBAWRAN9 CONDUCTAKCH REGULATOK; CODON KTIRW BIA-CMT-BI1 M3SSAGE 3440i 121 TO 4622OF HUMAN CFTIICDNA -MOM-303 3970 3W 3m 4010 A=7 TAACAACAOO AGAAWGA& OOMA&CG cOrATTATCCT OAC=rAmcC ATOAATATCA AT1TITG1~CC T~c1TcTr VcflCTCAAC CAYTAATAOC-A CTGAAATCOO TACTIATGI L 7 T G 310! Z G V r. I L T ILA, XMN J> CYST7I R13081 TRANS E4RAME COI1DUCTANE REGULATOR; CODON ;p Ii K"DXV RA.CFTR.EID IN8SAOR 3M___23TO 422 OFHUMAN CFPTRtCINA 35401 AM3 4040 4050 40o') 4m72 dwo TOA0rACA.Wr GCAc~nGOOCT GTAAACTCCA OCAXAGATOT OQATATT ATMOJTCT0 #.CTCATGTAA COCACA, CAT2TOAOGT CGrAICTACA CCTATCGAAC TACOCTA12AC M S T L Q W A V N s S I D V D 3 L MI R -CY=TIBXOSI3 TRANSMMERANS CONDUCTNCE REGULATOR: CODON h xWhRID Sl-CPT-D MESAGE ____2STO4620PHUMAN CPTCDNTi 36IOb, 4O4100 4110 d.120 4130 6140 TGAWCGAWr C1T~AA~rrC ATTIMACA1OC CAA&ACAAMO TAA.ACCTACC AAGrCAA=CA AcTcowTCA G&PATIcAO TAAc=0AcG GTToTCICC ATFTOamw TICAmXTGG V 3R IV F K P I1DM P TI 9 G KP T X 5 T> _CYS=KFIE1OSIS ThAMSZUBANE CONDUC7ANCP5 REGULATOR; COD ON BHYlkWID3A4C1TR-XIMSX5AGE I___b 123T04622OPHUMANCPTRCDNA &404160 4170 iLWO 42904= AACCATACAA 0AATOWCAA CTrGA0 TTAT0ATAT TGAWAITC& CACGrcIAAGA T17~N3AZGZT CITACCOGMT GAGOCTIC AATACTAA.TA ACTCITTAA~r LrGCiAr-TCT K? YK y r Q LS3 K v MI I BN Kz3 xv 1) _CYMrcP=OSIS TIANSU5KRA4 CONDUCTANCE REGULATOR. CODON ____YRIDEIA-CFTR-LbMD85Ar7B ___313i1 123 TO4612 OF HUMAN C PTI CIDNA 721 4210 4=0 4230 A240 4250 AAGATGACAT CT00GCCCA OOOO0cCAAA TOACTCOTCAA AOATCTCAC: OCA*AA TTCTACTGTA C0CCWA0Y CeC~ccX3rr ACTGACAGFI TWCA0Aar(3r CM=r&tci ID b I wI S 00a Q M Trv K D L T A K _CYS77CFEBRO 815 TRANSKMOR AN CONDUCTANCE REGULATOR; CODON ii KYRRDE1A-CFTI-3E1UMISSA02, 123 TO 4622 OF KUMAN CYTI CI)NA 3MWwup 4270 4230 '30 40 4310 432 CACAA=rX3 AAAT=CPTA T-IAGA"WAA TTTCrCCc ^AAMTCCr 43WCA0OAO0 GCTTCACC TrrAcoorAX AATCTCTTGT AAAOOAAGAG TTA7TCAWA CCGGrCTCCC E 10G N A I LI2 N 1 8SF S 1 8P G Q R> CY6TZCWWRO5I8 TRAN3hMRAM CONDUCTAKCS I)SGULATOR: C000J __1 h N?33m DIA-ClFrk-EE %SSA,0X h _L23 TO£622 OP HUMANI 68TX CDN.% saw 4330 4340 4370 4360 4370 4380 a TG=OCTCFI 00AGAC GOAAGG AGAMTACTVt MTATCA=c TrrTTGAQWa ACCCGOA0L C~t'TCrGA CCrArcC~ TCTCATGAA CAATACO&CI AAAA.C=cT v a LL 01RT? 91 K T L L 9A F L ll CYSTIC PMROSI TRA2Sfl1ZIAM CONDUCTASCIfI IEGULATORI. CODON __01 MMRRlEIA-CPT-I3 hOSAGE FUME_ -ei 173 TO 46220OF NUMA.N CPTh C-DNA__b 11

SSEC

104 uO4 77T 3- 2-98;11!:5B 29/ TABLE Hr-coghnued SEQ. m- No- 3 4w44M 4410 442 4430 4W4 TACTGAACAC TIA.AGGAGAA ATCCAOATCG AT~7rGTOTC TTOGATrCA ATAArTrroc ATGACTTGrG AC'rTCCTCrr TAWrCTA0C TACCACACAO AACCCTAAff TATrGAA-ACG L L N T 3 0 1 Q I DOG V S WD S I T 7- CYSTIC M3ROSIS TX&NSlWRVNRJ2 CONDUCTANCE R.EGULATOR; CODON KYDRlDEA-lC5TR-9IR cbMSAOE 13Eo462,2OF HUMXAN CICDKA ___3M6 ___3970t- 4450 4m4 4470 4410 44W 45W0 MACAGIOOGAG GAAAGCCTrT OoAGTaATAC CACAtL4AAr ArrrATITr TCTGG"CAT rTArCCrC CTTTC0GAAA CCTCACT.&TG OrGrcrrrvA TA&TAAAA.% AcAccrr~rA Q Q W R K A F 0 V I P Q KY v r F 3 01T> -CYSTI1CFIBROSIS ThAJISUB)ORAI4E CONDUCTANCE REGUJLATOR: COP ON__ KYREh E1A-CFT-E LD NESSAGE -3m I2S TO 462 2OF XV19AN CPTR CDNA 0_00 '510~~4 450 55 6i 45 TrAOAMA CT0GATCC TATOACAGT GGAGOATCA AGAA.ATATGG AAAFTQCAG AATCTrTITT GA4CCcTAa0 A.TACTTGTCA CCTCACTADTr TCTTTAhCC rrrCAACQrC P R X X L D P Y 3 Q w5 D Q E I W K VA>- -CYSrxcMrBOSIS TRANSMEEMBRANE CONDUCTANCE EGCULATOR; COI3ON -1 KYDRh!LA-CPTR-E~bSSAGI. _____L1ST0496220F5U5(ANCFTRtCDIIA 4MI~ am7 435 49 8 4610 46W0 ATclAGOTTOG GCTCAGATCT OTOATAQAAC AGITTCCT0O GAA&CTTGAC ThCTrCCAACC CG1AeCTA13A CACTA~crIG TCAAGACC CTICOAACTG AAACAGAAC D 3V 6 L RB V I Q P PG0 K LD F V Lv CYSTICFIBROSID TR NSMEBRANE CONDUCTANCE EGULATOR: CODON__ HbRIDUA-C~TR-E1IMZSAGB 41=J 12$ TO 4622OFHUMAN CPTRCDSA __41401 41210 4m2 4640 465) 4660 46M 4m1 TQOATCIDWo CTQI'GCCrA AOCCA.TOCC hCAA0CAJrr o.A=iorGr GCThf3ATCrO ACCACcCC GACACAOAT TCOOTACCGG T~rrC~rCAA CTACACGAAC CAfTCrAOa&C VDOG C V L 8 K H K Q L M CL AD R S0 CYSTIC PIUOSIS TkA if MB~iCRAX! CONDUCTANCE REGULATOR! COVOJI KYRRW RKA-CFT-EID MSSAGE h 41801 12 To4622 OPHUMAN CTCDN~ 421Q Ap4?M 4710 67D4730 0740 TZTCCA1GTAA OOCOAAATC TFTI1'OTIO ArOAACCCA0 TOCTCATrM Gh.TCCACGrAA AAGaGTCATT CcCITCTAO AACOACOAAC TACTTG=GC ACGAGTAAAC CTAGOTCAT V L 8K X KI L L L PB 2p A R L D P W~ -CYSTICF3ROSIS TRANSMHMBRANE CONDUCTANCE REGULATOR: CODON EY13RO ZIA.PTR-IS MESSAGE -123 TO46Z22OF HUMAN CFTR CDNA 42i 427M, 4750 4760) 4770 4710 4790 48W CATAccAAAT A&TTuflAfl ACTCTAAAAr- AA=ArITac ToAtTacA-A. arAaTTcTC GTATOGrFIA TrA&TCTTCT TGOAATTrO TTC=rAAco ACTAACG=G C&TTAAGA T YQ I K R TiL K Q APA D CT V I LI.

-CYMCV3?ROSIB TRAN(SMBOmWII CONDUCTANCE RBUUATOR; COD Oi hi HYDRID IA-CPTI-R1b gssAGR 1___23T4622FHMANCPTRCDXA __4320i asg1b 4810 4w2 4=044 4=85 4960 GTOAACACAU GATADAAGCA ATOCTOGAAT GCCAJCMAr 3TOMrCA CGWGAACA CAcTITGr7C CTo&TCrrCor TAC0AfCrTA COCK-ITrnAA AAACC~AQAT cTrCTCTrT c E H f IRBA, ML C QQFR L VI K E N> -CYSTICFIBROSXS TRAISIEMRANE CONDUCTANCE REGULATOR; CODON 43801 123 To 4 6220P HU)XAN C FTE CDWA I 430 S -9 4 NT 0~ 3- 2-98; 11:58 D/7 3 D/ 7 76 TABLE fl-antilued 4170 4m8 4= 4910 dm1 AAaGCGOCA rACGAreCC AICCAaAAAC TOcTOA6ACGA CAOCAOCCTC FYCCci3cAAG TTCACWOCT CATGCTAW TAWMMTTO ACGCr[OC CTCr-TC0OMA AAO0CC~f'TC K V RQ Y DS I Q X L L N E RSL p itQ> CYSTXC FIBROSIS TRAIISMEM3 RAMD CONDIICTANIC3 REGULATOK; CODON KYDID RA-Ffl.51IR MSSAGE 123TO46Z20FEUMdAXCPTRCDNA __4"M0 4450;0 4030 qmo49 4 0970 CC&TCAOCCC CTCCGACAOO OTOAAGCTc; T rCCCCACCG ClAACTCMOGC A~TOCM.O CIGTAGrCGGG GAG0CTOCC CACTTCGADA AAIWGGT0GC CTrG&MTCG TTCACGFICA IZ S p~ 6 bR V KL P P E R x1 S Kc C CY3TZCPIIXOSIS TRAN3U)IBRAZW CO1DVTANCE 130 ULATOR; CODO >1 HYRIM RIA.CFrTLElDMESSAGE Ii ___44Wi 123 TO4620 F UMANICPTRCDNA 45103 5= si 5030So S040 CTAAGDCCCA GATTGCTGCT CT13AAAOAGO AJ.CAGaAOA A0.400oCAA1 GATAAL'C OATTCO0OT CTAACcZACGA OACTFICTCC TC1~TTTCr TcrccAcarT cTAT~rCCG S X Q I A A L KR -3 ET iF R V Q 1) T R> CYSTICPIDRGSIS TRAlISMEB RAM CON DUCTANCE R.EGULATOR: CODON RTBREDN1A-CPTR-3IBXBSSAOF i S1 123 TO 62OF UMA FTIK CANA sam Swm 5wm Sm TTTAAGAAC ACCATAAATO TrOACATGOO ACATrrGCTC ATOGAWrFOO AOOIA9COGA AAATCTCTCO TCOTATTTAC AACT@1rACCC TarAAAcoAa TrA~CcrAAcc TccATcaCCT L h XYBRW lIA-CFTk-EIB IESSA132 ____4Si_23 TO422 OF HUMAN CPT CDNA -462Dt SLID Sim1 $130 5140 SLso 5 11GAOALr~ OA.ATGTGTG oocarGcrr maA0OOmGG AAGAATATA2 AA=OGcx AACTCCAIGA CT7TACACAC CCOACCGAA TrcCACCCT TTCTTATAXA TTCCACCCC h HYBRID ElA-CPTRf-32I19 MESSAGE b IODj R11331 UNTRANSLATED SEQUBNCES so lo0k BID VINTROIK k-40- k 5170 5180 5190 5200 5210 TCTCATGTAG TTTATCT GT7TGCMG A93COCCOCC ATGADCGCCA Ar'TWEFYA ACAWIACATC AAAACATAGA CAAAACGrCO TC4G0WCOG TACTC=coGr TOADCMJ.CT h( S A N S P D> IX PROTEN (RE, h HYBRID ZIA-CPT R-EIR MESSAGE b I I IXXRNA I 1 s IBT UNTRANSLATED SBQUNCIWZJ. 12 fl__ HiD R~ -go101 >l- 2AC5~ 5250 S260 0 5230 TOGAADCATT GrGOCTCAT AXTTGACAC GC0CATOCCC CCATGOOCCO OOUIOCGrCA ACCTrcGrAA CACTCGAOrA TAAACTUFEO COCOTACG OOAO CCCACAGr a I VS S Y LTT R XP P WA 0 V R Q IPROTEIN (EEXON-&aSOCZ&TRDPIOT~fl);CODOL-ST&RT= L HYDRED D1A-CYTR-ElD 1ZSAOX 13D~ "'WY NSA5 _gUCS J W 5290 szo 5310 OwZ OAATUOATO G=CCACE TTQGITC CCCCOTCCTO CCCOCAAACT CTcACTACTT CTRACACTAC CCG_%G=ccr AACTACC= GODCA00AC 0aa0*rWC%& CA~TA.A 11 vm cS 9s I D a R P V L p A 9 ITT Y UTRANSLATRED SEQUENCES 2 3- 2-98;11 .58 1/7 31/ 77 TAMLE U-contut~d ocAcAoAcc TcrrCcm tamcocc011 0GA0A~TCGCA oCCrCCOCCO ccacrrcwac CTOOATOCTC TG=CAGAAC CTTOCOOCAA CCTCTQACGI C30OW GC CGAAGrCO T YBE T VS T P L 15TA AS A AAS P> XIX PROTBIN (NEXON-ASSOCIATED PEOTEM;)CODONLSTART= L 250 EBD ThT RA2(BLATH EQUEi~fig m_3M 5410 545 Sdm S"C 545 5460 CGCTGCAMC A~CCCCG0G 00ATTOTOAC TOACTrOC TrCCTCAOCC CGCrr4CAAO GCOACMIC00 TGCMOC CCTAACACTO ACTMAACOA AAGGACTCOG GCOGAAFC 6%A A T AR 0 1 V T D P A F L S P LA S: IX PROTEIN (HBXON -ASSOCIATED PROTEIN); COD ONSTARtT~ I HYBRID 31 ACFR-31 DMS SAGE Kcm&N 310 EIB 3 1NTXA1NSLATED SEQUEN#CES 350 5470 Sm3 5400sm 5510 5520 CAGTGCAGCT TCCCarTCAT CCGCCCGCGA TGACAAG~rQ ACOCTCTTT TGGCACAA7T GCa~rCA AUGOAACMA GMGGCGC5 ACTCF1CAAC TCCAOAAA ACCGTrrA A A S R S S AID D XL T A L L A Q 1> _IX PROTBIN (KUXON-ASSQCIArsBDPRoTmfl),CODON-START-m I HYBID ZIA-CPTR-RB N=S~AOB h_ 1 1 EK ZXRNA. I 310BI T 1 UIITIAIS7.ATED SEQUENCYS 410 ~450__ 5530 5540 SSW $56o mosma aoGATTCrrro AeCCOGmAAC TrAATGrCGT TTCrCAOCAO CrOMATC TOQCGCAGCA CCTAAdh#LAC TOWCCrMT AATTACAOCA A&AAOCG1C OACAACCr^G ACWMMflGT D 8 L T R LYV I.vvv 6Q Q L LD L ,QR Q- _IX PKOII* (EXON soi=ATVV PROTEMl);CODON-START= I HRIRD I1A.CPTR-E1 WMSSAOZ 1~ 1 K blMA I Ve.. n NTkMABSE-QURZEs l- 7058 A Soo sm0 5610 %M3 CxnrrCr3cc CTraAADE2TT CCTCCCCTCC CAATCI3OIrT TAJ6AACATAA ATAAA CCAAA0AACQ0 GACTTCCAA OGAQGOGAOG GTA=COA ATTT1TAT TATrr A L K A 6 9P P NA V ";I EK PO~TEIN (jI2XO-ASSO~rATED PROTBI); C__ IX BI V 153 UWTIUISLAX3D SBQU3FCl~s 530 g-

~SEC

104W /V T O SEQUENCE LISTING GENERAL INFORMATION: CW APPLICANTS: Gregory, Armentano, Couture, L.A. Smith,

A.E.

(ii) TITLE OF INVENTION: GENE THERAPY FOR CYSTIC FIBROSIS (iii) NUMBER OF SEQUENCES: 9~ (iv) CORR.ESPONDENCE ADDRESS.

CA AD1DRESSEE- LAHI1W COCXFIELD STREET: 60 STATE S- 'TREET, SUJITE 510 CW) CVITY: BOSTON STATE: MASSACX1JS 'TS COUNTPY: USA Z1"g: 02109 Cv) COMPU'TERf~ YcIZAABLE FORM~ 1JUH TYE. Floppy disk COV PUTER B PC compatible (o C) OP3;RATiNG.,.:YE~EM: PC-DOS/MS-DOS (D SO'PTWARE: ASCT I (vi)~~WENTAPPLICATION DPTA: APP~r2AT~N C4FILING DATE, 2DC:9

CLASSIFICATIO'N

(vii) PR2OrA APPLICATION DATA:, APPLICATION NIMER: US 07/985,47F3 FILING DATE: 02-DEC-1992

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Hanley, Elizabeth A.

REGISTRATION NUMBER: 33,505 REFERENCE/DOCKET NUMBER: NZI-014CP2PC Cix) TELECOMMUNICATION INFORMATION: CA) TELEPHONE: C617) 227-7400 CE) TELEFAX: (617) 227-5941 C2) INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: CA) LENGTH: 6129 base pairs CE) TYPE: nucleic acid CC) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA SEC -11

~EC)

/'VT O' 79 (ix) FEATURE: NAME/KEY: CDS LOCATION: 133. .4572 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l1: AATTGGAAGC AAATGACATC ACAGCAGGTC AGAGAAAAAG GGTTGAGCGG CAGGCACCCA GAGTAGTAGG TCTTTGGCAT TAGGAGCTTG AGCCCAGACG GCCCTAGCAG GGACCCCAGC GCCCGAGAGA CC ATG CAG AGG TCG CCT CTG GAA AAG GCC AGC GTT GTC Met Gin Arg Ser Pro Leu Giu Lys Ala Ser Val: Val ii. 5' 10 120 :168 TCC AAA CTT TTT TTC AGC erLys Leu Phe Ph(- ,Ser TGG ACC AGA CCA ATT TTG AGG AAA GGA TAC Trp Thr Arg Pro Ile! Leu Arg Lys Giy Tyr 20 TCA GAO ATA TAO CAA ATO. CCT TCT GTT GAT Ser Asp Ile Tyr,.Gin 'Ile: Pro:-SerVal- Asp 216 AGA CAG Tr Gi Ser Ala CGC CTG GAA TTG Arg Leu Giu.Leu PAC ,AAT XCTA TCT Asp Asn Leu Ser 50 264 312 GAA AAA Tib GAX AGA: GAA TGG, GAT A Giu Lys Leu Giu'Arg Glu Trp: Asp-Arg

GAG

Giu CTG GCT TCA- AAG 320 ~,eu Ala Ser -Ly s AP.,PAAT CCT AAA CTC ATT -AAT GOC. -OTT *CGG.

Ly; :AsnPro Lys Lt'u lie Asn Ala Leu Arq CGA, TGT Arg -Cys TTT TTC -TGG AGA TTT -,ATG TTC TAT GGA ATC .TTT TTA -TAT Phe Phe Trp Arg ,Phe ,Met Phe Tyr Gly .11d Phe Leu. Tyr TTA.GCG GAA Leu Gly Glu ATA GOT TCC Ile Ala Ser GTC ACC AAA Vai Thr Lys 95 GCA GTA CAG OCT OTC TTA CTG OGA AGA Ala Vai Gin Pro Leu Leu Leu Gly Arg

ATO

Ile 105 TAT GAO Tyr Asp 110 OCG GAT AAC AAG GAG GAA CGC TCT ATC Pro Asp Asn Lys Glu Giu Arg Ser Ile

GCG

Al a 120 ATT TAT CTA GO-C Ile Tyr Leu Gly

ATA

Ile 125 GGC TTA TGC OTT Gly Leu Cys Leu

OC

Leu 130 TTT ATT GTG AGG ACA OTG CTC CTA CAC Phe Ile Val Arg Thr Leu Leu Leu His 135

CCA

Pro 1L40 GOC ATT TTT GG0 OTT CAT CAC ATT GGA ATG CAG ATG AGA ATA Ala Ile Phe Gly Leu His His Ile'Gly M~et Gin Met Arg Ile GCT ATG Ala Met 155 TTT AGT TTG Phe Ser Leu

ATT

Ile 160 TAT AAG AAG ACT Ty_- Lys Lys Thr TTA AAG Leu Lys 165 OTG TcA AGO Leu Ser Ser OGT GTT OTA Arg Val Leu 270

SSEC

-O i4 o 4s, T O GAT AAA ATA Asp Lys Ile 175 AGT ATT GGA CAA Ser Ile Gly Gin

CTT

Leu 180 GTT AGT CTC CTT TCC AAC AAC CTG Val Ser Leu Leu Ser Asn Asn Leu 285 AAC AIA Asn Lys 190 TTT GAT GAA GGA Phe Asp Giu Gly GCA TTG GCA CAT Ala Leu Ala His

TTC

Phe 200 GTG TGG ATC GCT Val. Trp Ile Ala

CCT

Pro 205 TTG CAA GTG GCA Leu Gin Val. Ala CTC ATG GGG-CTA Leu Met Gly Leu

ATC

Ile 215 TGG GAG TTG TTA Trp Giu Leu Leu

CAG

Gin 220 GCG TCT GCC TTC Ala Ser Ala Phe

TGT

Cys 225 GGA CTT GGT TTC Giy Leu Giy Phe

CTG

Leu 230 ATA GTC CTT GCC Ile Val Leu Ala CTT TTT Leu Phe 235 9 a a a..

aa. CAG G&T GGG Gin Ala Gly 20 GGG AAG ATC Gly Lys Ilie 255 ATc- CAAt TCT Ile Gin Ser 2*70

CTA

Leu 240 GGG AGA ATG ATG Gly Arg Met Met AAG TAC AGA GAT Lys Tyr Axg Asp AGT GAA AGA CTT Ser Giu Arg Leu

GTG

Val .260 ATT ACC ,C -le Thr Ser GAA ATG Giu Met 265 CAG AGA GCT Gin Arg t-la 250 ATT GAA AAT Ile Giu Asn- GAA AAA ATG Glu Lys Met GTT AAG GCA Val Lys Ala

TAC

Tyr 275 TGC TGG Cys Trp GAA GAA1.GCA ATG Giu. Giu.Ala. Met 2.80 30 ATT Ile 285

TAT

35 Tyr GAA AAC TTA AGA Glu Asn Leu Arg

CAA

Gin 290 ACA GAA CTG, AA Thr Giu Leu Lys.

CTG

Le u ACT -CGG AAG GCA Thr Arg Lys Ala,

GCC

Ala 3w 0 10-32 GTG AGA TAC Val Arg Tyr

TTC

Phe 305 AAT AGC TCA GCC Asn 5cr Ser Ala -TTC TTC TCA GGG Phe Phe Ser Gly TTC TTT Phe Phe 315 1080 GTG GTG TTT Val Val Phe CTC CGG AAA Leu Arg Lys 335

TTA

Leu 320 TCT GTG CTT CCC Ser Val Leu Pro

TAT

Tyr 325 GCA CTA ATC AAA Ala Leu Ile Lys GGA ATC ATC Gly Ile Ile 330 CTG CGC ATG Leu Arg Met ATA TTC ACC ACC Ile Phe Thr Thr

ATC

Ile 340 TCA TTC TGC ATT Ser Phe Cys Ile

GTT

Val 345 1128 1176 1224 12722 GCG GTC Ala Val 350 ACT CGG CAA TTT Thr Arg Gin Phe

CCC

Pro 355 TGG GCT GTA CAA Trp Ala Val Gin

ACA

Thr 360 TGG TAT GAC TCT Trp Tyr Asp Ser

CT

Leu 365 GGA GCA ATA AAC Gly Ala Ilie Asn

AAA

Lys 3-70 ATA CAG GAT TTC Ile Gin Asp Phe

TTA

Leu 375 CAA AAG CAA GAA Gin Lys Gin Giu

TAT

Tyr 380 AAG ACA TTG GAA TAT AAC TTA ACG ACT Lys Thr Leu Glu Tyr Asn Leu Thr Thr 385 GAA GTA GTG ATG G1u Val Vai Met GAG AAT Giu Asn 395 1320

~SEC

4 W C- o GTA ACA GCC Val Thr Ala AAA CAA AAC Lys Gin Asn 415

TTC

Phe 400 TGG GAG GAG GGA Trp, Giu Glu Gly TTT GOG Phe Gly 405 GAA TTA TTT Giu Leu Phe GAG AMA GCA Giu Lys Ala 41.0 GAC AGC CTC Asp Ser Leu 1368 1416 AAT MAC MAT AGA Asn Asn Asn Arg

MA

Lys 420 ACT TCT AAT GGT Thr Ser Asn GJly

GAT

Asp 425 TTc TTC Phe Phe 430 AGT MAT TTC Ser Asn Phe TCA ,C'I Ser Leu 435 CTT GGT ACT CCT GTC CTG AMA GAT ATT Leu Gly Thr Pro Val Leu Lys Asp Ile 440 1464

MAT

Asn 445 TTC MAG ATA GMA Phe Lys Ile Giu GGA CAG TTG TTG Gly Gin Leu Leu GCG GTT Ala Val 455 GCT GGA TCC Ala Gly Ser

ACT

Thr 460 1512 GGA GCA GGC MAG Gly Ala Gly Lys TCA CTT CTA Ser Leu Leu ATO ATG Met Met 470 ATT ATG GGA GMA Ile Met Gly Olu CTG GAG Leu Glu 475 0e 0.

.0 CCT TCA GAG Pro Ser Glu CAG TTT TCC Gin Phe Ser 495

GT

480 MAA ATT AMG CAC Lys Ile-Lys His

AGT

Ser 485 GGA AGA ATT TCA Gly Arg Ile Ser.

.TTCTOT TCT Phe ,Cyt Ser 490 1560 1608 1656 TGG ATT-ATG CCT -Trp Ilie Met Pro

GGC

Giy 500 ACC ATT AAA GMA MT ATLC:ATC TT Thr Ile Lys Giu Asn IleIle Phe 505 GGT GTT TCC .TAT GAT GMP TAT Gly-ValSer.,TyrAsv Giu Tyr 510 515 AGA TAC AGA Arg Tyr Arq AGC GTC Ser Val.

520 ATC AMA OCA TGC Ile :Lys. Ala Cys 1*704

CAA

Gin 525 CTA GM .GAG GAC Leu.Gilu Giu Asp

ATC

Ile 530 7CC MAG TTT GCA GAG AMA GAC M-T ATA Ser Lys Phe Ala Gili Lys Asp Asn Ile 535

OTT

Val 540 1752 1800 CTT GGA GMA GOT GGA ATC ACA CTG AGT Leu Gly Giu Giy Gly Ile Thr Leu Ser 545

GA

Gly 550 GGT CMA CGA GCA Gly Gin Arg Ala AGA ATT Arg Ile 555 TCT TTA GCA Ser Leu Ala TCT CCT TTT Ser Pro Phe 575

ACGA

Arg 560 GCA GTA TAC MA Ala Val Tyr Lys

OAT

Asp 565 GCT GAT T70 TAT Ala Asp Leu Tyr TTA TTA GAC Leu Leu Asp 570 184 S GGA TAC CTA OAT Giy Tyr Leu Asp

GTT

Val 580 TTA ACA GMA AMA GM ATA TTT GMA Leu Thr Giu Lays Glu Ile Phe Giu 585 1896 AGC TOT Ser Cys 590 GTC TOT MAA CTG Val Cys Lys Leu GCT MAC AMA ACT Ala Asn Lys Thr

AGO

Arg 600 ATT TTG GTC ACT Ile Leu Vai Thr 1944

TCT

Ser 605 AMA ATO GMA CAT Lys Met Giu His MOG AMA GCT GAC Lys Lys Ala Asp

AMA

Lys 615 ATA TTA ATT 770 Ile Lea Ile Leu

CAT

620 1992 'NT O GMA GGT AGC AGC Giu Giy Ser Ser

TAT

Tyr 625 TTT TAT GGG ACA Phe Tyr Gly Thr TTT TCA GAA CTC CAA MAT CTA Phe Ser Giu Leu Gin Asn Leti 630 635 CAG CCA GAC Gin Pro Asp TTT AGT GCA Phe Ser Ala 655

TTT

Phe 640 AGC TCA AMA CTC Ser Ser Lys Leti

ATG

Met 645 GGA TGT GAT TCT Gly Cys Asp Ser TTC GAC CAA Phe Asp Gin 650 TTA CAC CGT Leu His Arg 2040 2088 2136 2184 GMA AGA AGAAMT Glu Arg Arg Asn

TCA

Ser 660 ATC CTA ACT GAG Ile Leu Thr Glu

ACC

Thr 665 TTC TCA Phe Ser 670 TTA GMA GGA GAT Leu Giu Giy Asp

GCT

Ala 675 CCT GTC TCC TGG Pro Val Ser-Trp

ACA

Thr 680 GAA ACA AAA A Giti Thr Lys Lys

CAA

Gin 20 685 TCT TTT AMA CAG Ser Phe Lys Gin

ACT

Thr 690 GGA GAG TTT GGG Giy Giu Phe Giy

S

S.

S. S S GA MAA AGG Giu Lys Arg 695 TTT TCC ATI Phe Ser Ili

MAG

Lys

AAT

Asn

TCT

Ser 700 ATT CTC MAT CCA Ile Leu Asn Pro

ATC

Ile 705 MAC TCT ATA Asn Ser Ile CGA A Arg Lys 710 ACT CCC TTA CMA ATG MAT GGC ATC Thr Pro Leu Gin Met Asn Giy Ile

GMA

Giu 725 GAG GAT TCT GAT Ciii Asp Ser Asp GTG-CM MAG Val*Gin Lys 715 GAG CCT TTA Giu Pro Leix 730 GAG GCG' ATA Glu Ala Ile 2232 2280 2328 2376 GAG AGA AGG Giu Arg Arg 735 CTG TCC TTA GTA Leu Ser Leu Val

CCA

Pro 740 CAT TCT GAG CAG Asp Set- Giu -Gin

GGA

Gly 745 CTG CCT 35 Leu Pro 750 CGC ATC AGC GTG Arg Ile Ser Val AGC ACT GGC CCC Ser Thr Cly Pro CTT CAG GCA CGA Leu Gin Al a Arg 2424

AGG

Arg 40 765 AGG CAG TCT GTC Arg Gin Ser Val

CTG

Leti 770 MAC CTG ATG ACA Asn Leu Met Thr

CAC

His 775 TCA GTT MAC CAA Ser Val Asn Gin

GGT

Gly 780 2472 CAG MAC ATT CAC Gin Asn Ile His

CGA

Arg 785 MAG ACA ACA GCA TCC Lys Thr Thr Ala Ser 790 ACA CGA AMA Thr Arg Lys GTG TCA CTG Val Ser Leu 795 AGA AGG TTA Arg Arg Leu 810 2520 2568 GCC CCT CAG Ala Pro Gin

GCA

Ala 900 MAC TTG ACT GMA CTG CAT ATA TAT TCA Asn Leu Thr Giu Leu Asp Ile Tyr Ser 805 TCT CMA GMA Ser Gin Giu 815 ACT GGC TTG GMA Thr Giy Leu Glu AG? GAA GA ATT MAC GMA GMI GAC Ser Giu Glu Ile Asn Ciu Giu Asp 825 2616 TTA MAG Leu Lys 930

~SEC

"0 104 /'V-r GAG TGC CTT TTT Giti Cys Leu Phe GAT ATG GAG AGC Asp Met Giu Ser CCA GCA GTG ACT Pro Ala Val Thr 2664 p 404-

ACA

Thr 845 TGG AAC ACA TAC T-p, Asn Thr Tyr

CTT

Leu 650 CGA TAT ATT ACT Arg Tyr Ile Thr GTC CAC AAG AGC Val His Lays Ser 855 CTG GCA GAG GTG Leu Ala Gilu Val TTA ATT Leu Ile 860 2712 2760 TTT GTG CTA ATT Phe Val Let Ile TGC TTA GTA ATT Cys Leu Val Ile

TTT

Phe, 870 GCT GOT Ala Ala 875 TOT TTG GTT Ser Leu Val GGG AAT AGT G.1y Asn Ser 895

GTG

Val 880 CTG TGG CTC CTT Let Trp Leu Leu GGA AAC Gly Asn 885 ACT CCT CTT Thr Pro Let ACT CAT AGT AGA Thr His Ser Arg AAC AGC TAT Asn Ser Tyr GCA GTG Ala Val 905 GTG GGA Val Gly 920 CAA GAC AAA Gin Asp Lys 890 ATT ATC ACC Ile Ile Thr GTA GCC GAC Val Ala Asp 2808 2856 AGC ACC Ser Thr 910 AGT TCG TAT TAT Ser Ser Tyr Tyr

GTG

Val 915 TTT TAO ATT TAC Phe Tyr Ile Tyr SW S

S

S

S

*555

S

5555 555*

ACT

Thr 925 TTG CTT GCT ATG Leu Leu Ala Met

GGA

Gly 930 TTC TTC AGA GGT Phe Phe Arg Gly

CTA

Leu 935 CCA CTG GTG CAT Pro Leu Val His

ACT

Thr 940 2904 2952 3000 CTA ATC ACA GTG Leu Ile Thr Val AAA ATT TTA Lys Ile Leu CAC CAC His His 950 AAC ACG Asn Thr 965 AAA ATG TTA CAT Lys Met Leu His TCT GTT Ser Val 955 CTT CAAGCA Leu Gln Ala

CCT

Pro 960 ATG TCA ACC CTC Met Ser Thr Leu TTG AAA GCA Leu Lys Ala.

S. S* S CTT AAT AGA 35 Let Asn Arg 975 TTC TCC AAA GAT Phe Scr Lys Asp GCA AT? TTG AlaIle Leu GAT GAC Asp Asp 985 AT? GTG Ile Val 1000 GGT GGG ATT Gly Gly Ile 970 OTT CTG CCT Leu Leu Pro ATT GGA GCT Ile Gly Ala 3048 3096 OTT ACC Leu Thr 990 ATA GCA Ile Ala 1005 ATA TTT GAO TTC Ile Phe Asp Phe

ATC

Ile 995 CAG TTG TTA TTA Gin Leu Leu Leu GTT GTC GCA Val Val Ala GTT TTA Val Leu.

1010 CAA CCC TAO ATC TTT Gin Pro Tyr Ile Phe 1015 GTT GCA ACA Val Ala Thr

GTG

Val 1020 3144 3192 3240 CCA GTG ATA GTG Pro Val Ile Val GOT TTT ATT ATG TTG Ala Phe Ile Met Leu 1025 AGA GCA Arg Ala 1030 TAT TTC CTC CAA ACC Tyr Phe Leu G-in Thr 1.035 AGG AGT CCA ATT TT11C Arg Ser Pro Ile Phe 1050 TOA CAG CAA Ser Gin Gin CTC AAA CAA OTG GAA Let Lys Gin Leu Giu 1040 TOT GAA GGO Ser Giu Giy 1045 3288 ACT CAT CTT GTT ACA AGO TTA AAA GGA OTA TGG ACA CTT CGT GCC TTO Thr His Leu Val Thr Ser Leu Lys Gly Lej TI-p Thr Leu Arg Ala Phe 3336 1055 1060 1065 SSEC 11 /vT O 4k- GGA CGG CAG Gly Arg Gin 1070 CAT-ACT GCC His Thr Ala 1085 ATG AGA ATA Met Arg Ile CCT TAC TTT GAA ACT Pro Tyr Phe Glu Thr 1075 AAC TGG TTC TTG TAC Asn Trp Phe Leu Tyr 1090 GAA ATG AT? TTT GTC Giu Met Ile Phe Val 1105 ?TA ACA ACA GGA GAA Leu Thr Thr Gly Glu 1120 CTG TTC CAC MAA GC? Leu Phe His Lys Ala .1080 CTG TCA ACA CTG CGC Leu Ser Thr Leu Arg 1095 CTG MAT TTA Leu Asn Leu TGO TTC CAA Trp Phe Gin 1100 3384 3432 ATC TTC TTC ATT GCT GTT ACC TTC Ile Phe Phe Ile Ala Val Thr Phe 1110 1115 GGA GMA GGA AGA GTT GGT AT? ATC Gly Glu Gly Arg Val Gly le Ile 1125 1130 3480 3528 AT? TCC AT? Ile Ser Ile CTG ACT TTA GCC ATG MAT ATC ATG AG? ACA TTG CAG TGG GCT GTA MAC Leu Thr Leu Ala Met Asn Ile Met Ser Thr Leu Gin Trp Ala Val Asn

I

I

I*

I

I

I*

I

*1II I I

I

I I. I *1*I i 1135 1140 1145 TCC AGC ATA Ser Ser Ilie 1150 GAT GTG GAT Asp Vai Asp AGC TTG Ser Leu 1155 ATG CGA TCT Met Arg Ser GGT AMA CCT Gly Lys Pro .1175 GTG AGC CGA GTC TTT Val Ser Arg Val.Phe 1160 ACC MAG TCA ACC AMA Thr Lys Ser ?hr Lys 3576 3624 3672 MAG TTC Lys Phe 1165 AT? GAC ATG Ile Asp Met CCA ACA GMA Pro Thr Giu 1170 1180 30 CCA TAC MAG MAT Pro Tyr Lys Asn GGC CMA Giy Gin 1185 CTC ?CG MA Leu Ser Lys GTT ATG ATT Val Met Ile 1190 ATT GAG AAT TCA Ile Giu Asn Ser i195 3720 CAC GTG MAG 35 his Val Lys AMA GA? Lys Asp 1200 GAC ATC TGG Asp Ile Trp CCC TCA Pro Ser 1205 AMA GAT CTC ACA GCA Lys Asp Leu Thr Ala 1215 MAC AT? 7CC TTC TCA Asn Ile Ser Phe Ser 1230 AMA TAC ACA GMA GGT Lys Tyr Thr Giu Gly 1220 ATA AG? CCT GGC CAG Ile Ser Pro Gly Gin 1235 GGG GGC CMA ATG ACT GTC Gly Gly Gin Met Thr Val 1210 GGA MAT GCC ATA TTA GAG Gly Asn Ala Ile Leu Giu 1225 AGG GTG GGC CTC TTG GGA Arg Val Gly Leu Leu Giy 1240 3768 3816 3864 3912. AGA ACT GGA TCA GGG MAG AG? ACT TTG TTA Azg Thr Gly Ser Gly Lys Ser Thr Leu Leu 1245 1250 TCA GCT TTT TTG Ser Ala Phe Leu 1255 AGA CTA Arg Leu 1260 GAT TCA Asp Ser 1275 CTG MAC ACT Leu Asn Thr GMA GGA GMA AC CAG Glu Gly Giu Ile Gin 1265 CMA CAG TGG AGG AAM Gin Gin Trp Arg Lys 1280 AC GAT GGT GTG TCT ?GG Ile Asp Gly Val Ser Trp 1270 3960 4008 ATA ACT TTG Ilie Thr Leu GCC I'TT GGA GTG ATA CCA CAG A Ala Phe Gly Val Ile Pro Gin Lys 1285 1290 sRAk,

SSEC

A1O4J Wr 0 GTA TTT ATT 'TT Val Phe Ile Phe 1295 CAG TGG AGT GAT Gin Trp Ser Asp 1310 TCT GGA ACA TTT A-GA Ser Gly Thr Phe A-rg 1300 CA-A GAA A-TA TGG A-A-A Gin Glu Ile Trp Lys 1315 A-AA A-AC TTG GAT CCC TA-_ GA-A Lys A-sn Leu Asp Pro Tyr Giu 1305 GTT GCA GAT GAG GTT GGG CTC Val A-la Asp Glu Val Giy Leu 1320 4056 4104 AGA TCT Arg Ser 1325 GTG A-TA GA-A CAG TTT Val Ile Glu Gin Phe 1330 CCT GGG A-AG CTT.GA-C Pro Gly Lys Leu Asp 1335 TTT GTC CIT Phe Val Leu

GTG

Val 1.340 4152 GAT GGG GGC TGT Asp Gly Gly Cys GTC CTA Val Leu 1345 AGC CAT GGC Ser His Gly CAC A-AG His Lys 1350 CAG TTG ATG Gin Leu Met TGC TTG Cl's Leu 1355 4200 GCT AGA TCT GTT CTC A-GT A-AG GCG A-AG ATC TTG CTG CTT GAT GA). CCC Ala A-rg Ser Val Leu Ser Lys Ala Lys Ile Leu Leu Leu Asp Giu Pro 1360 1365 1370 a a 6*4* a a a S C

S

AGT GCT CAT TTG Ser Ala His Leu 1375 GAT CCA GTA Asp Pro Val A-CA TAC Thr Tyr- 1390 CAA A-TA ATT..1GA A-GA ACT' CTA.

Gin Ile ie~tArq ArgThr- Leu 3135 4248 4296 4344 AAA CAA GCA Lys Gin Ala 1390 TTT GCT GAT Phe A-la Asp TGC A-CA GTA ATT Cys Thr Val Ile 1395 CTC TGT GAA.

Leu Cys'Glu 1400 CAC A-GG A-TA His Arg le 30GAA GCA A-TG CTG GA-A TGC CAA CA-A TTT TTG GTC A-TA *GA-A GAG A-AC A-AA Glu A-la Met Leu Glu Cys Gin Gin Phe Leu Val -le Glu GlU A-sn Lys 1405 1410 1415 1420 4392 GTG CGG CAG 35 Val Arg Gin TAC GAT TCC Tyr Asp 5cr 1425 ATC CAG AAA CTG CTG AA-C GAG Ile Gin Lys Leu Leu Asn Glu 1430 AGG AGC CTC Arg Ser Leu~ 1435 TTT CCC CAC Phe Pro H-1is 1450 4440 4488 TTC CGG CAA Phe A-rg Gin GCC A-TC Ala Ile 1440 AGC CCC TCC Scr Pro Ser GAC A-GG Asp A-rg 1445 GTG A-AG CTC Val Lys Leu CGG A-A-C TCA AGC A-AG Aig A-sn Ser Ser Lys 1455 TGC A-AG TCT A-AG CCC Cys Lys Ser Lys Pro 1460 CA-C ATT GCT GCT CTG AAA Gin Ile Ala A-la Leu Lys 1465 GAG GAG ACA GA-A GA-A GAG GTG CAA GAT ACA AGG CTT TAGAGA-GCAG Glu Glu Thr Glu Giu Glu Val Gin Asp Thr Arg Leu 1470 1475 1480 CATA-AATGTT GACATGGGAC.ATTTGCTCAT GGAATTGGAG CTCGTGGGAC AGTCACCTCA TGGAATTGGA GCTCGTGGAA CAGTTA-CCTC TGCCTCAGAA A-ACAAGGA-TG AA-TTAAGT'7.

TTTTTaA-AAA AAGAA.ACATT TGGTAAGGGG AATTGAGGAC ACTGATATGG GTCTTGATAA A-GCTTCCT GGCAATAGTC AAATTGTGTG AAA-GGTA-CTT CAAATCCTTG A-AGATTTACC ACTTGTGTTT TGCAAGCCAG ATTTTICCTC-A AA-ACCCTTGC CATGTGCTAG TAATTGGAAA 4536 4582 4642 4702 4762 4822 4882 sRA/,

SSEC

/VT 0~ .e

S

S

S S

S

5* 0 0

S.

0* 0 5 0 *00* b000 0@ .5.5 0 5*00 e~ S. 0

GGC.AGCTCTA

TTTGTAGTGT

ACTGGAAACT

GTTTAGAAAC

ATTAGAATAC

TGAGCAGTCA

TACCAAAAAT

TATAATCTTT

CAACTCCAGA

TTAGTGC.AAA

TAAGTAGATA

ATAGGTTGAT

25 GAGAGAXTGA

TTTATAATTT

AA.CATATATT

ATTTTTATAT

AA.AACTGGGA

GTCTGGAGGG

AGACACAGCC

TCAAGGGTAC

TTACTGTAAG

AATGTCAATC

TGGAGAAGAA

TCAGCGGTTT

ACAACTATAT

CACAGGAACC

GGAAAGAGAA

CTCAATATTT

CACAGGGGAC

AAGTGACAAG

TTGTCACAGG

GGCCATGGGC

GGTGGTATGT

GAGACACACT

TGTGAAGCAA

ACAATGCTGT

TTGAAATATT

CAGGGGAGAA

AAGCCTTGGG

TCTTAGATGC

ACTGCCTTCT

AAAATATCAC

AGCCTAGTTG

CTGAAATCAT

ATATAAGCTT

TGTTTGCTAA

ACAAGACTGC

CTTCCAGATC

CAGATAATCA

AGGATGGTTC

CTCACAGACC

ACAGCCCTTC

ACTGTGGGTA

TTTCAGGCTA

GAAGAAGCAC

AATTTTTTCT

ATTTTAA)IAG

GACTTTTTAT

CCTAGGGTGA

GCTGATCGAG

AGTTCTGAAG

CAACTCCAAA

TTGTCAATAA

ATCAGCTTAT

ACTTCTTAGG

GTATTCCTTT

GCATTCCAAC

ACATCAAAAT

CTGGAAATCA

CAATACATCC

CCTTGATGAA

TTTGAACTAG

TTTCCACAGA

GACACACATG

GATGTATGTA

CAATCATGAA

CTAGGAAATA

AATGATTATG

GGCACTAGTAI

TATTAACCAG

TTGTTGCCCA

AAGATGGTAC

CTGACTCTTA

AATCCATACA

TGTCTAGTGA AAC'TCGTTAA GTTATGATTA AGTAATGATA TTCTCTCCTC TCCCCATGAT TATCTCATTT CCAAGCAAGT ATGCCCCATT CAACATCTAG GGGTTAGTAT TGTCCAGGTC CTTACCTGGG AAAGGGCTGT GAAGTTGATA TGCCTTTTCC AGTTTAGCTG GAAAAGTATG AGCTCCAGGT AGAGGGTGTG AAGTCCAAGC ATTTAGATGT

.CTTCATGCTG-TCTACACTAA

TTAGTTTTAT'. ATGCTTCTGT TTTATTTTAA TAATGTTTCA AATTACATTT GTATAAAATA

TTTTTATGAA'ATATTATGTT

GGGCCATGAA .TCACCTTTTG CAGCTGTATG ATTCCCAGCC CACCAGTCTG ACTGTTTCCA AGAAGACTGC ATTATATTTA

TTTGTGT

4942 5002 5062 5122 5182 5242 5302 5362 5422 5482 5542.

5602.''' 5 66 2 5722- 5782 5842 5902 5962 6022 6082 6129

S

S S St 55 5 0

S

INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1480 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Gln Arg Ser Pro Leu Glu Lys Ala Ser Val Val Ser Lys Leu Phe 1 5 10 Phe Ser Trp Thr Axg Pro Ile Leu Arg Lys Giy Tyr Arg Gin Arg Leu 25 Giu Leu Ser Asp Ile Tyr Gin Ile Pro Se- Val Asp Ser Ala Asp Asn 40 Leu Ser Glu Lys Leu Giu Arg Giu Trp Asp AZ-g Giu Leu Ala Ser Lys so 55 Lys Asn Pro Lys Leu Ile Asn Ala Leu Arg Arg Cys Phe Phe Trp Arg 70 75 Phe Met Phe Tyr Giy Ile Phe Leu Tyr Leu Gly Giu Val Thr Lys Ala 85 90 Val. Gin Pro Leu Leu Leu Gly Arg Ile Ile Ala Ser Tyr Asp Pro Asp 100 105 110 Asn Lys Giu Giu Arg Ser Ile Ala Ile Tyr Leu Giy Ile Giy Lieu Cys *115 120 125 Leu Leu Phe Ile Val Arg Thr-Leu Leu Leu. His Pro Ala Ile Phe Gly 1 30 135 140 Leu His-s Ile Gly Met Gin Met Arg le-Ala Met Phe Ser Leu Ile 145 IS0 155 160 Tyr. Lys, Lys Thr Leu Lys Leu Ser Ser Arg Val Leu Asp Lys Ile Ser 165 3.70 175 Ile Gij' Gin Leu Val Ser Leu Leu Ser Asn.Asn Leu Asn Lys Phe Asp 180 185 190 Glu Gly Leu Ala Leu Ala His Phe Val Trp le Ala Pro Leu Gin Val 195 200 205 Ala Leu Leu Met Gly Leu Ile Trp Giu Leu Leu Gin Ala Ser Ala Phe 210 215 220 40LeGyPhLeliVaLeAl ePhGiAlGyLe Cys.Gly e l h eIlVa eAl e hGiAl yLu 225 230 235 240 Gly Arg Met Met Met Lys Tyr Arg Asp Gin Arg Ala Gly Lys Ile Ser 245 -250 255 Glu Arg Leu Val Ile Thr Ser Giu Met le Giu Asn Ile Gin Ser Val 260 265 270 Lys Ala Tyr Cys Trp Giu Giu Ala Met Giu Lys Met Ile Glu Asn Leu 275 280 285 Arg Gin Thr Glu Leu Lys Leu Thr Arg Lys Ala Ala Tyr Val Arg Tyr 290 295 300 Phe Asn Ser Ser Ala Phe Phe Phe Ser Gly Phe Phe Val Val Phe Leu 305 310 315 320 S SEC _0104 'UJ Al4

I,

Ser Val Leu Pro Tyr Ala Leu Ile Lys Phe Gin Asn Tyr 385 Trp 20 Asn Phe Giu Thr .30 465 Lys Ile Asp Asp Giy 545 Al a Tyr Lys His Thr Phe Lys 370 Asn GiU Asn Ser Arg 450 5cer Ile Met Giu Ile 530 Ile Val Leu Leu Leu 610 Thr Pro 355 Ile Leu Glu Arg Leu 435 Gly Lei! Lys Pro Tyr 515 Ser Thr Tyr Asp Met 595 Lys Ile 340 Trp Gin Thr Gly L.ys 420 Leu Gin Leu His Gly 500 Arg Lys Leu Lys Val 580 Aia Lys 325 Ser Ala Asp Thr Phe 405 Thr Gly Leu Met Ser 485 Thr Tyr Phe Ser Asp 565 Leu Asn Ala Phe Val Phe Thr 390 Gly Ser Thr Leu Met 470 Gly Ile Arg Ala G ,ly 550 Al a Thr Lys Asp Cys Gin Leu 375 Giu Glu Asn Pro Ala 455 Ile Arg Lys Ser Giu 535 Gly Asp Giu Thr Lys 615 Ile Thr 360 Gin Val.

Leu Gly Val 440 Vai Met Ile Glu Val 520 Lys Gin Leu Lys Arg 600 Ile Val 345 Trp Lys Val Phe Asp 425 Leu Ala Gly Ser Asn Ile.

Asp Arg Tyr Glu Ile Leu Gly Ile 330 Leu Arg Tyr Asp Gin Giu Met Giu 395 Glu Lys 410 Asp Ser Lys Asp Gly Ser Glu Leu 475 Phe Cys 490 Ile Ile Lys Ala Asn Ile Al a Arg 555 Leu Leu 570 Ile Phe Leu Val I Ie Leu Ile Met Ser Tyr 380 Asn Ala Leu Ile Thr 460 Giu Ser Phe Cys Val 540 Ile Asp Giu Th r His 620 Leu Arg Ala Val 350 Leu Gly 365 Lys Thr Val Thr Lys Gin Phe Phe 430 Asn !Phe 445 Gly Ala Pro Ser Gin Phe- Gly Val 510 Gin Leu 525 Leu Gly Ser Leu Ser Pro Ser Cys 590 Ser Lys 605 Glu Gly Lys 335 Thr .Aia Leu Ala Asn 415 Ser Lys Gly Giu Ser 495 Ser Giu Glu Al a Phe 575 Val Met Scr Ile Arg Ile Giu Phe 400 Asn Asa Ile Lys Gly 480 Trp Tyr Glu Gly Arg 560 Gly Cys Glu Ser Tyr Phe Tyr Giy Thr Phe Ser Glu Leu Gin Asn Leu Gin Pro Asp. Phe 625 630 635 640 Ser Ser Lys Leu Met Gly Cys Asp Ser Phe Asp Gin Phe Ser Ala Giu 645 650 655 Arg Arg Asn Ser Ile Leu Thr Giu Thr Leu His Arg Phe Ser Leu Giu 660 665 670 Gly Asp Ala Pro Val Scr Trp Thr Giu Thr Lys Lys Gin Ser Phe Lys 675 68*0 685 Gin Thr Giy Giu Phe Giy Glu Lys Arg Lys Asn Ser Ile Leu Asn Pro 690 695 700 Ile Asn Ser Ile Arg Lys Phe Ser Ilie Val Gin Lys Thr Pro Leu Gin 705 710 715 720 Met Asn Giy Ile Giu Giu Asp Ser Asp Giiu Pro Leu Giu Arg Arg Leu :::725 730 735 Ser Leu Val Pro Asp Ser Giu Gin Gly Giu AlaIle Leu Pro Arg Ile 740 745 750 Ser Vai Ile Ser Thr Giy Pro Thr Leu Gin Ala Arg Arg Arg Gin Ser 755 760 765 Val Leu Asn Leu Met Thr His Ser Vai Asn Gin Gly Gin Asn Ile His 770 775 780 Arg Lys Thr Thr Ala Ser Thr Arg Lys Val Ser Leu Ala Pro Gin Ala 785 790 795 Boo 35 Asn Leu Thr Giu Leu Asp Ile Tyr Ser Arg Arg Leu Ser Gin Giu Thr 805 810 815 Gly Leu Giu Ile Ser Giu Giu Ilie Asn Glu Giu Asp Leu Lys Giu Cys 820 825 830 Leu Phe Asp Asp Met Giu Ser Ilie Pro Aia Val Thr Thr Trp Asn Thr 835 840 845 Tyr Leu Arg Tyr Ile Thr Val His Lys Ser Leu Ile Phe Val Leu Ile 850 855 860 Trp Cys Leu Vai Ile Phe Leu Ala Giu Val.Ala Ala Scr Leu Vai Val 865 870 875 880 Leu Trp Leu Leu Gly Asn Thr Pro Lieu Gin Asp Lys Gly Asn Ser Thr 885 890 895 His Ser Arg Asn Asn 5cr Tyr Ala Val Ilie Ilie Thr Ser Thr Ser Ser 900 905 910 Tyr Tyr Val Phe Tyr Ile Tyr Vai Gly Vai Ala Asp Thr Leu Leu Ala 915 920 925

RAI..

SEC

t /"VT O Met Gly Phe Phe Arg Gly Leu Pro Leu Val His Thr Leu Ile Thr Val 930 935 940 Ser Lys lie Leu His His Lys Met Leu His Ser Val Leu Gin Ala Pro 945 950 955 960 Met Ser Thr Leu Asn Thr Leu Lys Ala Gly Gly Ile Leu Asn Arg Phe 965 970 975 Ser Lys Asp Ile Ala Ile Leu Asp Asp Leu Leu Pro Leu Thr Ile Phe 980 985 990 Asp Phe Ile Gin Leu Leu Leu Ile Val Ile Gly Ala Ile Ala Val Val 995 1000 1005 Ala Val Leu Gin Pro Tyr Ile Phe Val Ala Thr Val Pro Val Ile Val 1010 1015 1020 Ala Phe lie Met Leu Arg Ala Tyr Phe Leu Gin Thr Ser Gin Gin Leu 1025 1030 1035 1040 Lys Gin Leu Giu Ser Giu Gly Arg Ser Pro, -Ile Phe Thr His Leu Val 1045 1050 1055 Thr Ser Leu Lys Giy Leu Trp Thr Leu Arg Ala. Phe Gly Arg Gin Pro 1060 1065 1070 Tyr Phe Giu Thr Leu Phe His Lys Ala Leu Asn Leu His Thr Ala Asn 1075 1080 1085 Trp Phe Leu Tyr Leu Ser Thr Leu Arg Trp Phe Gin Met Arg Ile Glu 1090 1055 .1100 Met Ile Phe Val Ile Phe Phe Ile Ala Val Thr Phe Ile Ser Ile Leu 1105 1110 1115 1120 Thr Thr Gly Giu Gly Giu Gly Arg Val Giy Ile Ile Leu Thr Leu Ala 1125 1130 1135 f Met Asn Ile-Met Ser Thr Leu Gin Trp Ala Val Asn Ser Ser Ile Asp 1140 1145 1150 Val Asp Ser Leu Met Arg Ser Val Ser Arg Val Phe Lys Phe Ile Asp 1155 1160 1165 Met Pro Thr Giu Gly Lys Pro Thr Lys Ser Thr Lys Pro Tyr Lys Asn 1170 1175 1180 Gly Gin Leu Ser Lys Val Met Ile Ile Glu Asn Ser His Val Lys Lys 1185 1190 1195 1200 Asp Asp Ile TrD Pro Ser Giy Gly Gin Met Thr Val Lys Asp Leu Thr 1205 1210 1215 Ala Lys Tyr Thr Glu Gly Gly Asn Ala Ile Leu Glu Asn Ile Ser Phe 8 (RA/ 1220 1225 1230

SEC

104

TO'

Ser Ile Ser Pro Gly Gin Arg Val Gly Leu Leu Gly Arg Thr Gly Ser 1235 1240 1245 Gly Lys Ser Thr Leu Leu Ser Ala Phe Leu Arg Leu Leu Asn Thr Giu 1250 1255 1260 Gly Giu Ile Gin Ile Asp Gly Val Ser Trp Asp Ser Ile Thr Leu Gin 1265 1270 1275 1280 Gin Trp Arg Lys Ala P"he 'Giy Val Ile Pro Gin Lys Val Phe Ile Phe 1285 1290 1295 Ser Gly Thr Phe Arg Lys Asn Leu Asp Pro Tyr Giu Gin Trp, Ser Asp 1300 1305 1310 Gin Giu Ile Trv Lys Val Ala Asp Giu Val Gly Leu Arg Ser Val Ile 1315 1320 1325 Giu Gin Phe Pro Gly Lys Leu Asp Phe Val Leu Val Asp Gly Gly Cys 1330 1335 1340 *Val Leu Ser His Gly His Lys Gin Leu Met Cys Leu Ala Arg Ser Val :.1345 1350 1355 16 *Leu Ser Lys Ala Lys Ile Leu Leu Leu Asp Giu Pro Ser Ala His Leu 1.365 1370 1375 Asp Pro Val Thr Tyr Gin Ile Ile Arg Arg Thr Leu Lys Gin Ala Phe 1380 1385 1390 Ala Asp Cys Thr Vai Ile Leu Cys Giu His Arg Ile' Glu Ala Met Leu '1395 1400 1405 Giu Cys Gin Gin Phe Leu Val Ile Glu Glu Asn Lys Val Arg Gin Tyr 6 .1410 1415 1420 Asp Ser Ile Gin Lys Leu Leu Asn Glu Arg Sex, Leu Phe Arg Gin Ala 1425 1430 1435 1440 Sle Ser Pro Ser Asp Arg Val Lys Leu Phe Pro His Arg Asn Ser Ser 1445 1450 1455 Lys Cys Lys Ser Lys Pro Gin Ile Ala Ala Leu Lys Glu Glu Thr Glu 1460 -1465 1470 Glu Giu Val Gin Asp) Thr Arg Leu 1475 1480 !NFOP-MATION -FOR SE-Q ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 5635 base pairs TYPE: nucleic acid STR.AIDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA SSEC

S

104 'AIT O 0 II (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CATCATAT AATATACCTT ATTTTGGATT GAAGCCAATA TGATAATGAG GGGGTGGAGT je.

TTGTGACGTG

GATGTTGCAA

GTGTGCGCCG

TAAATTTGGG

AGTGAAATCT

GACTTTGACC

CGGGTCAAAG

TGAGTTCCTC

TCCGAGCTAG

CCATGCAGAG

CCAGACCAAT

TCCCTCTGT

30 AG CTGGCTTC

GATTTATGTT

TCTTACTGGG

CGATTTATCT

CAGCCATTTT

40 TTTATAAGAA

TTGTTAGTCT

TCGTC-TGGAT

AGGCGTCTGC

TAGGGAGAAT

TGATTACCTC

CAATGGAAAA

CCTATGTGAG

TATCTGTGCT

>\TCTCATTCTG

GCGCGGGGCG

GTGTGGCGGA

GTGTATACGG

CGTAACCAAG

GAATAATTCT

GTTTACGTGG

TTGGCGTTTT

AAGAGGCCAC

TAACGGCCGC

GTCGCCTCTG

TTTGAGGAA.A

TGATTCTGCT

AAAGAAAAAT

CTATGGAATC

AAGAATCATA

AGGCATAGGC

TGGCCTTCAT

GACTTTAAAG

CCTTTCCAAC

CGCTCCTTTG

CTTCTGTGGA

GATGATGAAG

AGAAATGATT

AATGATTGAA

ATACTTCAAT

TCCCTATGCA

CATTGTTCTG

TGGGAACGGG

ACACATGTAPA

GAAGTGACAA

TAATGTTTGG

GTGTTACTCA

AGACTCGCCC

ATTATTATAG

TCTTGAGTGC

CAGTGTGCTG

GAAAAGGCCA

GGATACAGAC

GACAATCTAT

CCTAAACTCA

TTTTTATATT

GCTTCCTATG

TTATGCCTTC

CACATTGGAA

CTGTCA;AGCC

AACCTGAACA

CAAGTGGCAC

CTTGGTTTCC

TACAGAGATC

GAAAACATCC

AACTTAAGAC

AGCTCAGCCT

CTAATCAAAG

CGCATGGCGG

GCGGGTGACG

GCGCCGGATG

TTT'TCGCGCG

CCATTTTC GC

TAGCGCGTAA

AGGTGTTTTT

TCAGCTGACG

CAGCGAGTAG

CAGATATCAA

GCGTTGTCTC

AGCGCCTGGA

CTGAAXAATT

TTAATGCCCT

T.AGGGGAAGT

ACCCGGATAA

TCTTTATTGT

TGCAGATGAG

GTGTTrCTAGA

AATTTGATGA

TCCTCATGGG

TGATAGTCCT

AGAGAGCTGG

AATCTGTTAA

AAACAGAACT

TCTTCTTCTC

GAATCATCCT

TCACTCGGCA

TAGTAGTGTG

TGGTAAAAGT

GTTTTAGGCG

GGGAAAACTG

TATTTGTCTA

CTCAGGTGTT

CGCAGTGTAT

AGTTTTCTCC

AGTCGAC GGT CAAACtTTT'

ATTGTCAGAC

GGAAAGAGAA

TCGGCGATGT

CACCAAAGCA

CA.AGGAGGAA

GAGGACACTG

AATAGCTATG

TAAAATAAGT

AGGACTTGCA

GCTAATCTGG

TGCCCTTTTT

GAAGATCAGT

GGCATACTGC

GAAA.CTGACT

AGGGTTCTTT

CCGGAAAA

ATTT.CCCTGG

GCGGAAGTGT

GACGTTTTTG

GATGTTGTAG

AATAAGAGGA

GGGCCGCGGG

TTCCGCGTTC

TTATACCCGG

TCCGAGCCGC

ACCCGAGAGA

TTCAGCTGGA

ATATACCAAA

TGGGATAGAG

TTTTTCTGGA

GTACAGCCTC

CGCTCTATCG

CTCCTACACC

TTTAGTTTGA

ATTGGACAAC

TTGGCAC.ATT

GAGTTGTTAC

CAGGCTGGGC

GAAAGACTTG

TGGGAAGAAG

CGGAAGGCAG

GTGGTGTTTT

TTCACCACCA

GCTGTACAAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 16B0 i CATGGTATGA CTCTCTTGGA ATAAGACATT GGAATATAAC TCTGGGAGGA Gr.GATTTGGG ~AACTTCTAA TGGTGATGAC TCCTGAAAGA TATTAATTTC CTGGAGCAGG CAAGACTTCA GTAAAATTAA GCACAGTGGA GCACCATTAA AGAAAATATC TCATCAAAGC ATGCCAACTA TTCTTGGAGA AGGTGGAATC GAGCAGTATA CAAAGATGCT T1'TTAACAGA AAAAGAAATA 25 GGATTTTG.GT CACTTCTAAA ATGAAGGTAG CAGCTATTTT TTAGCTCAAA, ACTCATGGGA 310 CAATCCTAAC TGAGACCTTA CAGA.AACAAA AAAACAATCT 35 CTATTCTCAA TCCAATCAAC AAATGAATGG CATCGAAGAG CAGATTCTGA GCAGGGAGAG CGCTTCAGGC ACG)4AGGAGG GTCAGAACAT TCACCGAAAG CAAACTTGAC TGAACTGGAT TAAGTGAAGA AATTAACGAA TACCAGCAGT GACTACATGG TTTTTGTGCT AATTTGGTGC TGCTGTGGCT CCTTGGAAAC ATAACAGCTA TGCAGTGATT TGGGAGTAGC CGACACTTTG >\CTCTAATCAC AGTGTCGAAA

GCAATAAACA

TTAACGACTA

GAATTATTTG

AGCCTCTTCT

AAGATAGAAA

CTTCTAA.TGA

AGAATTTCAT

ATCTTTGGTG

GAAGAGGACA

ACACTGAGTG

GATTTGTATT

TTTGAAAGCT

ATGGAACATT

TATGGGACAT

TGTGATTCTT

CACCGTTTCT

TTTAXACAGA

TCTATACGAA

GATTCTGATG

GCGATACTGC

CAGTCTGTCC

ACAACAGCAT

'ATATATTCAA

GAAGACTTAA

AACACATACC

TTAGTAATTT

ACTCCTCTTC

ATCACCAGCA

CTTGCTATGG

ATTTTACACC

TGATTATGGG

TCTGTTCTCA

TTTCCTATGA

TCTCCAAGTT

GAGGTCAACG

TATTAGACTC

GTGTCTGTAA

TAALAGAAAGC

TTTCAGAACT

TCGACCAATT

AGAACTGGAG

GTTTTCCTGG

TGAATATAGA

TGCAGAGAAA

AGCAAGAATT

TCCTTTTGGA

ACTGATGGCT

TGACAAAATA

CCAAAATCTA

TAGTGCAGAA

AAATACAGGA T&TTCTTACAA CAGAAGTAGT GATGGAGAAT AGAAAGCAAA ACAAAACAAT TCAGTAATTT CTCACTTCTT GAGGACAGTT GTTGGCGGTT

AAGCAAGAAT

GTAACAGCCT

AACAATAGAA

GGTACTCCTG

GCTGGATCCA

CCTTCAGAGG

ATTATGCCTG

TACAGAAGCG

GACAATATAG

TC7TTAGCAA

TACCTAGATG

AACAAAACTA

TTAATTTTGC

CAGCCAGACT

AGAAGAAATT

GTCTCCTGGA

AGGAAGAATT

ACTCCCTTAC

TCCTTAGTAC

ACTGGCCCCA

GTTAACCAAG

GCCCCTCAGG

GGCTTGGAAA

ATGGAGAGCA

AAGAGCTTAA

TCTTTGGTTG

CATAGTAGAA

TACATTTACG

CTGI-TGCA7A

CTT-CAAGCAC

1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 246 0 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 CATTAGAAGG AGATGCTCCT CTGGAGAGTT TGGGGAAAAA 6.9 a

AATTTTCCAT

AGCCTTTAGA

CTCGCATCAG

TGAACCTGAT

CCACACGAAA

GAAGGTTATC

AGGAGTGCCT

TTCGXIATAT

TTCTGGCAGA

AAGACAAAGG

CCAGTTCGTA

GA'rTCTTCAG

ACAAAATGTT

TGTGCAAA-AG

GAGAAGGCTG

CGTGATCAGC

GACACACTCA

AGTGTCACTG

TCAAGA;AACT

TTTTGATGAT

TACTGTCCAC

GGTGGCTGCT

GAATAGTACT

TTATGTGTTT

AGGTCTACCA

ACATTCTGTT

.JRA

*-Y

/VdT O kl a a a 4 a.

i. a a 4.

a

CTATGTCAAC

TAGCAATTTT

TTGTGATTGG

TGCCAGTGAT

TCAAACAACT

AAGGACTATG

AAGCTCTGA

AAZATGAGAAT

TAACAACAGG

20 TGAGTACATT

TGAGCCGAGT

AACCATACA

AAGATGACAT

CAGAAGGTGG

TGGGCCTCTT

TACTGAACAC

AACAGTGGAG

TTAGAAAAAA

ATGAGGTTGG

TGGATGGGGG

TTCTCAGTAA

C-ATACCAA.AT

GTGA.ACACAG

AAGTGCGGCA

CCATCAGCCC

CTAAGCCCCA

TTTAGAGAGC

TTGAGGTACT

TCCTGA

CCTCAACACG

GGATGACCTT

AGCTATAGCA

AGTGGCTTTT

GGAATCTGAA

GACACTTCGT

TTTACATACT

AGAAATGATT

AGAAGGAGAA

GCAGTGGGCT

CTTTAAGTTC

GAATGGCCAA

CTGGCCCTCA

AAATGCCATA

GGGAAGAACT

TGAAGGAGAA

GAAAGCCTTT

CTTGGATCCC

GCTCAGATCT

CTGTGTCCTA

GGCGAAGATC

AATTAGAAGA

GATAGAAGCA

GTACGATTCC

CTCCGACAGG

GATTGCTGCT

AGCATAAATG

GAAATGTGTG

TTTTGTATCT

TTGAAAGCAG

CTGCCTCTTA

GTTGTCGCAG

ATTATGTTGA

GGCAGGAGTC

GCCTTCGGAC

GCCAACTGGT

TTTGTCATCT

GGAAGAGTTG

GTAAACTCCA

ATTGACATGC

CTCTCGAAAG

GGGGGCCAAA

TTAGAGAACA

GGATCAGGGA

ATCCAGATCG

GGAGTGATAC

TATGAACAGT

GTGATAGAAC

AGCCATGGCC

TTGCTGCTTG

ACTCTAAAAC

ATGCTGGAAT

ATCCAGAAAC

GTGAAGCTCT

CTGAAAGAGG

TTGACATGGG

GGCGTGGCftT

GTTTTGCAGC

GTGGGATTCT

CCATATTTGA

TTTTACAACC

GAGCATATTT

CAATTTTCAC

GGrCAGCCTTA

TCTTGTACCT

TCTTCATTGC

GTATTATCCT

GCATAGATGT

CAACAGAAGG

TTATGATTAT

TGACTGTCAA

TTTCCTtCTC

AGAGTACTTT

ATGGTGTGTC

CACAGAAAGT

GGAGTGATCA

AGTTTCCTGG

ACA.AGCAGTT

ATGAACCCAG

AAGCATTTGC

GCCAACAATT

TGCTGAACGA

TTCCCCACCG

AGACAGAAGA

ACATTTGCTC

AAGGGTGGGA

AGCCGCCGCC

TAATAGATTC

CTTCATCCAG

CTACATCTTT

CCTCCAAACC

TCATCTTGTT

CTTTGAAACT

GTCAACACTG

TGTTACCTTC

GACTTTAGCC

GGATAGCTTG

TAAACCTACC

TGAGAATTCA

AGATCTCACA

AATAAGTCCT

GTTATCAGCT

TTGGGATTCA

ATTTA-TTTT

AGAAATATGG

GAAGCTTGAC

GATGTGCTTG

TGCTCATTTG

TGATTGCACA

TTTGGTCATA

GAGGAGCCTC

GAACTCAAGC

AGAGGTGCAA

ATGGAATTGG

AAGAATATAT

ATGAGCGCCA

T%-CXZAGATA

TTGTATTAA

GrGCAAltAG

TCACAGCAAC

ACXLAGCTTAA

CTGTTCCACA

CGCTGGTTCC

ATTCCATTT

ATGAATATCA

ATGCGATCTG

AAGTCAACCA

CACGTGAAGA

GCAAAATACA

GGCCAGAGGG

TTTTTGAGAC

ATAACTTTGC

TCTGGAACAT

AAAGTTGCAG

TTTGTCCTTG

GCTAGATCTG

GATCCAGTAA

GTAATTCTCT

GAAG.AGAACA

TTCCGGCAAG

AAGTGCAAGT

GATACAAGGC

AGGTAGCGGA

AAGG7GGGGG

ACTCGTTTGA

3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320.

4380 .4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 M'F4(

'SEC

'/VT Oq. 0

OS..

*0 SO 9 *0a* -TG'GAAGCATT GTGAGCTCAT ATTTGACAAC GCGCATGCCC GAATGTGATG GGCTCCAGCA TTGATGGTCG CCCCGTCCTG GACCTACGAG ACCGTGTCTG GAA~CGCCGTT GGAGACTOCA CGCTGCAGCC ACCGCCCGCG GGATTGTGAC TGACTTTGCT CAGTGCAGCT TCCCGTTCAT CCGCCCGCGA TGACAAGTTG GGATTCTTTG ACCCGGGAAC TTAATGTCGT TTCTCAGCAG GGTTTCTGCC CTGA).GGCTT CCTCCCCTCC CAATGCGGT INFORMATION FOR SEQ ID NO: 4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRA1NDEDN"ESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ACTCTTGAGT GCCAGCGAGT AGAGTTTTCT CCTCCG INFORMATION FOR SEQ ID NO:S: Wi SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear Ci)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GCAAAGGAGC GATCCACACG AAATGTGCC INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: s0 LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA CCATGGGCCG GGGTGCGTCA CCCGCAAACT CTACTACCTT GCC TCCGCCG CCGCTTCAGC TTCCTGAGCC CGCTTGCAAG ACGGCTCTTT TGGCACAATT CTGTTGGATC TGCGCCAGCA TAAAACATAA ATAAA 5280 5340 5400 5460 5520 5580 5635 36 eq...

SSEC$

0 O' g (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6; CTCCTCCGAG CCGCTCCGAG CTAG 24 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCAAAAATGG CTGGGTGTAG GAGCAGTGTC C 31 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid 25 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGGATCCTTT ATTATAGGGG AAGTCCACGC CTAC 34 35 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs 40 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGGGATCCAT CGATGAAATA TGACTACGTC CG

Claims (38)

1. An adenoviral vector comprising an adenovirus genome from which the El region and DNA sequence of the E4 region that is non-essential to virus replication in vitro have been deleted and which contains one or more open reading frames of the E3 region, and additionally comprising a DNA sequence of interest operably linked to expression control sequences and inserted into said adenoviral genome.

2. The vector of claim 1, in which the open reading frame of the E3 region contained in the vector is gpl9K.

3. The vector of claim 1, in which the open reading frame of the E3 region contained in the vector is 14.7K.

4. The vector of claim 1, in which the open reading frame of the E3 region contained in the vector is 10.4K. The vector of claim 1, in which the open reading frame of the E3 region contained in the vector is 14.5K.

6. The vector of any of claims 1 to 5, in which the E4 region is comprised of ORF6.

7. The vector of any one of claims 1 to 6, in which the DNA sequence of interest is the gene encoding cystic fibrosis transmembrane regulator. *see

8. The vector of any one of claims 1 to 7, in which the expression control sequence is the PGK promoter. Dated this 21st day of August 2000 Genzyme Corporation 4/ kRA By its Patent Attorneys Davies Collison Cave P:\OPERJMS7349-94.CLM 31/10/97 8. The adenoviral vector of any of claims 1 to 5 from which the E3 region of the adenovirus genome has been deleted.

9. The adenoviral vector of any one of claims 1 to 8 wherein the DNA sequence of interest encodes cystic fibrosis transmembrane regulator protein. A method for providing cystic fibrosis transmembrane conductance regulator protein to airway epithelial cells of a cystic fibrosis patient comprising administering directly to airway epithelial cells of said patient an adenoviral vector, said vector comprising an adenovirus genome from which E4 DNA sequence that is non-essential to virus replication in vitro has been deleted, but retaining sufficient E4 sequence to promote virus replication in vitro, and additionally comprising a DNA sequence encoding cystic fibrosis transmembrane regulator protein operably linked to expression control sequences and inserted into said adenoviral genome, under conditions whereby the DNA sequence encoding cystic fibrosis transmembrane regulator protein is expressed and a functional chloride ion channel is produced in the airway epithelial cells of the patient.

11. The method of claim 10 wherein open reading frame 6 of the E4 region of the St adenovirus genome is retained in the vector.

12. The method of claim 10 wherein the expression control sequences operably linked to the DNA sequence encoding cystic fibrosis transmembrane regulator protein comprise a PGK promoter.

13. A method for providing cystic fibrosis transmembrane conductance regulator protein to airway epithelial cells of a cystic fibrosis patient comprising administering directly to airway epithelial cells of said patient an adenoviral vector, said vector comprising San adenovirus genome from which E4 DNA sequence that is non-essential to virus replication in vitro has been deleted, and wherein open reading frame 3 of the E4 -m 104 m Tt/r O P:\OPERUMS\57349-94.CLM 31/10/97 -vw- region is retained, and additionally comprising a DNA sequence encoding cystic fibrosis transmembrane regulator protein operably linked to expression control sequences and inserted into said adenoviral genome, under conditions whereby the DNA sequence encoding cystic fibrosis transmembrane regulator protein is expressed and a functional chloride ion channel is produced in the airway epithelial cells of the patient.

14. The method of claim 13 wherein the expression control sequences operably linked to the DNA sequence encoding cystic fibrosis transmembrane regulator protein comprise the cytomegalovirus immediate early promoter.

15. The method of any one of claims 10 to 14 in which the Ela and Elb regions of the adenovirus genome of the vector have been deleted.

16. The method of claim 15 wherein the DNA sequence encoding cystic fibrosis transmembrane regulator protein is inserted into the deleted Ela and Elb regions of the adenoviral genome.

17. The method of any one of claims 10 to 14 in which the E3 region of the adenovirus genome of the vector has been deleted.

18. An adenoviral vector of claim 1 or claim 4, substantially as hereinbefore described.

19. A method of claim 10 or claim 13, substantially as hereinbefore described. Y SEC -o 104 /T O0 P:\OPERUMS\57349DIV.CLM 31/10197 too An adenoviral vector comprising an adenovirus genome from which the El region and DNA sequence of the E4 region that is non-essential to virus replication in vitro have been deleted and which contains one or more open reading frames of the E3 region, and additionally comprising a DNA sequence of interest operably linked to expression control sequences and inserted into said adenoviral genome.

21. The vector of claim 1, in which the open reading frame of the E3 region contained in the vector is gpl9K.

22. The vector of claim 1, in which the open reading frame of the E3 region contained in *the vector is 14.7K. o

23. The vector of claim 1, in which the open reading frame of the E3 region contained in the vector is 10.4K.

24. The vector of claim 1, in which the open reading frame of the E3 region contained in the vector is 14.5K.

25. The vector of any of claims 1 to 5, in which the E4 region is comprised of ORF6. a. a

26. The vector of any one of claims 1 to 6, in which the DNA sequence of interest is the gene encoding cystic fibrosis transmembrane regulator.

27. The vector of any one of claims 1 to 7, in which the expression control sequence is the PGK promoter.

28. A pseudo-adenoviral vector, comprising the 5' and 3' inverted terminal sequences, the packaging sequence, and the EIA enhancer sequence of the adenovirus genome. SEC 104 /VT O P:\OPERUMS57349DIV.CLM 31/10/97 l4

29. The vector of claim 9, further comprising at least one DNA sequence of interest operably linked to expression control sequences and inserted into said vector. The vector of claim 9 or claim 10, further comprising a nuclear localisation signal and a polyadenylation signal.

31. The vector of claim 11, in which the expression control sequences comprise the adenovirus major late promoter and the adenovirus tripartite leader, the nuclear localisation signal comprises the SV40 nuclear localisation signal, and the polyadenylation signal comprises the SV40 polyadenylation signal. *4

32. The vector of claim 12, which is PAV I.

33. The vector of claim 12, which is PAV II. 4* 4

34. The vector of claim 11, further comprising an internal initiation signal. The vector of claim 15, in which the internal initiation signal is the EMC internal i: ribosomal entry signal.

36. The vector of claim 10, in which the DNA sequence of interest is a gene coding for cystic fibrosis transmembrane regulator.

37. The vector of claim 10, in which the DNA sequences of interest are a gene coding for cystic fibrosis transmembrane regulator and a gene coding for a protein selected from the group consisting of antiprotease alpha-1-antitrypsin, superoxide dismutase, and DNAse. SEC S 104 g /V T O' P:AOPERJMSX57349D1V.CLM 31/10/97

38. A method for producing pseudo-adenoviral vectors, comprising introducing the DNA encoding the pseudo-adenoviral vector and a helper adenovirus into a cell line capable of producing the pseudo-adenoviral vectors.

39. The method of claim 19, in which the helper adenovirus is replication-defective. The method of claim 20, in which the cell line is the 293 cell line.

41. The method of claim 19, in which the helper adenovirus is packaging defective.

42. The method of claim 19, in which the helper adenovirus comprises an adenovirus genome from which an essential gene has been deleted, and in which said essential gene is inserted into the pseudo-adenoviral vector.

43. The method of claim 19, in which the helper adenovirus exceeds 100% of the adenovirus genome length and is deleted fro protein IX. b*

44. The method of claim 24, in which the helper adenovirus and the pseudo-adenoviral vector are introduced into a protein IX-containing cell line, and in which the viruses produced in said cell line are further introduced into a cell line lacking protein IX, such that the helper adenovirus cannot be packaged and pseudo-adenoviral vectors are produced. Dated this 31st day of October 1997. Genzyme Corporation By its Patent Attornyes Davies Collison Cave SEC S104W /V T O%$