Gene therapy for cystic fibrosis

Gene Therapy vectors, which are especially useful for cystic fibrosis, and methods for using the vectors are disclosed.

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 born 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 95% 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 by 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; Rommens, J. M. et al. (1989) 
Science 245:1059-1065)). U.S. Ser. No. 07/488,307 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, R. J. et al. (1990) Nature 
347:382-386; Rich, D. P. et al. (1990) Nature 347:358-362). The co-pending 
patent application 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-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 in response to phosphorylation by cyclic AMP-dependent 
protein kinase or protein kinase 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; Hwang, T-C. et al. (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 (.DELTA.F508), 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 .DELTA.F508, 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 plasma 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 cAMP-stimulated Cl.sup.- 
channels in cells expressing CFTR .DELTA.F508 (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 
complications. Because CF mucus consists of a high concentration of DNA, 
derived from lysed neutrophils, one approach has been to develop 
recombinant human DNase (Shak, S. et al. (1990) Proc. Nati. 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 damage caused by anexcess 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 ranspot 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) FASEB J. 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 chloride 
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 work 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, (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 vectors for transferring 
selected genetic material of interest (e.g., 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 E1a and E1b regions of the 
genome, which are involved in early stages of viral replication have been 
deleted and replaced by genetic material of interest (e.g., 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 tetninal repeats and the minimal sequences of 
a wild-type 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. 
In a further embodiment, the adenovirus-based gene therapy vector contains 
the open reading frame 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 include deletions in the E1 and/or E3 regions. 
Alternatively, the adenovirus-based gene therapy vector contains the open 
reading frame 3 (ORF3) of adenoviral E4 from the E4 promoter and is 
deleted for all other E4 open reading frames. Again, optionally, this 
vector can include deletions in the E1 and/or E3 regions. The deletion of 
non-essential open reading frames of E4 increases the cloning capacity by 
approximately 2 kb without significantly reducing the viability of the 
virus in cell culture. In combination with deletions in the E1 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.

DETAILED DESCRIPTION AND BEST MODE 
Gene Therapy 
As used herein, the phrase "gene therapy" refers to the transfer 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 interest 
encodes a product (e.g., a protein polypeptide, peptide or functional RNA) 
whose production in vivo is desired. For example, the genetic material 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 transmembrane regulator (CFTR), Factor VIII, 
low density lipoprotein receptor, beta-galactosidase, alpha-galactosidase, 
beta-glucocerebrosidase, insulin, parathyroid hormone, and 
alpha-1-antitrypsin. 
Although the potential for gene therapy to treat genetic diseases has been 
appreciated for many years, it is only recently that such approaches have 
become practical with the treatment of two patients with adenosine 
deamidase deficiency. The protocol consists of removing lymphocytes from 
the patients, stimulating them to grow in tissue culture, infecting them 
with an appropriately engineered retrovirus followed by reintroduction of 
the cells into the patient (Kantoff, P. et al. (1987) J. Exp. Med. 
166:219). Initial results of treatment are very encouraging. With the 
approval of a number of other human gene therapy protocols for limited 
clinical use, and with the demonstration 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 sequences 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 epithelial cells are preferred 
target cells for CF gene therapy. The first generation of CF gene therapy 
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 stem cell to air epithelial cells becomes 
known. If DNA were incorporated into airway stem cells, all subsequent 
generations of such cells would make authentic CFTR from the integrated 
sequences and would correct the physiological defect almost irrespective 
of the biochemical basis of the action of CFTR. 
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 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 of retroviruses in CF 
will probably require damaging the airways (by agents such as SO.sub.2 or 
O.sub.3) 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 saall fraction of which 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 (R. A. Weinberg (1989); 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 may alleviate this problem somewhat, because the vectors will not 
lead to productive viral life cycles generating infected cells, cell lysis 
or large numbers 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 tppe 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 good animal model for the disease nor a simple clinical 
end point to measure the is efficacy of treatment. 
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 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 showm to result in 
expression of the DNA in lung (Brigham, K. et al. (1989) Am. J. Med. Sci. 
298:278). 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 of plasmid 
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 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, (e.g., 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 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. Natl. 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, 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 10. 
FIG. 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 E1a and E1b 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 
E1a/E1b region and transcription of the CFTR sequence is driven by the 
endogenou E1a 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 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 E1a 
and E1b functions. 
The E3 region of the Ad2/CFTR-1 encodes a variety of proteins. One of these 
proteins, gp19, is believed to interact with and pmeyent presentation of 
class 1 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, the large size of the viral DNA renders it doubly 
defective for replication (i.e., 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 E1 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. 
The adenovirus vector (Ad2/CFTR-1) and a related virus encoding the marker 
.beta.-galactosidase (Ad2/.beta.-gal) have been constructed and grown in 
human 293 cells. These cells contain the E1 region of adenovirus and 
constitutively express E1a and E1b, 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-1 virus has been shown to encode CFTR by demonstrating the 
presence of the protein in 293 cells. The Ad2/.beta.-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, hum an HeLa, human CF cells (see Example 8) 
and airway epithelial cells from CF patients (Rich, O. et al. (1990) 
Nature 347:358). 
Ad2/CFTR-1 is constructed from adenovirus 2 (Ad2) DNA sequences. Other 
varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) may also prove useful as 
gene therapy 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 (.gtoreq.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 
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 
non-dividing human target cell types. 
The PAV vector can be maintained as either a plasmid-borne 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 FIG. 
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 E1a 
enhancer. Various convenient restriction sites have been incorporated into 
the fragments, allowing the insertion of promoter/gene cassettes which can 
be packaged in the PAV virion and used for gene transfer (e.g. for gene 
therapy). The construction and propagation of PAV is described in detail 
in the following Example 1l. 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 (e.g., CFTR (7.5 kb)); Factor VIII (8 kb); Factor 
IX (9 kb)), 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 (e.g., antiprotease 
alpha-1-antitrypsin) tissue inhibitor of metaloproteinase, antioxidants 
(e.g., superoxide dismutase), enhancers of local host defense (e.g., 
interferons), mucolytics (e.g., DNase); and proteins which block 
inflammatory cytokines. 
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. 
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 E1 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 vector 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 of E4, 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 factor 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 (e.g., 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 (e.g., 293 cells), but the absence of ORF617 would 
prevent the potential activation of 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) 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 cAMP-stimulated 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 
Na+ absorption. 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 inhaled 
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 CFTRR 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 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. (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 response. 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. 
e. 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 immnunoprecipitation 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 (10.sup.6 -10.sup.7 ions/sec) (Hille, B. 
(1984) Sinauer Assoc. Inc., Sunderland, Mass. 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. Nati. 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 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 1 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 instability 
to explain the apparent inability to recover clones encoding fulll 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 FIG. 2, 
partial CFTR clone T16-4.5 was cleaved with restriction enzymes Sph 1 and 
Pst 1 and the resulting 3.9 kb restriction fragment containing exons 11 
through mist of exon 24 (including an 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 1 and Pst 
1 sites of the pMB1 based vector pkk223-3 (Brosius and Holy, (1984) Proc. 
Natl. Acad. Sci. 81:6929). It was hoped that the pMB1 origin contained 
within this plasmid would allow it and plasrnids 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 pkk-4.5. 
Partial CFTR clone T11 was cleaved with Eco R1 and Hinc II 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 R1 
site and Sma 1 restriction site of the plamid Bluescript Sk- (Stratagene, 
catalogue number 212206), such that the CFTR sequences were now flanked on 
the upstream (5') side by a Sal 1 site from the cloning vector. This 
clone, designated T11-R, was cleaved with Sal 1 and Sph 1 and the 
resultant 1.8 kb band isolated by agarose gel purification. Plasmid 
pkk-4.5 was cleaved with Sal 1 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-CFTR-1 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-CFTR-1 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 CFTR1 derived from partial 
cDNA clone T11 and the published CFTR cDNA sequence. These undesired 
differences included a 1 base-pair deletion at position 995 and a 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 FIG. 3, pkk-CFTR1 was cleaved with Xba I and Hpa I, and 
dephosphorylated with calf intestinal alkaline phosphatase. In addition, 
to reduce the likelihood of recovering the original clone, the small 
unwanted Xba I/Hpa I restriction fragment from pKK-CFTR1 was digested with 
Sph I. T16-1 was cleaved with Xba I and Acc I and the 1.15 kb fragment 
isolated by agarose gel purification. T16-4.5 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-CFTR1 were ligated to produce pKK-CFTR-2. 
Alternatively, pKK-CFTR-2 could have been constructed using corresponding 
restriction fragments from the partial CFTR cDNA clone C1-1/5. pKK-CFR2 
contains the uninterrupted CFTR protein coding sequence and conferred slow 
growth upon E. coli host cells in which it was inserted, whereas pKK-CFTR1 
did not. The origin of replication of pKK-CFTR-2 is derived from pMB1 and 
confers a plasmid copy number of 15-20 copies per host cell. 
Example 2 
Improving Iost 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 pSC101 replication origin of the low copy 
number plasmid pLG338 (Stoker et al., Gene 18, 335 (1982)) and the 
ampicillin resistance gene and polylinker of pGEM-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 NI, 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 c al.). 
With additional reference to FIG. 4, pKK-CFTR2 was cleaved with Eco RV, Pst 
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 Sal I to Eco RV restriction fragment which was retained within 
the column. pSC-3Z was digested with Sma I and Pst I and also passed over 
a Sephacryl S400 spun column to remove the small Sma I/Pst I restriction 
fiagment which was retained within the column. The column eluted fractions 
from the pKK-CFTR2 digest and the pSC3Z digest were mixed and ligated to 
produce pSC-CFTR2. A map of this plasmid is presented in FIG. 5. Host 
cells containing CFTR cDNAs at this and similar gene dosages grow well and 
have stably maintained the recombinant 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 atposition 1990, where there 
is C in place of the reported A. See Gregory, R. J. 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 advantageous 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, 1196RG, 1197RG and 1198RG) 
collectively extending from the Sph I cleavage site at position 1700 to 
the Hinc II cleavage site at position 1785 and including the additional 83 
nucleotides between 1716 and 1717 (see FIG. 6, SEQ ID NO:10). These 
oligonucleotides were phosphorylated with T4 polynucleotide kinase as 
described by Sambrook et al., mixed together, heated to 95.degree. 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 FIGS. 7A and 7B, a subclone of 
plasmid T11 was made by cleaving the Sal I site in the polylinker, 
repairing the recessed 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 Eco RV and Nru I and 
religated. The resulting plasmid T16-.DELTA.5' 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 Sph I and Hinc II at positions corresponding to 
nucleotides 1700 and 1785 of the CFTR cDNA. T16-.DELTA.5' plasmid was 
cleaved with Sph I and Hinc II and the large fragment was isolated by 
agarose gel purification. The annealed synthetic oligonucleotides were 
ligated into this vector fragment to generate T16-intron. 
T16-intron was then digested with Eco RI and Sma I and the large fragment 
was isolated by agarose gel purification. T16-4.5 was digested with Eco RI 
and land the 790 bp fragment was also isolated by agarose gel 
purification. The purified T16-intron and T16-4.5 fragments 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 Sca I site at 
position 2818, and 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 a 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-CFTR-3, shown in FIG. 8. The CFTR cDNA within pKK-CFTR-3 is 
identical to that within pSC-CFTR-2 and pKK-CFTR-2 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-CFTR-3 relative to cells containing the unmodified 
CFTR cDNA in pKK-CFTR-2. 
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-CFTR-2 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 25 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 .sup.35 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-HCl, 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. .sup.35 S labelled proteins were detected by 
fluorgraphy. A band of approximately 180 kD was detected, consistent with 
translation of the full length CFTR insert. 
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, Mass.). 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 
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 trascription 
and translation could also be advantageously altered and/or deleted by the 
same method. 
Further 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 AT to a C 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) 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-1 virus (shown as SEQ ID NO:3) was 
accomplished as follows: The CFTR cDNA was excised from the plasmid 
pCMV-CFTR-936C using restriction enzymes Spe1 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 Spe1/EcII361 restriction 
fragment contains 47 bp of 5' sequence derived from synthetic linkers and 
the multiple cloning site of the vector. 
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 Nhe1 and SnaB1 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 5' 10680 bp of 
Ad2 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 SnaBl site. The construct also contains the 5' 
inverted terminal repeat and viral packaging signals, the E1a enhancer and 
promoter, the E1b 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 A2/CFTR-1 adenovirus, the vector pBR-Ad2-7/CFTR 
was cleaved with BstB1 at the site corresponding to the unique BstB1 site 
at 10670 in Ad2. The cleaved plamid DNA was ligated to BstB1 restricted 
Ad2 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 development 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-1 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 E1 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 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.times.10.sup.7 pfu of MVSS onto approximately 1-2.times.10.sup.7 Wcb 
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 
mls) to each flask. Batches of 50-60 flasks constitute a lot. 
Following 40-48 hours incubation at 37.degree. C., the cells are shaken 
loose from the flask and transferred with medium to a 250 ml centrifuge 
bottle and spun at 1000.times.g. The cell pellet is resuspended in 4 ml 
phosphate buffered saline containing 0.1 g/l CaCl.sub.2 and 0.1 g/l 
MgCl.sub.2 and the cells subjected to cycles of freeze-thaw to release 
virus. Cellular debris is removed by centrifugation at 1000.times.g for 15 
min. The supernatant from this centrifugation is layered on top of the 
CsCl step gradient: 2 ml 1.4 g/ml CsCl and 3 ml 1.25 g/ml CsCl in 10 mM 
Tris, 1 mM EDTA (TE) and spun for 1 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 CsCl 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 CsCl 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.degree. C. for 
several weeks or can be stored for longer periods at -80.degree. 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 
SDS polyacrylarnide 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 
CsCl 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. 
6. Contaminating Materials 
The material to be admi nistered to patients wil be 2.times.10.sup.6 pfu, 
2.times.10.sup.7 pfu and 5.times.10.sup.7 pfu of purified Ad2/CFTR-1. 
Assuming a minimum particle to pfu ratio of 500, this corresponds to 
1.times.10.sup.9, 1.times.10.sup.10 and 2.5.times.10.sup.10 viral 
particles, these correspond to a dose by mass of 0.25 .mu.g, 2.5 .mu.g and 
6.25 .mu.g assuming a molecular mass for adenovirus of 150.times.10.sup.6. 
The origin of the materials from which a production lot of the purified 
Ad/CFTR-1 is derived was described in detail above and is illustrated as a 
flow diagram in FIG. 6. All the starting materials from which the purified 
virus is made (i.e., 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 CsCl centrifugation, dialyzed, and tested. A 
production lot should constitute 1-5.times.10.sup.10 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 raised against the proteins purified in a mock 
purification run. Development of such an assay has not yet been attempted 
for the CsCl purification scheme for Ad2/CFTR-1. 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 serun albumin 
(BSA), transferin and IgG heavy and light chain derived from the serun 
added to the growth medium. Tests using such reagents to examine research 
batches of Ad2/CFTR-1 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-1 vector or other contaminating agents. For the test, 
it is proposed that 10 dishes of Rat 1 cells containing 2.times.10.sup.6 
cells (the number of target cells in the patient) with 4 times the highest 
human dose of Ad2/CFTR-1 (2.times.10.sup.8 pfu) will be infected. 
Following infection, the cells will be plated out in agar and examined for 
the 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/.beta.Gal Virus to Enter 
Airway Epithelial Cells 
a. Hamster Studies 
Initial studies involving the intratracheal instillation of the 
Ad-.beta.Gal 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-.beta.Gal 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.times.10.sup.11 particles; 3.times.10.sup.8 pfu), and 
8 high dose virus (1.7.times.10.sup.12 particles; 5.times.10.sup.9 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. 
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 
time-dependent effects were apparent. First, no damage to airway 
epitheliun 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 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. 
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-.beta.Gal virus 
was used. The epithelial cell density in the nasal cavity of the Rhesus 
monkey is estimated to be 2.times.10.sup.6 cells/cm (based on an average 
nasal epithelial cell diameter of 7 .mu.m) and the surface near 25-50 
cm.sup.2. Thus, there are about 5.times.10.sup.7 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 
10.sup.9 -10.sup.10 pfu were used. 
In the first pilot study the right nostril of Monkey A was infected with 
Ad-.beta.-Gal (.about.1 ml). This viral preparation was purified by CsCl 
gradient centrifugation and then by gel filtration chromatography one week 
later. Adenoviruses are typically stable in CsCl at 4.degree. C. for one 
to two weeks. However, this viral preparation was found to be defective 
(i.e., it did not produce detectable .beta.-galactosidase activity in the 
permissive 293 cells). Thus, it was concluded that there was no live viral 
activity in the material. .beta.-galactosidase activity in nasal 
epithelial cells from Monkey A was also not detected. Therefore, in the 
next study, two different preparations of Ad-.beta.-Gal virus: one that 
was purified on a CsCl gradient and then dialyzed against Tris-buffered 
saline to remove the CsCl, and a crude unpurified one was used. Titers of 
Ad-.beta.-Gal viruses were .about.2.times.10.sup.10 pfu/ml and 
&gt;1.times.10.sup.13 pfu/ml, respectively, and both preparations produced 
detectable .beta.-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 
.beta.-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 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 (.about.5 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-.beta.-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 CsCl-purified virus (.about.1.5 ml) and Monkey B 
received the crude virus (.about.6 ml). (note that this was the second 
exposure of Monkey A to the recombinant adenovirus). 
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 
.about.10.sup.6 cells/ml. Cells were then collected on slides 
(approximately 2.times.10.sup.4 cellstslide using a Cytospin 3 (Shandon, 
Pa.)) for subsequent staning (see below). 
To determine viral efficacy, nasal, pharyngeal, and tracheal cells were 
stained for .beta.-galactosidase using X-gal (5 
bromo-4chloro-3-indolyl-.beta.-D-galactoside). Cleavage of X-gal by 
.beta.-galactosidase produces a blue color that can be seen with light 
microscopy. The Ad-.beta.-gal vector included a nuclear-localization 
signal (NLS) (from SV40 large T-antigen) at the amino-terminus of the 
.beta.-galactosidase sequence to direct expression of this protein to the 
nucleus. Thus, the number of blue nuclei after staining was determined. 
RT-PCR (reverse trnnscriptase-polymerase chain reaction) was also used to 
determine viral efficacy. This assay indicates the presence of 
.beta.-galactosidase MRNA in cells obtained by brushings or swabs. PCR 
primers were used in both the adenovirus sequence and the LacZ sequence to 
distinguish virally-produced niRNA 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 
.beta.-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 of neutrophils 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 supernatant of the cell suspension from 
swabs or brushes. Each supernatant 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 .beta.-galactosidase. Cytopathic effects and blue-stained 
cells indicated the presence of live virus. 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.degree. C. (normal for Rhesus 
monkey 38.8.degree. C.) but had no other abnormalities on 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 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 .beta.-Gal probe, consistent with .beta.-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. 
c. Human Explant Studies 
In a second type of experiment, epithelial cells from a nasal polyp of a CF 
patient nwere 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 (1 sc) across the 
monolayer was measured. cAMP agonists did not increase the lsc, 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 pfulcell) express functional CFTR; cAMP agonists stimulated 1 sc, 
indicating stimulation of Cl.sup.- 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 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 Deliver to and Expression of CFTR in Cotton Rat and Rhesus Monkey 
Epithelium 
MATERIALS AND METHODS 
Adenovirus Vector 
Ad2/CFTR1 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 E1a promoter 
was used for CFTR cDNA. Termination/polyadenylation occurs at the site 
normally used by the E1b and 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 E1 early viral promoters. The 
cells were frozen and thawed three times to release the virus and the 
preparation was purified on a CsCl gradient, then dialyzed against 
Tris-buffered saline (PBS) to remove the CsCl, 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 .mu.l solution 
containing 4.1.times.10.sup.9 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.times.10.sup.8 pfu of the Ad2/CFTR-1 virus on day 0 and 9 of the rats 
received a second dose of 3.2.times.10.sup.8 pfu of Ad2/CFTR1 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 brochoaveolar lavage (BAL) 
was performed with 3 ml aliquots of phosphate-buffered saline. A median 
stenotomy 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.degree. 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). 
Monkys. 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 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.times.10.sup.9 pfu the 
first time, 2.3.times.10.sup.9 pfu the second time, and 2.8.times.10.sup.9 
pfu the third time. We estimated that the cell density of the nasal 
epithelia to be 2.times.10.sup.6 cells/cm.sup.2 and a surface area of 25 
to 50 cm.sup.2. This corresponds to a multiplicity of infection (MOI) of 
approximately 25. 
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, only 
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 of 
ketamine (15 mg/kg). To obtain nasal epithelial cells, 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. 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 consistency was also recorded to check for the 
possibility of diarrhea At each of the evaluation time points, we measured 
rectal temperature, respiratory rate, and heart rate. We visually 
inspected the nasal mucosa, conjunctivas, and pharynx. 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. 
Serology 
Sera were obtained and anti-adenoviral antibody titers were measured by an 
enzyme-linked immunoadsorbant assay (ELISA). For the ELISA, 50 ng/well of 
filled adenovirus (Lee Biomolecular Research Laboratories, San Diego, 
Calif.) in 0.1 M NaHCO.sub.3 were coated on 96 well plates at 4.degree. 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 ImnmunoResearch 
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.sub.2 SO.sub.4 and read 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&gt;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)] were added in two-fold serial dilutions to 
a 96 well plate. Adenovirus (2.5.times.10.sup.5 pfu was added and 
incubated for 1 hour at 37.degree. C. The 293 cells were then added to all 
wells and the plates were incubated until the serum-free control wells 
exhibited &gt;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 &gt;95% cytopathic effect. 
Bronchoalveolar Lavage and Nasal Brushings for Cytology 
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 10.sup.6 
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 
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 (H&E). Sections were reviewed by at 
least one of the study personnel and by a pathologist who was unaware of 
the treatment each rat received. 
Pieces of lung and trachea of the cotton rats and nasal biopsies were 
frozen in liquid 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, Calif.) was added. After 
removal of the secondary antibody, streptavidin FITC (Biomeda, Foster 
City, Calif.) 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 
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 .mu.l sterile water, boiled for 
5 min., and centrifuged. A 5 .mu.l aliquot of the supernatant 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-1 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 NO:5) 
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 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM 
MgCl.sub.2, 0.001% (w/v) gelatin, 400 .mu.M each dNT?, 0.6 .mu.M each 
primer (first set), and 2.5 units AmpliTaq (Perkin Elmer) was aliquoted 
into separate tubes. A 5 .mu.l aliquot of each sample prep was then added 
and the mixture was overlaid with 50 .mu.l of light mineral oil. The 
samples were processed on a Barnstead/Thermolyne (Dubuque, Iowa) thermal 
cycler programmed for 1 min. at 94.degree. C., 1 min. at 65.degree. C., 
and 2 min. at 72.degree. C. for 40 cycles. Post-run dwell was for 7 min. 
at 72.degree. C. A 5 .mu.l aliquot was removed and added to a second PCR 
reaction using the nested set of primers and cycled as above. A 10 .mu.l 
aliquot of the final 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 .mu.l aliquot of 
guanidine isothiocyanate solution (4 M guanidine isothiocyanate, 25 mM 
sodium citrate t)H 7.0, 0.5% sarcosyl, and 0.1 M .beta.-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 Wis.) at 37.degree. C. for 20 min., denatured at 
99.degree. C. for 5 min., precipitated with ammonium acetate and ethanol, 
and redissolved in 4 .mu.l diethylpyrocarbonate treated water containing 
20 units RNase Block 1 (Stratagene, La Jolla Calif.). A 2 .mu.l aliquot of 
the purified RNA was reverse transcribed using the 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 .mu.l 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 .mu.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 et al.). A fragment of CFTR cDNA (aminoacids #1-525) was labeled 
with [.sup.32 P]-dCTP (ICN Biomedicals, Inc. Irvine Calif.) using an 
oligolabeling kit (Pharmacia, Piscataway, N.J.) and purified over a NICK 
column (Pharmacia Piscataway, N.J.) for use as a hybridization probe. The 
labeled probe was denatured, cooled, and incubated with the prehybridized 
filter for 15 hours at 42.degree. C. The hybridized filter was then 
exposed to film (Kodak XAR-5) for 10 min. 
Culture of Ad2/CFTR-1 
Viral cultures were performed on the permissive 293 cell line. For culture 
of virus from lung tissue, 1 g of lung was frozenahawed 3-6 times and then 
mechanically disrupted in 200 .mu.l 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 .mu.l of the supernatant was added in 
duplicate to 293 cells grown in 96 well plates at 50% confluence. The 293 
cells were incubated for 72 hr at 37.degree. C., then fixed with a mixture 
of equal parts of methanol and acetone for 10 min. and incubated with 
FITC-labeled antiadenovirus monoclonal antibodies (Chemicon, Light 
Diagnostics, Temecuca, Calif.) for 30 min. Positive nuclear 
immunofluorescence was interpreted as positive culture. The sensitivity of 
the assay was evaluated by adding dilutions of Ad2/CFTR-1 to 50 .mu.l 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-1 to transfer CFTR cDNA to the 
intrapulmonary airway epithelium, several studies were performed. 
4.times.10 pfu-I.U. of Ad2/CFTR-1 in 100 .mu.l s adminstered to seven 
cotton rats; three control rats received 100 .mu.i 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 procedure did 
not detect endogenous rat CFTR. The lungs of animals which received 
Ad2/CFTR-1 were positive for virally-encoded CFTR mRNA. The lungs of all 
control rats were negative. 
To detect the protein, lung sections were immunostained with antibodies 
specific to CFTR. CFTR was detected at the apical membrane of bronchial 
epithelium from all rats exposed to Ad2/CFTR-1, but not from control rats. 
The location of recombinant CFTR at the apical membrane is consistent with 
the location of endogenous CFTR in human airway epithelium. Recombinant 
CFTR was detected above background levels because endogenous levels of 
CFTR in airway epithelia are very low and thus, difficult to detect by 
immunocytochemistry (Trapnell, B. et al. (1991) Proc. Natl. Acad. Sci. USA 
88:6565-6569; Denning, G. M. et al. (1992) J. Cell Biol. 118:551-59). 
These results show that Ad2/CFTR-1 directs the expression of CFTR mRNA in 
the lung of the cotton rat and CFTR protein in the intrapulmonary airways. 
Safety of Ad2/CFTR-1 in Cotton Rats 
Because the E1 region of Ad2 is deleted in the Ad2/CFTR-1 virus, the vector 
was expected to be replication-impaired (Berkner, K. L. (1988) 
BioTechniques 6:616-629) and that it would be unable to shut off host cell 
protein synthesis (Basuss, L. E. et al. (1989) J. Virol. 50:202-212). 
Previous in vitro studies have suggested that this is the case in a 
variety of cells including primary cultures of human airway epithelial 
cells (Rich, D. P. et al. (1993) Human Gene Therapy 4:461-476). However, 
it is important to confirm this in vivo in the cotton rat, which is the 
most permissive animal model for human adenovirus infection (Ginsberg, H. 
S. et al. (1989) Proc. Natl. Acad Sci. USA 86:3823-3827; Prince, G. A. et 
al. (1993) J. Virol. 67:101-111). Although dose of virus of 
4.1.times.10.sup.10 pfus per kg was used, none of the rats dies. More 
importantly, extracts from lung homogenates from each of the cotton rats 
were cultured in the permissive 293 cell line. With this assay 1 pfu of 
recombinant virus was detected in lung homogenate. However, virus was not 
detected by culture in the lungs of any of the treated animals. Thus, the 
virus did not appear to replicate in vivo. 
It is also possible that administration of Ad2/CFTR-1 could cause an 
inflammatory response, either due to a direct effect of the virus or as a 
result of administration 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 inflamunatory response. To assess the pulmonary 
inflammatory response more directly, bronchoalveolar lavage was performed 
on each of the rats. FIG. 18A 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. There was no 
evidence of viral inclusions or any other changes characteristic of 
adenoviral infection (Prince, G. A. et al. (1993) J. Virol. 67:101-111). 
When coded lung sections were evaluated by a skilled reader 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 fromt 
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 CFTR in the intestinal epithelium, 
there was no obvious adverse consequence. 
Repeat Administration of Ad2/CFTR-1 to Cotton Rats 
Because adenovirus DNA integration into chromosomal DNA is not necessary 
for gene expression and only occurs at very low frequency, expression 
following any given treatments was anticipated to be finite and that 
repeated administration of recombinant adenovirus would be required for 
treatment of CF airway disease. Therefore, the effect of repeated 
administration of Ad2/CFTR-1 cotton rats was examined. Twelve cotton rats 
received 50 .mu.l of Ad2/CFTR-1. Two weeks later, 9 of the rats received a 
second dose of 50 .mu.l of Ad2/CFTR-1 and 3 rats received 50 .mu.l of TBS. 
Rats were sacrificed on day 3, 7, or 14 after virus administration. At the 
time of the second vector administration all cotton rats had an increased 
antibody titer to adenovirus. 
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 animals receiving adenovirus for the first 
time. Viral cultures 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 
differential white blood count at any of the time points. The lungs were 
evaluated by histologic sections stained with H&E; and found no observable 
differences between the control and treated rats when sections were read 
by us or by a blinded skilled reader. When organs were examined for viral 
DNA using PCR, viral DNA was found only in the small 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 
immunocytochemistry of sections stained with CFTR antibodies) similar to 
that seen in animals that received a single administration was observed. 
These results suggest that prior administration of Ad2/CFTR-1 and the 
development of an antibody response did not cause an inflammatory response 
in the rats nor did it prevent virus-dependent production of CFTR. 
Evidence that Ad2/CFTR-1 Expresses CFTR in Primate Airway Epithelium 
The cells lining the respiratory tract and the immune system of primates 
are similar to those of humans. To test the ability of Ad2/CFTR-1 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 epithelium 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 detecable for 18 days after virus 
administration. An example of the results are shown in FIG. 18A. The 
presence of a positive reaction in cells from the left nostril most likely 
represents some virus movement to the left side due to drainage, or 
possibly from the monkey moving the virus from one nostril to the other 
with its fingers after it recovered from anesthesia. 
The specificity of the RT-PCR is shown in FIG. 23B. A Southern blot with a 
probe to CFTR hybridized with the RT-PCR product from the monkey infected 
with Ad2/CFTR-1. As a control, one monkey received a different virus 
(Ad2/.beta.Gal-1) which encodes .beta.-galactosidase. When different 
primers were used to reverse transcribe the .beta.-galactosidase mRNA 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 amplification of the 
adenovirus-directed CFTR transcript. 
The failure to detect evidence of adenovirus-encoded CFTR mRNA at 18 days 
or beyond suggests that the sensitivity of the RT-PCR may be low because 
of limited efficacy of the reverse transcriptase 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 mRNA was not 
detected after 2 weeks, viral DNA was present for a prolonged period and 
may have been transcriptionally active. 
To assess the presence of CFTR proteins directly, cells obtained by 
brushing were plated onto slides by cytospin and stained with antibodies 
to CFTR. A positive reaction was clearly evident in cells exposed to 
Ad2/CFTR-1. The cells were scored as positive by immunocytochemistry when 
evaluated by a reader uninformed to the identity of the samples. 
Immunocytochemistry remained positive for five to six weeks for the three 
monkeys, even after the second administration of Ad2/CFTR-1. On occasion, 
a few positive staining cells were observed from the contralateral 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 immunofluorescence from the surface epithelium was observed, but 
the submucosal glands showed significant staining of CFTR. These 
observations are consistent with results of previous studies (Engelhardt, 
J. F. and Wilson, J. M. (1992) Nature Gen. 2:240-248.) In contrast, 
sections from monkeys that received Ad2/CFTR-1 revealed increased 
immunofluorescence at the apical membrane of the surface epithelium. The 
submucosal glands did not appear to have greater immunostaining than was 
observed under control conditions. These results indicate that Ad2/CFTR-1 
can transfer the CFTR cDNA to the airway epithelium of Rhesus monkeys, 
even in seropositive animals (see below). 
Safety of Ad2/CFTR-1 Administered to Monkeys 
FIG. 20 shows that all three treated monkeys developed antibodies against 
adenovirus. 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 neutralizing antibodies were also 
performed. After the first administration, neutralizing antibodies were 
not observed, but they were detected after the second administration and 
during the third viral administration (FIG. 20). 
To detect virus, supernatants from nasal brushings and swabs were cultured 
on 293 cells. All monkeys had positive cultures on day 1 and on day 3 or 4 
from the infected nostril. Cultures remained positive in one of the 
monkeys at seven days after admninistration, but cultures were never 
positive beyond 7 days. Live virus was occasionally detected in swabs from 
the contra lateral 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 
inflammation. Visual inspection of the nasal epithelium revealed slight 
erythema in all three monkeys in both nostrils on the first day after 
infection; but similar erythema was observed in the control monkey and 
likely resulted from the instrumentation. There was no visible 
abnormalities at days 3 or 4, or on weekly inspection thereafter. Physical 
examination revealed no fever, lymphadenopathy, conjunctivitis, tachypnea, 
or tachycardia at any of the time points. No abnormalities were found in a 
complete blood count or sedimentation rate, nor were abnormalities 
observed in serum electrolytes, transaminases, or blood urea nitrogen and 
creatinine. 
Examination of Wright-stained cells from the nasal brushings showed that 
neutrophils and lymphocytes accounted for less than 5% of total cells in 
all three monkeys. Administration of the Ad2/CFTR-1 caused no change in 
the distribution or number of inflammatory 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 differentiated from that of 
the experimental monkey when the specimens were reviewed by an independent 
pathologist. 
These results demonstrate 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 ofhumans with cystic fibrosis. 
Two methods were used to show that Ad2/CFTR-1 expresses CFTR in the airway 
epithelium of cotton rats and primates: CFTR mRNA was detected using 
RT-PCR and protein was detected by immunocytochemistry. Duration of 
expression as assessed immunocytochemically was five to six weeks. Because 
very little protein is required to generate Cl.sup.- secretion (Welsh, M. 
J. (1987) Physiol. Rev. 67:1143-1184; Trapnell, B. C. et al. (1991) Proc. 
Natl. Acad. Sci. USA 88:6565-6569; Denning, G. M. et al. (1992) J. Cell 
Biol. 118:551-559), it is likely that functional expression of CFTR 
persists substantially longer than the period of time during which CFTR 
was detected by immunocytochemistry. Support for this evidence comes from 
two considerations: first, it is very difficult to detect CFTR 
immuncytochemically in the airway epithelium, yet the expression of an 
apical membrane Cl.sup.- permeability due to the presence of CFTR 
Cl.sup.- 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 number of ions (10.sup.6 
-10.sup.7 ions/sec). Thus, ion channels are not usually abundant proteins 
in epithelia. Second, previous work suggests that the defective 
electrolyte transport of CF epithelia can be corrected when only 6-10% of 
cells in a CF airway epithelium overexpress wild-type CFTR (Johnson, L. G. 
et al. (1992) Nature Gen. 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-1 at relatively low multiplicities of 
infection generated a cAMP-stimulated Cl.sup.- secretory response in CF 
epithelia (Rich, D. P. et al. (1993) Human Gene Therapy 4:461-476). 
This study also provides the first comprehensive data on the safety of 
adenovirus vectors for gene taansfer to airway epithelium. Several aspects 
of the studies are encouraging. There was no evidence of viral 
replication, rather infectious viral particles were rapidly cleared 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 adenovirus vector in the treatment of CF 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 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-1 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-1 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 
Adenovirus vector 
The recombinant adenovirus Ad2/CFTR-1 was used to deliver CFTR cDNA. The 
construction and preparation of Ad2/CFTR-1, 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 E1a promoter was used for CFTR cDNA; 
this is a low to moderate strength promoter. Termination/polyadenylation 
occurs at the site normally used by E1b and protein IX trnscripts. The E3 
region of the virus was conserved. 
Patients 
Three patients with CF were studied. Genotype was determined by IG Labs 
(Framingham, Mass.). All three patients had mild CF as defined by an NIH 
score&gt;70 (Taussig, L. M. et al. (1973) J. Pediatr. 82:380-390), a normal 
weight for height ratio, a forced expiratory volume in one second (FEV1) 
greater than 50% of predicted and an arterial PO.sub.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 E1 
region were negative. Patients were evaluated at least mice by FEV1, 
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 .DELTA.F508 mutation. Her NIH 
score was 90 and her FEV1 was 83% predicted. The second patient is a 36 
year old man who was diagnosed at the age of 13 when he presented with 
symptoms of pancreatic insufficiency. A sweat chloride test revealed a 
chloride concentration of 70 mEq/l. He is a heterozygote with the 
.DELTA.F508 and G55ID mutations. His NIH score was 88 and his FEV1 was 66% 
predicted. The third patient is a 50 year old woman, diagnosed 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 homozygous 
for the .DELTA.F508 mutation. Her NIH score was 73 and her FEV1 was 65% 
predicted. 
Transepithelial Voltage 
The transepithelial electric potential difference across the nasal 
epithelium was measured using techniques similar to those previously 
described (Alton, E. W. F. W. et al (1987) Thorax 42:815-817; Knowles, M. 
et al. (1981) N. Eng. J. Med. 305:1489-1495). A 23 gauge subcutaneous 
needle connected with sterile normal saline solution to a silver/silver 
chloride pellet (E. W. Wright, Guilford, Conn.) was used as a reference 
electrode. The exploring electrode was a size 8 rubber catheter (modified 
Argyle.sup.R Foley catheter, St. Louis, Mo.) with one side hole at the 
tip. The catheter was filled with Ringer's solution containing (in mM), 
135 NaCl, 2.4 KH.sub.2 PO.sub.2, K.sub.2 HPO.sub.4, 1.2CaCL.sub.2, 1.2 
MgCl.sub.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 
(eithley Instruents Inc., Cleveland, Ohio) connected to a strip chart 
recorder (Servocorder, Watanabe Instruments, Japan). Prior to the 
measurements, the silver/silver chloride pellets were connected in series 
with the Ringer's solution; the pellets were changed if the recorded Vt 
was greater than .+-.4 mV. The rubber catheter was introduced into the 
nostril under telescopic guidance (Hopkins Telescope, Karl Storz, 
Tuttlingen West Germany) and the side hole of the catheter was placed next 
to the study area in the medical aspect of the inferior nasal turbinate. 
The distance from the anterior tip of the inferior turbinate and the 
spatial relationship with the medial turbinate, the maxillary sinus 
ostium, and in one patient a small polyp, were used to locate the area of 
Ad2/CFTR-1 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 .mu.l/min infusion of the Ringer's 
solution. Once a stable baseline was achieved, 200 .mu.l of a Ringer's 
solution containing 100 .mu.M amiloride (Merck and Co. Inc., West Point, 
Pa.) was instilled through the catheter and changes in Vt were recorded 
until no further change were observed after intermittent instillations. 
Finally, 200 .mu.l Ringer's solution containing 100 .mu.M amiloride plus 
10 .mu.M terbutaline (Geigy Pharmaceuticals, Ardsley, N.Y.) was instilled 
and the changes in Vt were recorded. 
Measurements of basal Vt were reproducible over time: in the three treated 
patients, the coefficients of variation before administration of 
Ad2/CFTR-1 were 3.6%, 12%, and 12%. The changes induced by terbutaline 
were also reproducible. In 30 measurements in 9 CF patients, the 
terbutaline-induced changes in Vt (.DELTA.Vt) ranged from 0 mV to +4 mV; 
hyperpolarization of Vt was never observed. In contrast, in 7 normal 
subjects .DELTA.Vt ranged from -1 mV to -5 mV; hyperpolarization was 
always observed. 
Ad2/CFTR-1 Application and Cell Acquisition 
The patients were taken to the operating room and monitoring was commenced 
using continuous EKG and pulse oximetry recording as well as automatic 
intermittent blood pressure measurement. After mild sedation, the nasal 
mucosa was anesthetized by atomizing 0.5 ml of 5% cocaine. The mucosa in 
the area of the inferior turbinate was then packed with cotton pledgets 
previously soaked in a mixture of 2 ml of 0.1% adrenaline and 8 ml of 1% 
tetracaine. The pledgets remained in place for 10-40 min. Using endoscopic 
visualization with a television monitoring system, the applicator was 
introduced through the nostril and positioned on the medial aspect of the 
inferior turbinate, at least three centimeters from its anterior tip 
(FIGS. 21A-21I). The viral suspension was infused into the applicator 
through connecting 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 nasal 
epithelium for thirty 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 
endoscopic guidance until the side hole of the catheter was in contact 
with the area of interest in the inferior turbinate. The viral solution 
was infused slowly until a drop of solution was seen with the telescope. 
The catheter was left in place for thirty minutes and then removed. 
Cells were obtained from the area of virus administration approximately 2 
weeks before treatment and then at weekly intervals after treatment. The 
inferior turbinate was packed for 10 minutes with cotton pledgets 
previously soaked in 1 ml of 5% cocaine. Under endoscopic control, the 
area of administration was gently brushed for 5 seconds. The brushed cells 
were dislodged in PBS. Swabs of the nasal epithelia were collected using 
cotton tipped applicators without anesthesia. Cytospin slides were 
prepared and stained with Wright's 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 inspection and identification of 
the site to be biopsied, the submucosa was injected with 1% xylocaine, 
with 1/100,000 epinephrine. The area of virus application on the inferior 
turbinate was removed. The specimen was fixed in 4% formaldehyde and 
stained. 
Results 
On day one after Ad2/CFTR-1 administration and at all subsequent time 
points, Ad2/CFTR-1 from the nasal epithelium, pharynx, blood, urine, or 
stool could not be cultured. As a control for the sensitivity of the 
culture assay, samples were routinely spiked with 10 and 100 I.U. 
Ad2/CFTR-1. In every case, the spiked samples were positive, indicating 
that, at a minimum, 10 I.U. 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 arterial blood gases performed before and 
after Ad2/CFTR-1 administration. An increase in antibodies to adenovirus 
was not detectable by ELISA or by neutralization for 35 days after 
treatment. 
Three to four hours after Ad2/CFTR-1 administration, at the time that local 
anesthesia and localized vasoconstriction abated, all patients began to 
complain of nasal congestion and in one case, mild rhinorrhea 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 erythema, 
edema, and exudate (FIGS. 21A-21C). These physical findings followed a 
time course similar to the symptoms. The physical findings were not 
limited to the site of virus application, even though preliminary studies 
using the applicator showed that marker methylene blue was limited to the 
area of application. In two additional patients with CF, the 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 (FIGS. 21G-21I). Moreover, the local anesthesia 
and vasoconstriction generated similar changes even when the applicator 
was not used, suggesting 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 percent neutrophils in patients treated with Ad2/CFTR-1 or with 
saline. One week after application, the neutrophilia had resolved in both 
groups. Respiratory epithelial 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 possible ischemia (probably secondary to the 
anesthetic/vasoconstrictors used before virus administration), but there 
were no abnormalities suggestive of virus-mediated damage. 
Because the 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 or 
vasoconstriction and which was thus less likely to cause injury, but which 
was also less certain in its ability to constrain Ad2/CFTR-1 in a 
precisely defined area. On the right side, Ad2/CFTR-1 was administered as 
in the first two patients, and on the left side, the virus was 
administered without anesthesia or the applicator, instead using a small 
Foley catheter to apply and maintain Ad2/CFTR-1 in a relatively defined 
area by surface tension (FIG. 21E). On the right side, the symptoms and 
physical findings were the same as those observed in the first two 
patients. By contrast, on the left side there were no symptoms and on 
inspection the nasal mucosa appeared normal (FIGS. 21O-21F). Nasal swabs 
obtained 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 (FIG. 22), showed 
morphology consistent with CF--a thickened basement membrane and 
occasional polymorphonuclear cells in the submucosa--but no abnormalities 
that could be attributed to the adenovirus vector. 
The first patient developed symptoms of a sore throat and increased cough 
that began three weeks after treatment and persisted for two days. Six 
weeks after treatment she developed an exacerbation of her 
bronchitis/bronchiectasis and hemoptysis that required hospitalization. 
The second patient had a transient episode of minimal hemoptysis three 
weeks after treatment; 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 pretreatment clinical history, evaluation of the 
episodes, and viral cultures, no evidence could be discerned that linked 
these episodes to administration of Ad2/CFTR-1. Rather the episodes 
appeared consistent with the normal course of disease in each individual. 
The loss of CFTR Cl.sup.- channel function causes abnormal ion transport 
across affected epithelia, which in turn contributes to the pathogenesis 
of CF-associated airway disease (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) FASEB J. 4:2709-2717). In airway epithelia, 
ion transport is dominated by two electrically conductive processes: 
amiloride-sensitive absorption of Na.sup.+ from the mucosal to the 
submucosal surface and cAMP-stimulated Cl.sup.- secretion in the opposite 
direction. (Quinton, P. M. (1990) FASEB J. 4:2709-2717; Welsh, M. J. 
(1987) Physiol. Rev. 67:1143-1184). These two transport processes can be 
assessed noninvasively by measuring the voltage across the nasal 
epithelium (Vt) in vivo (Knowles, M. et al (1981) N. Eng. J. Med. 
305:1489-1495; Alton, E. W. F. W. et al.(1987) Thorax 42:815-817). FIG. 23 
shows an example from a normal subject. Under basal conditions, Vt was 
electrically negative (lumen referenced to the submucosal surface). 
Perfusion of amiloride (100 .mu.M) onto the mucosal surface inhibited Vt 
by blocking apical Na.sup.+ channels (Knowles, M. et al (1981) N. Eng. J. 
Med. 305:1489-1495; Quinton, P. M. (1990) FASEB J. 4:2709-2717: Welsh M. 
J. (1992) Neuron 8:821-829). Subsequent perfusion of with terbutaline (10 
.mu.M) a .beta.-adrenergic agonist, hyperpolanzed Vt by increasing 
cellular levels of cAMP, opening CFTR Cl.sup.- channels, and stimulating 
chloride secretion (Quinton, P. M. (1990) FASEB J. 4:2709-2717; Welsh, M. 
J. et al. (1992) Neuron 8:821-829). FIG. 24A shows results from seven 
normal subjects: basal Vt was -10.5.+-.1.0 mV, and in the presence of 
amiloride, terbutaline hyperpolarized Vt by -2.3.+-.0.5 mV. 
In patients with CF, Vt was more electrically negative than in normal 
subjects (FIG. 24B), as has been previously reported (Knowles, M. et al. 
(1981) N. Eng. J. Med. 305:1489-1495). Basal Vt was -37.0.+-.2.4 mV, much 
more negative than values in normal subjects (P&lt;0.001). (Note the 
difference in scale in FIG. 24A and FIG. 24B). Amiloride inhibited Vt, as 
it did in normal subjects. However, Vt failed to hyperpolarize when 
terbutaline was perfused onto the epithelium in the presence of amiloride. 
Instead, Vt either did not change or became less negative: on average Vt 
depolarized by +1.8.+-.0.6 mV, a result very different from that observed 
in normal subjects. (P&lt;0.001). 
After Ad2/CFTR-1 was applied, basal Vt became less negative in all three CF 
patients FIG. 25A shows an example from the third patient before (FIG. 
25A) and after (FIG. 25B) treatment and FIGS. 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 
electolyte transport defect in nasal epithelium of all three patients. 
Additional evidence came from an examination of the response to 
terbutaline. FIG. 25B shows that in contrast to the response before 
Ad2/CFTR-1 was applied, after virus replication, in the presence of 
amiloride, terbutaline stimulated Vt. FIGS. 26B, 26D, and 26F show the 
time course of the response. These data indicate that Ad2/CFTR-1 corrected 
the CF defect in Cl.sup.- transport. Correction of the Cl.sup.- 
transport defect cannot be attributed to the anesthesia/application 
procedure because it did not occur in patients treated with saline instead 
of Ad2/CFTR-1 FIG. 27). Moreover, the effects of the anesthesia were 
generalized on the nasal mucosa, but basal Vt decreased only in the area 
of virus administration. Finally, similar changes were observed in the 
left nasal mucosa of the third patient (FIGS. 26E and 26F), which had no 
symptomatic or physical response after the modified application procedure. 
Unsuccessful attempts were made to detect CFTR transcripts by reverse 
trnsciptase-PCR and by immunocytochemistry in cells from nasal brushings 
and biopsies. Although similar studies in animals have been successful 
(Zabner, J. et al. (1993) Nature Gen. (in press)), those studies used much 
higher doses of Ad2/CFTR-1. The lack of success in the present case likely 
reflects the small amount of available tissue, the low MOI, the fact that 
only a fraction of cells may have been corrected, and the fact that 
Ad2/CFTR-1 contains a low to moderate strength promoter (E1a) which 
produces much less mRNA and protein than comparable constructs using a 
much stronger CMV promoter (unpublished observation). The E1a promoter was 
chosen because CFTR normally expressed at very low levels in airway 
epithelial cells (Trapnell, B. C. et al. (1991) Proc. Natl. Acad. Sci. USA 
88:6565-6569). It is also difficult to detect CFTR protein and mRNA in 
normal human airway epithelia, although function is readily detected 
because a single ion channel can conduct a very large number of ions per 
second and thus efficiently support Cl.sup.- transport. 
With time, the electrical changes that indicate correction of the CF defect 
reverted toward pretreatment values. However, the basal Vt appeared to 
revert more slowly than did the change in Vt produced by terbutaline. The 
significance of this difference is unknown, 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 area 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 of Adenovirus-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 Cl.sup.- transport that is characteristic of CF epithelia. 
Complementation of the Cl.sup.- 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 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.sup.- 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 publication): at an MOI of less than 1, 
cAMP-stimulated Cl.sup.- 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 .beta.-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 2.times.10.sup.6 cells/cm.sup.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.sup.2 (Weibel, E. R. Morphometry of the Human 
Lung (Springer Verlag, Heidelberg, 1963) then there would be approximately 
3.times.10.sup.9 potential target cells. Assuming a particle to I.U. ratio 
of 100, this would correspond to approximately 3.times.10.sup.11 particles 
of adenovirus with a mass of approximately 75 .mu.g. While obviously only 
a crude estimate, such information is useful in designing animal 
experiments to establish the likely safety profile of a human dose. 
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.sup.- 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 Cl.sup.- channel is capable of transporting large 
numbers of ions/sec) and the low activity of the E1a promoter used to 
transcribe CFTR, the inability to detect CFTR protein and CFTR nmRNA are 
perhaps not surprising. Although CFTR mRNA could not be detected by 
reverse transcriptase-PCR, Ad2/CFTR-1 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 tracriptase. Although there is little doubt that the changes in 
electrolyte tranport measured here result from expression of CFTR, it 
remains to be seen whether this will lead to measurable clinical changes 
in lung function. 
Safety Considerations 
Application of the adenovirus vector to the nasal epithelium in these three 
patients was well-tolerated. Although mild inflammation was observed in 
the nasal epithelium of all three patients following administration of 
Ad2/CFTR-1, 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-1. 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 E1 region of adenovirus, such as 293 cells, or under-conditions where 
the E1 region is provided by coinfection with or recombination with an 
E1-containing adenovirus (Graham, F. L. and Prevec, L. Vaccines: New 
Approaches to Inmmunological Problems (R. W. Ellis, ed., Boston, 
Butterworth-Heinermann, 1992); Berkner, K. L. (1988) Biotechniques 
6:616-629). The patients studied here where seropositive for adenovirus 
types 2 and 5 prior to the study were negative for adenovirus upon culture 
of nasal swabs prior to administration of Ad2/CFTR-1, and were shown by 
PCR methods to lack endogenous E1 DNA sequences such as have been reported 
in some human subjects (Matsuse T. et al. (1992) Am. Rev. Respir. Dis. 
146:177-184). 
Example 11 
Construction and Packaging of Pseudo Adenoviral Vector (PAV) 
With reference to FIG. 16, the PAV construct was made by inserting the Ad2 
packaging signal and E1 enhancer region (0-358 nt) in Bluescript II SK- 
(Stratagene, LaJolla, Calif.). A variation of this vector, known as PAV II 
was constructed similarly, except the Ad2 packaging signal and E1 enhancer 
region contained 0-380 nt. The addition of nucleotides at the 5' end 
results in larger PAVs, which may be more efficiently packaged, yet would 
include more adenoviral sequences and therefore could potentially be more 
immunogenic or more capable of replicating. 
To allow ease of manipulation for either the insertion of gene coding 
regions or complete excision and use in transfections for the purpose of 
generating infectious particles, a complementary plasmid was also built in 
p Bluescript SKII-. This complementary plasmid contains the Ad2 major late 
promoter (MLP) and tripartite leader CrPL) DNA and an SV40 T-antigen 
nuclear localization signal (NLS) and polyadenylation signal (SVpA). As 
can be seen in FIG. 16, this plasmid contains a convenient restriction 
site for the insertion of genes of interest between the MLP/TPL and SV40 
poly A. This construct is engineered such that the entire cassette may be 
excised and inserted into the former PAV I or PAV II construct. 
Generation of PAV infectious particles was performed by excision of PAV 
from the plasmid with the Apa I and Sac II restriction endonucleases and 
co-transfection into 293 cells (an E1/E1b expressing cell line) (Graham, 
F. L. et al, (1977) J. Gen Virol 36:59-74) with either wild-type Ad2, or 
packaging/replication deficient helper virus. Purification of PAV from 
helper can be accompanied by CsCl gradient isolation as PAV 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 
virion free from contaminating helper virus. The primary advantage of PAV 
over standard adenoviral vectors is the ability to package large DNA 
inserts into virion (up to about 36 kb). However, 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 proportion of 
PAV in viral preparation several approaches can be employed. For example, 
one can use a helper virus which is partially defective for packaging into 
virions (either by virtue of mutations in the packaging sequences (Grable, 
M. and Hearing P. (1992) J. Virol. 66: 723-731)) or by virtue of its 
size--viruses with genome sizes greater than approximately 37.5 kb package 
inefficiently. In mixed infections with packaging defective virus, PAV 
would be expected to be represented at higher levels in the virus mixture 
than would occur with non-packaging defective helper viruses. 
Another approach is to make the helper virus dependent upon PAV for its own 
replication. This may most easily be accomplished by deleting an essential 
gene from the helper virus (e.g. IX or a terminal protein) and placing 
that gene in the PAV vector. In this way neither PAV nor the helper virus 
is capable of independent replication--PAV and the helper virus are 
therefore co-dependent. This should result in higher PAV representation in 
the resulting virus preparation. 
A third approach is to develop a novel packaging cell line, which is 
capable of generating significant quantities of PAV virion free from 
contaminating helper virus. A novel protein IX, (pIX) packaging system has 
been developed. This system exploits several documented features of 
adenovirus molecular biology. The first is that adenoviral defective 
particles are known to comprise up to 30% or more of standard wild-type 
adenoviral preparations. These defective or incomplete particles are 
stable and contain 15-95% of the adenoviral genome, typically 15-30%. 
Packaging of a PAV genome (15-30% of wild-type genome) should package 
comparably. Secondly, stable packaging of full-length Ad genome but not 
genomes &lt;95% required the presence of the adenoviral gene designated pIX. 
The novel packaging system is based on the generation of an Ad protein pIX 
expressing 293 cell line. In addition, an adenoviral helper virus 
engineered such that the E1 region is deleted but enough exogenous 
material is inserted to equal or slightly exceed the full length 36 kb 
size. Both of these two 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 vector) and 
used to infect standard 293 (lacking pix) at a multiplicity of infection 
of 1 relative to PAV. Since research with this line as well as from 
incomplete or defective particle research indicates that fildl length 
genomes have a competitive packaging advantage, it is expected that 
infection with an MOI of 1 relative to PAV will necessarily equate to an 
effective MOI for helper of greater than 1. All cells will presumably 
contain both PAV (at least 1) and helper (greater than 1). Replication and 
viral capsid production in this cell should occur normally but only PAV 
genomes should be packaged. Harvesting these 293/pIX cultures is expected 
to yield essentially helper-free PAV. 
Example 12 
Construction of Ad2-E4/ORF 6 
Ad2-E4/ORF6 (FIG. 17 shows the plasmid construction of Ad2-E4/ORF6) is an 
adenovirus 2 based vector deleted for all Ad2 sequences between nucleotide 
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 mRNA intact. 
In place of the deleted sequences, a DNA fragment encoding ORF6 (Ad2 
nucleotides 34082-33178) which was derived by polymerase chain reaction of 
Ad2 DNA with ORF6 specific DNA primers (Genzyme oligo. 
#2371-CGGATCCTTTATTATAGGGGAAGTCCACGCCTAC (SEQ. ID NO:8) and oligo. 
#2372-CGGGATCCATCGATGAAATATGACTACGTCCG (SEQ. ID NO:9) were inserted). 
Additional sequences supplied by the oligonucleotides included a cloning 
site at the 5' and 3' ends of the PCR fragment (Clal and BamHl 
respectively) and a polyadenylation sequence at the 3' end to ensure 
correct polyadenylation of the ORF6 mRNA. As illustrated in FIG. 17, the 
PCR fragment was first ligated to a DNA fragment including the inverted 
terminal repeat (ITR) and E4 promoter region of Ad2 (Ad2 nucleotides 
35937-35577) and cloned in the bacterial plasmid pBluescript (Stratagene) 
to create plasmid ORF6. After sequencing to verify the integrity of the 
ORF6 reading frame, the fragment encompassing the ITR and ORF6 was 
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 28562-35937) and is 
deleted for all E4 sequences (promoter to poly A site Ad2 positions 
32815-35641) using flanking restriction sites. In this second plasmid, 
virus expressing only E4 ORF6, pAdORF6 was cut with restriction enzyme 
PacI and ligated to Ad2 DNA digested with PacI. This PacI site corresponds 
to Ad2 nucleotide 28612. 293 cells were transfected with the ligation 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 ORF6. 
A cell line could in theory be established that would fully complement E4 
functions deleted from a recombinant virus. The problem with this approach 
is that E4 functions in the regulation of host cell protein synthesis and 
is therefore toxic to cells. Our current recombinant adenoviruses are 
deleted for the E1 region and must be grown in 293 cells which complement 
E1 functions. The E4 promoter is activated in by the E1a gene product, and 
therefore to prevent inadvertent toxic expression of E4 transcription of 
E4 must be tightly regulated. The requirements of such a promoter or 
transactivating system is that in the uninduced state expression must be 
low enough to avoid toxicity to the host cell, but in the induced state 
must be sufficiently activated to make enough E4 gene product to 
complement the E4 deleted virus during virus production. 
Example 13 
An adenoviral vector is prepared as described in Example 7 while 
substituting the PGK promoter for the E1a promoter. 
Example 14 
An adenoviral vector is prepared as described in Example 11 while 
substituting the PGK promoter for the Ad2 major late promoter (MLP). 
Example 15 
Generation of Ad2-ORF6/PGK-CFTR 
This protocol uses a second generation adenovirus vector named 
Ad2-ORF6/PGK-CFTR. This virus lacks E1 and in its place contains a 
modified trascription unit with the phosphoglycerate kinase (PGK) 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 tissue culture. This has the effect of 
generating a genome of 101% the size of wild type Ad2 and renders the 
vector more easy to grow in culture than Ad2-ORF6/PGK-CFTR. 
The DNA construct comprises a full length copy of the Ad2 genome from which 
the early region 1 (E1) genes (present at the 5' end of the viral genome) 
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 Ad2 has been 
deleted and replaced with only open reading frame 6 (ORF6) of the Ad2 E4 
region. The Adenovirus vector is referred to as AD2-ORF6/PGK-CFTR and is 
illustrated schematically in FIG. 28. The entire wild-type Ad2 genome has 
been previously sequenced (Roberts, R. J., (1986) In Adenovirus DNA, W. 
Oberfler, editor, Matinus Nihoff Publishing, Boston) and we have adopted 
the existing numbering system when referring to the wild type genome. Ad2 
genomic regions flanking E1 and E4 deletions, and insertions into the 
genome are being completely sequenced. 
The Ad2-ORF6/PGK-CFTR construct differs from the one used in our earlier 
protocol (Ad2/CFTR-1) in that the latter utilized the endogenous E1a 
promoter, had no poly A addition signal directly downstream of CFTR and 
retained an intact E4 region. The properties of Ad2/CFTR-1 in tissue 
culture and in animal studies h have been reported (Rich et al., (1993) 
Human Gene Therapy, 4:461-467; and Zabner et al. (1993) Nature Genetics, 
In Press). 
At the 5' end of the genome, nucleotides 357 to 3328 of Ad2 have been 
deleted 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 the published CFTR sequence (Riordan et al. (1989) Science, 
245:1066-1073), 21 nucleotides of linker, and a 32 nucleotide synthetic 
BGH poly A addition signal followed by a final 11 nucleotides of linker. 
The topology of the 5' end of the recombinant molecule is illustrated in 
FIG. 28. 
At the 3' end of the genome of Ad2-ORF6/PGK-CFTR, Ad2 sequences between 
nucleotides 32815 and 35577 have been deleted to remove all open reading 
frames of E4 but retain the E4 promoter, the E4 cap sites and first 32-37 
nucleotides of E4 mRNA. The deleted sequences were replaced with a 
fragment derived by PCR which contains open reading frame 6 of Ad2 
(nucleotides 34082-33178) and a synthetic poly A addition signal. The 
topology of the 3' end of the molecule is shown in FIG. 28. The predicted 
sequence of this region of the molecule is given at the end of this 
appendix. The sequence of this segment of the molecule will be confirmed. 
The remainder of the Ad2 viral DNA sequence is published in Roberts, R. J. 
in Adenovirus DNA. (W. Oberfler, Matinus Nihoff Publishing, Boston, 1986). 
The overall size of the Ad2-ORF6/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 
transcriptional start sites in the cloned PGK promoter (Singer-Sam et al. 
(1984) Gene, 32:409-417) at nucleotides 828, 829 and 837 of the 
recombinant vector (Singer-Sam et al. (1984) Gene, 32:409-417). A hybrid 
5' untranslated region is comprised of 72, 80 or 81 nucleotides of PGK 
promoter region, 86 nucleotide of linker sequence, and 10 nucleotides 
derived from the CFTR insert. Transcriptional termination is expected to 
be directed by the BGH poly A addition signal at recombinant vector 
nucleotide 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-BGH mRNA 
respectively, depending on which transcriptional initiation site is used. 
Within the CFTR 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, 347:382-386; and Cheng et al. (1990) Cell, 
63:827-834). The 3' untranslated region of the predicted CFTR transcript 
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 
immunocytochemically, 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. coli .beta. 
galactosidase protein fused to a nuclear localization signal derived from 
the SV40 T-antigen was therefore constructed. Reporter gene transcription 
is driven by the powerful CMV early gene constitutive promoter. 
Specifically, the E1 region of wild type Ad2 between nucleotides 357-3498 
has been deleted and replaced it with a 515 bp fragment containing the CMV 
promoter and a 3252 bp fragment encoding the .beta. galactosidase gene. 
Regulatory Characteristics of the Elements of the AD2-ORF6/PGK-CFTR 
In general terms, the vector is similar to several earlier adenovirus 
vectors encoding CFTR but it differs in three specific ways from our 
earlier Ad2/CFTR-1 construct. 
PGK Promoter 
Transcription of CFTR is from 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. It may also be less likely 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 we believe 
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) Blood, 78:310-317). 
Polyadenylation 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 
E1 region. Since protein is believed to be involved in packaging of 
virions, we retained this coding region. Furthermore, since protein IX is 
synthesized from a separate transcript with its own promoter, to prevent 
possible promoter occlusion at the protein IX promoter, we inserted the 
BGH poly A addition signal. We have indirect evidence that promoter 
occlusion can be problematic in that AdCMV .beta.Gal grows to lower viral 
titers on 293 cells than does Ad2/.beta.gal-1. These constructs are 
identical except for the promoter used for .beta. galactosidase 
expression. Since the CMV promoter is much stronger than the E1a promoter 
we assume that abundant transcription from the CMV promoter through the 
.beta. 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 of Ad2/CMV-.beta.gal we 
obtain. 
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 E1 and E4 regions in 
Ad2-ORF6/PGK-CFTR reduce the genome size to 101% of wild type. In practice 
we find that it is straightforward to prepare high tier lots of this 
virus. 
The second reason to remove E4 sequences relates to the safety of 
adenovirus vectors. It is our goal 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 E1 region already 
renders such vectors disabled, in part because E1a, 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 E1-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 cell shut off and late mRNA accumulation in the normal 
adenovirus life cycle. The E1-E4-ORF6.sup.+ 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 E1a for activation and 
contains E1a 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 E1 activity. 
Steps Used to Derive the DNA Construct 
Construction of the recombinant Ad2-ORF6/PGK-CFTR virus was accomplished by 
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 E1 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 
ClaI and BamHI sites of 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 ClaI and SpeI 
sites at the region of the E1 deletion and is designated pBRAd2/CMV. The 
plasmid also contains the Ad2 5' ITR, packaging and replication sequences 
and E1 enhancer. The E1 promoter, E1a and most of E1b coding region has 
been deleted. The 3' terminal portion of the E1b coding region coincides 
with the pIX promoter which was retained. The CMV promoter was removed and 
replaced with the PGK promoter as a ClaI and SpeI fragment from the 
plasmid PGK-GCR. The resulting plasmid, pBRAd2/PGK, was digested with 
AvrlI and BstBI and the excised fragment replaced with the SpeI to BstBI 
fragment from the plasmid construct pAd2E1a/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 SpeI and Ecl136II. 
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 supplied by the oligonucleotides include 
cloning sites at the 5' and 3' ends of the PCR fragment. (ClaI and BamHI 
respectively) and a poly A addition sequence AATAAA at the 3' end to 
ensure correct polyadenylation of ORF6 mRNA. The PCR fragment was cloned 
into pBluescript (Stratagene) along with an Ad2 fragment (nucleotides 
35937-35577) containing the inverted terminal repeat, E4 promoter, E4 mRNA 
cap sites and first 32-37 nucleotides of E4 mRNA to create pORF6. A 
SalI-BamHI fragment encompassing the ITR and ORF6 was used to replace the 
SalI-BamHI fragment encompassing the ITR and E4 deletion in pAd.DELTA.E4 
contains the 3' end of Ad2 from a SpeI site to the 3' ITR (nucleotides 
27123-35937) and is deleted for all E4 sequences including the promoter 
and poly A signal (nucleotides 32815-35641). The resulting construct, 
pAdE4ORF6 was cut with PacI and ligated to Ad2 DNA digested with PacI 
nucleotide 28612). 293 cells were transfected with the ligation reaction 
to generate virus containing only open reading frame 6 from the E4 region. 
In Vitro Studies with Ad2-ORF6/PGK-CFTR 
The ability of Ad2-ORF6/PGK-CFTR to express CFTR in several cell lines, 
including human HeLa cells, human 293 cells, and primary cultures of 
normal and CF human airway epithelia. 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 cDNA and express CFTR 
as assessed by inmmunoprecipitation and by fuinctional assays of halide 
efflux. Gregory, R. J. et al. (1990) Nature 347:382-386; Cheng, S. H. et 
al. (1990) Cell 63:827-834. More specifically, procedures for preparing 
cell lysates, immunoprecipitation of proteins using anti-CFTR antibodies, 
one-dimensional peptide analysis and SDS-polyacrylamide 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-stimulated CFTR 
chloride channel activity using the halide sensitive fluorophore SPQ in 
293 cells treated with 500 IU/cell Ad2-ORF6/PGK-CFTR. Stimulation of the 
infected cells with forskolin (20 .mu.M) and IBMX (100 .mu.m) increased 
SPQ fluorescence indicating the presence of functional chloride channels 
produced by the vector. 
Additional studies using primary cultures of human airway (nasal polyp) 
epithelial cells (from CF patients) infected with Ad2-ORF6/PGK-CFTR 
demonstrated that Ad2-ORF6/PGK-CFTR infection of the nasal polyp 
epithelial cells resulted in the expression of cAMP dependent Cl.sup.- 
channels. FIG. 29 is an example of the results obtained from such studies. 
Primary cultures of CF nasal polyp epithelial cells were infected with 
Ad2-ORF6/PGK-CFTR at multiplicities of 0.3, 3, and 50. Three days post 
infection, monlayers were mounted in Ussing chambers and short-circuit 
current was measured. At the indicated times: (1) 10 .mu.M amiloride, (2) 
cAMP agonists (10 .mu.M forskolin and 100 .mu.M IBMX), and (3) 1 mM 
diphenylamine-2-carboxylate were added to the mucosal solution. 
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 CsCl gradient and then dialyzed against 
tris-buffered saline to remove the CsCl. The preparation for the first 
administration (lot #2) had a titer of 2.times.10.sup.10 IU/ml. The 
preparation for the second administration (lot #6) had a titer of 
4.times.10.sup.10 IU/ml. 
Animals 
Three female Rhesus monkeys, Macaca 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) 
has been done 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 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. 
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.times.10.sup.10 IU/ml. and each monkey received approximately 0.3 ml. 
Thus the total dose applied to each monkey was approximately 
6.5.times.10.sup.9 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. 
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 (15 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. 
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, we 
measured rectal temperature, respiratory rate, and heart rate. 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 antiadenoviral antibody titers were 
measured by ELISA. For the ELISA, 50 ng/well of killed adenovirus (Lee 
Biomolecular Research Laboratories, San Diego, Calif.) was coated in 0.1 M 
NaHCO.sub.3 at 4.degree. 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 H.sub.2 
SO.sub.4 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 dilution in the last well with an 
OD&gt;0.100. Nasal washings from the monkeys were obtained and antiadenoviral 
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 as 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 supernatant was used for anti-adenoviral, and neutralizing 
antibody measurement. 
Cytology 
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 of PBS. The cell suspension was 
spun at 5000 rpm for 5 min. and resuspended in 293 media at a 
concentration of 10.sup.6 cells/ml. Forty .mu.l 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-CFTRB 
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 .mu.L 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.degree. C., then fixed with a 
mixture of equal parts of methanol and acetone for 10 min and incubated 
with an FITC label antiadenovirus monoclonal antibodies (Chernicon, Light 
Diagnostics, Temecuca, Calif.) for 30 min. Positive nuclear 
inmmunofluorescence was interpreted as positive culture. 
Immunocytochemistry for the Detection of CFTR 
Cells were obtained by brushing. Eighty .mu.l of cell suspension were spun 
onto gelatin-coated 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, Ill.) and then blocked for 60 minutes with 5% 
goat serum (Sigma, Mo.). A pool of monoclonal antibodies (M13-1, M1-4, and 
M6-4) (Gregory et al., 1990; Denning et al., 1992b; Denning et al., 1992a) 
were added and incubated for 12 hours. The primary antibody was washed off 
and an antimouse biotinylated antibody (Biomeda, Foster City, Calif.) was 
added. After washing, the secondary antibody, streptavidin FITC (Biomeda, 
Foster City, Calif.) was added and the slides were observed with a laser 
scanning confocal rnicroscope. 
Biopsies 
To assess for histologic evidence of safety, nasal medial turbinate 
biopsies were obtained 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 H&E 
stained sections were reviewed. 
Results 
Studies of Efficacy 
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-ORF6/PGK-CFTR. The cells were scored as positive by 
immunocytochemistry 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. 
Studies of Safety 
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, 
lymphadenopathy, conjunctivitis, ocryza, tachypnea, or tachycardia at any 
of the time points. There was no cough, sneezing or diarrhea. The monkeys 
had no fever. Appetites and weights were not affected by virus 
administration in either monkey. The data are summarized in FIGS. 30A-30C. 
The presence of live virus was tested in the supernatant of cell 
suspensions from swabs and brushes from each nostril and the pharynx. Each 
supematant was used to infect the virus-sensitive 293 cell line. Live 
virus was never detected at any of the time points. The rapid loss of live 
virus suggests that there was no viral replication. 
The results of complete blood counts, sedimentation rate, and clinical 
chemistries are shown in FIGS. 31A-31C. There was no evidence of a 
systemic inflammatory 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 
epithelium. The percentage of neutrophils and 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. Neutrophils and 
lymphocytes accounted for less than 5% of total cells at all time points. 
The data are shown in FIGS. 32A-32C. The data indicate that administration 
of Ad2-ORF6/PGK-CFTR caused no change in the distribution or number of 
inflammatory cells at any of the time points following virus 
administration, even during a second administration of the virus. The 
biopsies slides obtained after the second Ad2-RF6/PGK-CFTR administration 
were reviewed by an independent pathologist, who found no evidence of 
inflammation or any other cytopathic effects. 
FIGS. 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 antiadenoviral 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 epitheliun 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. We found no evidence of 
viral replication; 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, 
we observed no evidence of a systemic or local inflammatory response. The 
cells obtained by brushings and swabs were not altered by virus 
application. Since these Monkeys had been previously exposed three times 
to Ad2/CFTR-1, these data suggests 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 expression is safe in primates. 
Equivalents 
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 described herein. Such equivalents are 
intended to be encompassed by the following claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 10 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6129 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 133..4572 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - AATTGGAAGC AAATGACATC ACAGCAGGTC AGAGAAAAAG GGTTGAGCGG CA - 
#GGCACCCA 60 
- - GAGTAGTAGG TCTTTGGCAT TAGGAGCTTG AGCCCAGACG GCCCTAGCAG GG - 
#ACCCCAGC 120 
- - GCCCGAGAGA CC ATG CAG AGG TCG CCT CTG GAA AAG - # GCC AGC GTT GTC 
168 
Met Gln Ar - #g Ser Pro Leu Glu Lys Ala Ser Val Val 
1 - # 5 - # 10 
- - TCC AAA CTT TTT TTC AGC TGG ACC AGA CCA AT - #T TTG AGG AAA GGA TAC 
216 
Ser Lys Leu Phe Phe Ser Trp Thr Arg Pro Il - #e Leu Arg Lys Gly Tyr 
15 - # 20 - # 25 
- - AGA CAG CGC CTG GAA TTG TCA GAC ATA TAC CA - #A ATC CCT TCT GTT GAT 
264 
Arg Gln Arg Leu Glu Leu Ser Asp Ile Tyr Gl - #n Ile Pro Ser Val Asp 
30 - # 35 - # 40 
- - TCT GCT GAC AAT CTA TCT GAA AAA TTG GAA AG - #A GAA TGG GAT AGA GAG 
312 
Ser Ala Asp Asn Leu Ser Glu Lys Leu Glu Ar - #g Glu Trp Asp Arg Glu 
45 - # 50 - # 55 - # 60 
- - CTG GCT TCA AAG AAA AAT CCT AAA CTC ATT AA - #T GCC CTT CGG CGA TGT 
360 
Leu Ala Ser Lys Lys Asn Pro Lys Leu Ile As - #n Ala Leu Arg Arg Cys 
65 - # 70 - # 75 
- - TTT TTC TGG AGA TTT ATG TTC TAT GGA ATC TT - #T TTA TAT TTA GGG GAA 
408 
Phe Phe Trp Arg Phe Met Phe Tyr Gly Ile Ph - #e Leu Tyr Leu Gly Glu 
80 - # 85 - # 90 
- - GTC ACC AAA GCA GTA CAG CCT CTC TTA CTG GG - #A AGA ATC ATA GCT TCC 
456 
Val Thr Lys Ala Val Gln Pro Leu Leu Leu Gl - #y Arg Ile Ile Ala Ser 
95 - # 100 - # 105 
- - TAT GAC CCG GAT AAC AAG GAG GAA CGC TCT AT - #C GCG ATT TAT CTA GGC 
504 
Tyr Asp Pro Asp Asn Lys Glu Glu Arg Ser Il - #e Ala Ile Tyr Leu Gly 
110 - # 115 - # 120 
- - ATA GGC TTA TGC CTT CTC TTT ATT GTG AGG AC - #A CTG CTC CTA CAC CCA 
552 
Ile Gly Leu Cys Leu Leu Phe Ile Val Arg Th - #r Leu Leu Leu His Pro 
125 1 - #30 1 - #35 1 - 
#40 
- - GCC ATT TTT GGC CTT CAT CAC ATT GGA ATG CA - #G ATG AGA ATA GCT 
ATG 600 
Ala Ile Phe Gly Leu His His Ile Gly Met Gl - #n Met Arg Ile Ala Met 
145 - # 150 - # 155 
- - TTT AGT TTG ATT TAT AAG AAG ACT TTA AAG CT - #G TCA AGC CGT GTT CTA 
648 
Phe Ser Leu Ile Tyr Lys Lys Thr Leu Lys Le - #u Ser Ser Arg Val Leu 
160 - # 165 - # 170 
- - GAT AAA ATA AGT ATT GGA CAA CTT GTT AGT CT - #C CTT TCC AAC AAC CTG 
696 
Asp Lys Ile Ser Ile Gly Gln Leu Val Ser Le - #u Leu Ser Asn Asn Leu 
175 - # 180 - # 185 
- - AAC AAA TTT GAT GAA GGA CTT GCA TTG GCA CA - #T TTC GTG TGG ATC GCT 
744 
Asn Lys Phe Asp Glu Gly Leu Ala Leu Ala Hi - #s Phe Val Trp Ile Ala 
190 - # 195 - # 200 
- - CCT TTG CAA GTG GCA CTC CTC ATG GGG CTA AT - #C TGG GAG TTG TTA CAG 
792 
Pro Leu Gln Val Ala Leu Leu Met Gly Leu Il - #e Trp Glu Leu Leu Gln 
205 2 - #10 2 - #15 2 - 
#20 
- - GCG TCT GCC TTC TGT GGA CTT GGT TTC CTG AT - #A GTC CTT GCC CTT 
TTT 840 
Ala Ser Ala Phe Cys Gly Leu Gly Phe Leu Il - #e Val Leu Ala Leu Phe 
225 - # 230 - # 235 
- - CAG GCT GGG CTA GGG AGA ATG ATG ATG AAG TA - #C AGA GAT CAG AGA GCT 
888 
Gln Ala Gly Leu Gly Arg Met Met Met Lys Ty - #r Arg Asp Gln Arg Ala 
240 - # 245 - # 250 
- - GGG AAG ATC AGT GAA AGA CTT GTG ATT ACC TC - #A GAA ATG ATT GAA AAT 
936 
Gly Lys Ile Ser Glu Arg Leu Val Ile Thr Se - #r Glu Met Ile Glu Asn 
255 - # 260 - # 265 
- - ATC CAA TCT GTT AAG GCA TAC TGC TGG GAA GA - #A GCA ATG GAA AAA ATG 
984 
Ile Gln Ser Val Lys Ala Tyr Cys Trp Glu Gl - #u Ala Met Glu Lys Met 
270 - # 275 - # 280 
- - ATT GAA AAC TTA AGA CAA ACA GAA CTG AAA CT - #G ACT CGG AAG GCA GCC 
1032 
Ile Glu Asn Leu Arg Gln Thr Glu Leu Lys Le - #u Thr Arg Lys Ala Ala 
285 2 - #90 2 - #95 3 - 
#00 
- - TAT GTG AGA TAC TTC AAT AGC TCA GCC TTC TT - #C TTC TCA GGG TTC 
TTT 1080 
Tyr Val Arg Tyr Phe Asn Ser Ser Ala Phe Ph - #e Phe Ser Gly Phe Phe 
305 - # 310 - # 315 
- - GTG GTG TTT TTA TCT GTG CTT CCC TAT GCA CT - #A ATC AAA GGA ATC ATC 
1128 
Val Val Phe Leu Ser Val Leu Pro Tyr Ala Le - #u Ile Lys Gly Ile Ile 
320 - # 325 - # 330 
- - CTC CGG AAA ATA TTC ACC ACC ATC TCA TTC TG - #C ATT GTT CTG CGC ATG 
1176 
Leu Arg Lys Ile Phe Thr Thr Ile Ser Phe Cy - #s Ile Val Leu Arg Met 
335 - # 340 - # 345 
- - GCG GTC ACT CGG CAA TTT CCC TGG GCT GTA CA - #A ACA TGG TAT GAC TCT 
1224 
Ala Val Thr Arg Gln Phe Pro Trp Ala Val Gl - #n Thr Trp Tyr Asp Ser 
350 - # 355 - # 360 
- - CTT GGA GCA ATA AAC AAA ATA CAG GAT TTC TT - #A CAA AAG CAA GAA TAT 
1272 
Leu Gly Ala Ile Asn Lys Ile Gln Asp Phe Le - #u Gln Lys Gln Glu Tyr 
365 3 - #70 3 - #75 3 - 
#80 
- - AAG ACA TTG GAA TAT AAC TTA ACG ACT ACA GA - #A GTA GTG ATG GAG 
AAT 1320 
Lys Thr Leu Glu Tyr Asn Leu Thr Thr Thr Gl - #u Val Val Met Glu Asn 
385 - # 390 - # 395 
- - GTA ACA GCC TTC TGG GAG GAG GGA TTT GGG GA - #A TTA TTT GAG AAA GCA 
1368 
Val Thr Ala Phe Trp Glu Glu Gly Phe Gly Gl - #u Leu Phe Glu Lys Ala 
400 - # 405 - # 410 
- - AAA CAA AAC AAT AAC AAT AGA AAA ACT TCT AA - #T GGT GAT GAC AGC CTC 
1416 
Lys Gln Asn Asn Asn Asn Arg Lys Thr Ser As - #n Gly Asp Asp Ser Leu 
415 - # 420 - # 425 
- - TTC TTC AGT AAT TTC TCA CTT CTT GGT ACT CC - #T GTC CTG AAA GAT ATT 
1464 
Phe Phe Ser Asn Phe Ser Leu Leu Gly Thr Pr - #o Val Leu Lys Asp Ile 
430 - # 435 - # 440 
- - AAT TTC AAG ATA GAA AGA GGA CAG TTG TTG GC - #G GTT GCT GGA TCC ACT 
1512 
Asn Phe Lys Ile Glu Arg Gly Gln Leu Leu Al - #a Val Ala Gly Ser Thr 
445 4 - #50 4 - #55 4 - 
#60 
- - GGA GCA GGC AAG ACT TCA CTT CTA ATG ATG AT - #T ATG GGA GAA CTG 
GAG 1560 
Gly Ala Gly Lys Thr Ser Leu Leu Met Met Il - #e Met Gly Glu Leu Glu 
465 - # 470 - # 475 
- - CCT TCA GAG GGT AAA ATT AAG CAC AGT GGA AG - #A ATT TCA TTC TGT TCT 
1608 
Pro Ser Glu Gly Lys Ile Lys His Ser Gly Ar - #g Ile Ser Phe Cys Ser 
480 - # 485 - # 490 
- - CAG TTT TCC TGG ATT ATG CCT GGC ACC ATT AA - #A GAA AAT ATC ATC TTT 
1656 
Gln Phe Ser Trp Ile Met Pro Gly Thr Ile Ly - #s Glu Asn Ile Ile Phe 
495 - # 500 - # 505 
- - GGT GTT TCC TAT GAT GAA TAT AGA TAC AGA AG - #C GTC ATC AAA GCA TGC 
1704 
Gly Val Ser Tyr Asp Glu Tyr Arg Tyr Arg Se - #r Val Ile Lys Ala Cys 
510 - # 515 - # 520 
- - CAA CTA GAA GAG GAC ATC TCC AAG TTT GCA GA - #G AAA GAC AAT ATA GTT 
1752 
Gln Leu Glu Glu Asp Ile Ser Lys Phe Ala Gl - #u Lys Asp Asn Ile Val 
525 5 - #30 5 - #35 5 - 
#40 
- - CTT GGA GAA GGT GGA ATC ACA CTG AGT GGA GG - #T CAA CGA GCA AGA 
ATT 1800 
Leu Gly Glu Gly Gly Ile Thr Leu Ser Gly Gl - #y Gln Arg Ala Arg Ile 
545 - # 550 - # 555 
- - TCT TTA GCA AGA GCA GTA TAC AAA GAT GCT GA - #T TTG TAT TTA TTA GAC 
1848 
Ser Leu Ala Arg Ala Val Tyr Lys Asp Ala As - #p Leu Tyr Leu Leu Asp 
560 - # 565 - # 570 
- - TCT CCT TTT GGA TAC CTA GAT GTT TTA ACA GA - #A AAA GAA ATA TTT GAA 
1896 
Ser Pro Phe Gly Tyr Leu Asp Val Leu Thr Gl - #u Lys Glu Ile Phe Glu 
575 - # 580 - # 585 
- - AGC TGT GTC TGT AAA CTG ATG GCT AAC AAA AC - #T AGG ATT TTG GTC ACT 
1944 
Ser Cys Val Cys Lys Leu Met Ala Asn Lys Th - #r Arg Ile Leu Val Thr 
590 - # 595 - # 600 
- - TCT AAA ATG GAA CAT TTA AAG AAA GCT GAC AA - #A ATA TTA ATT TTG CAT 
1992 
Ser Lys Met Glu His Leu Lys Lys Ala Asp Ly - #s Ile Leu Ile Leu His 
605 6 - #10 6 - #15 6 - 
#20 
- - GAA GGT AGC AGC TAT TTT TAT GGG ACA TTT TC - #A GAA CTC CAA AAT 
CTA 2040 
Glu Gly Ser Ser Tyr Phe Tyr Gly Thr Phe Se - #r Glu Leu Gln Asn Leu 
625 - # 630 - # 635 
- - CAG CCA GAC TTT AGC TCA AAA CTC ATG GGA TG - #T GAT TCT TTC GAC CAA 
2088 
Gln Pro Asp Phe Ser Ser Lys Leu Met Gly Cy - #s Asp Ser Phe Asp Gln 
640 - # 645 - # 650 
- - TTT AGT GCA GAA AGA AGA AAT TCA ATC CTA AC - #T GAG ACC TTA CAC CGT 
2136 
Phe Ser Ala Glu Arg Arg Asn Ser Ile Leu Th - #r Glu Thr Leu His Arg 
655 - # 660 - # 665 
- - TTC TCA TTA GAA GGA GAT GCT CCT GTC TCC TG - #G ACA GAA ACA AAA AAA 
2184 
Phe Ser Leu Glu Gly Asp Ala Pro Val Ser Tr - #p Thr Glu Thr Lys Lys 
670 - # 675 - # 680 
- - CAA TCT TTT AAA CAG ACT GGA GAG TTT GGG GA - #A AAA AGG AAG AAT TCT 
2232 
Gln Ser Phe Lys Gln Thr Gly Glu Phe Gly Gl - #u Lys Arg Lys Asn Ser 
685 6 - #90 6 - #95 7 - 
#00 
- - ATT CTC AAT CCA ATC AAC TCT ATA CGA AAA TT - #T TCC ATT GTG CAA 
AAG 2280 
Ile Leu Asn Pro Ile Asn Ser Ile Arg Lys Ph - #e Ser Ile Val Gln Lys 
705 - # 710 - # 715 
- - ACT CCC TTA CAA ATG AAT GGC ATC GAA GAG GA - #T TCT GAT GAG CCT TTA 
2328 
Thr Pro Leu Gln Met Asn Gly Ile Glu Glu As - #p Ser Asp Glu Pro Leu 
720 - # 725 - # 730 
- - GAG AGA AGG CTG TCC TTA GTA CCA GAT TCT GA - #G CAG GGA GAG GCG ATA 
2376 
Glu Arg Arg Leu Ser Leu Val Pro Asp Ser Gl - #u Gln Gly Glu Ala Ile 
735 - # 740 - # 745 
- - CTG CCT CGC ATC AGC GTG ATC AGC ACT GGC CC - #C ACG CTT CAG GCA CGA 
2424 
Leu Pro Arg Ile Ser Val Ile Ser Thr Gly Pr - #o Thr Leu Gln Ala Arg 
750 - # 755 - # 760 
- - AGG AGG CAG TCT GTC CTG AAC CTG ATG ACA CA - #C TCA GTT AAC CAA GGT 
2472 
Arg Arg Gln Ser Val Leu Asn Leu Met Thr Hi - #s Ser Val Asn Gln Gly 
765 7 - #70 7 - #75 7 - 
#80 
- - CAG AAC ATT CAC CGA AAG ACA ACA GCA TCC AC - #A CGA AAA GTG TCA 
CTG 2520 
Gln Asn Ile His Arg Lys Thr Thr Ala Ser Th - #r Arg Lys Val Ser Leu 
785 - # 790 - # 795 
- - GCC CCT CAG GCA AAC TTG ACT GAA CTG GAT AT - #A TAT TCA AGA AGG TTA 
2568 
Ala Pro Gln Ala Asn Leu Thr Glu Leu Asp Il - #e Tyr Ser Arg Arg Leu 
800 - # 805 - # 810 
- - TCT CAA GAA ACT GGC TTG GAA ATA AGT GAA GA - #A ATT AAC GAA GAA GAC 
2616 
Ser Gln Glu Thr Gly Leu Glu Ile Ser Glu Gl - #u Ile Asn Glu Glu Asp 
815 - # 820 - # 825 
- - TTA AAG GAG TGC CTT TTT GAT GAT ATG GAG AG - #C ATA CCA GCA GTG ACT 
2664 
Leu Lys Glu Cys Leu Phe Asp Asp Met Glu Se - #r Ile Pro Ala Val Thr 
830 - # 835 - # 840 
- - ACA TGG AAC ACA TAC CTT CGA TAT ATT ACT GT - #C CAC AAG AGC TTA ATT 
2712 
Thr Trp Asn Thr Tyr Leu Arg Tyr Ile Thr Va - #l His Lys Ser Leu Ile 
845 8 - #50 8 - #55 8 - 
#60 
- - TTT GTG CTA ATT TGG TGC TTA GTA ATT TTT CT - #G GCA GAG GTG GCT 
GCT 2760 
Phe Val Leu Ile Trp Cys Leu Val Ile Phe Le - #u Ala Glu Val Ala Ala 
865 - # 870 - # 875 
- - TCT TTG GTT GTG CTG TGG CTC CTT GGA AAC AC - #T CCT CTT CAA GAC AAA 
2808 
Ser Leu Val Val Leu Trp Leu Leu Gly Asn Th - #r Pro Leu Gln Asp Lys 
880 - # 885 - # 890 
- - GGG AAT AGT ACT CAT AGT AGA AAT AAC AGC TA - #T GCA GTG ATT ATC ACC 
2856 
Gly Asn Ser Thr His Ser Arg Asn Asn Ser Ty - #r Ala Val Ile Ile Thr 
895 - # 900 - # 905 
- - AGC ACC AGT TCG TAT TAT GTG TTT TAC ATT TA - #C GTG GGA GTA GCC GAC 
2904 
Ser Thr Ser Ser Tyr Tyr Val Phe Tyr Ile Ty - #r Val Gly Val Ala Asp 
910 - # 915 - # 920 
- - ACT TTG CTT GCT ATG GGA TTC TTC AGA GGT CT - #A CCA CTG GTG CAT ACT 
2952 
Thr Leu Leu Ala Met Gly Phe Phe Arg Gly Le - #u Pro Leu Val His Thr 
925 9 - #30 9 - #35 9 - 
#40 
- - CTA ATC ACA GTG TCG AAA ATT TTA CAC CAC AA - #A ATG TTA CAT TCT 
GTT 3000 
Leu Ile Thr Val Ser Lys Ile Leu His His Ly - #s Met Leu His Ser Val 
945 - # 950 - # 955 
- - CTT CAA GCA CCT ATG TCA ACC CTC AAC ACG TT - #G AAA GCA GGT GGG ATT 
3048 
Leu Gln Ala Pro Met Ser Thr Leu Asn Thr Le - #u Lys Ala Gly Gly Ile 
960 - # 965 - # 970 
- - CTT AAT AGA TTC TCC AAA GAT ATA GCA ATT TT - #G GAT GAC CTT CTG CCT 
3096 
Leu Asn Arg Phe Ser Lys Asp Ile Ala Ile Le - #u Asp Asp Leu Leu Pro 
975 - # 980 - # 985 
- - CTT ACC ATA TTT GAC TTC ATC CAG TTG TTA TT - #A ATT GTG ATT GGA GCT 
3144 
Leu Thr Ile Phe Asp Phe Ile Gln Leu Leu Le - #u Ile Val Ile Gly Ala 
990 - # 995 - # 1000 
- - ATA GCA GTT GTC GCA GTT TTA CAA CCC TAC AT - #C TTT GTT GCA ACA GTG 
3192 
Ile Ala Val Val Ala Val Leu Gln Pro Tyr Il - #e Phe Val Ala Thr Val 
1005 1010 - # 1015 - # 1020 
- - CCA GTG ATA GTG GCT TTT ATT ATG TTG AGA GC - #A TAT TTC CTC CAA ACC 
3240 
Pro Val Ile Val Ala Phe Ile Met Leu Arg Al - #a Tyr Phe Leu Gln Thr 
1025 - # 1030 - # 1035 
- - TCA CAG CAA CTC AAA CAA CTG GAA TCT GAA GG - #C AGG AGT CCA ATT TTC 
3288 
Ser Gln Gln Leu Lys Gln Leu Glu Ser Glu Gl - #y Arg Ser Pro Ile Phe 
1040 - # 1045 - # 1050 
- - ACT CAT CTT GTT ACA AGC TTA AAA GGA CTA TG - #G ACA CTT CGT GCC TTC 
3336 
Thr His Leu Val Thr Ser Leu Lys Gly Leu Tr - #p Thr Leu Arg Ala Phe 
1055 - # 1060 - # 1065 
- - GGA CGG CAG CCT TAC TTT GAA ACT CTG TTC CA - #C AAA GCT CTG AAT TTA 
3384 
Gly Arg Gln Pro Tyr Phe Glu Thr Leu Phe Hi - #s Lys Ala Leu Asn Leu 
1070 - # 1075 - # 1080 
- - CAT ACT GCC AAC TGG TTC TTG TAC CTG TCA AC - #A CTG CGC TGG TTC CAA 
3432 
His Thr Ala Asn Trp Phe Leu Tyr Leu Ser Th - #r Leu Arg Trp Phe Gln 
1085 1090 - # 1095 - # 1100 
- - ATG AGA ATA GAA ATG ATT TTT GTC ATC TTC TT - #C ATT GCT GTT ACC TTC 
3480 
Met Arg Ile Glu Met Ile Phe Val Ile Phe Ph - #e Ile Ala Val Thr Phe 
1105 - # 1110 - # 1115 
- - ATT TCC ATT TTA ACA ACA GGA GAA GGA GAA GG - #A AGA GTT GGT ATT ATC 
3528 
Ile Ser Ile Leu Thr Thr Gly Glu Gly Glu Gl - #y Arg Val Gly Ile Ile 
1120 - # 1125 - # 1130 
- - CTG ACT TTA GCC ATG AAT ATC ATG AGT ACA TT - #G CAG TGG GCT GTA AAC 
3576 
Leu Thr Leu Ala Met Asn Ile Met Ser Thr Le - #u Gln Trp Ala Val Asn 
1135 - # 1140 - # 1145 
- - TCC AGC ATA GAT GTG GAT AGC TTG ATG CGA TC - #T GTG AGC CGA GTC TTT 
3624 
Ser Ser Ile Asp Val Asp Ser Leu Met Arg Se - #r Val Ser Arg Val Phe 
1150 - # 1155 - # 1160 
- - AAG TTC ATT GAC ATG CCA ACA GAA GGT AAA CC - #T ACC AAG TCA ACC AAA 
3672 
Lys Phe Ile Asp Met Pro Thr Glu Gly Lys Pr - #o Thr Lys Ser Thr Lys 
1165 1170 - # 1175 - # 1180 
- - CCA TAC AAG AAT GGC CAA CTC TCG AAA GTT AT - #G ATT ATT GAG AAT TCA 
3720 
Pro Tyr Lys Asn Gly Gln Leu Ser Lys Val Me - #t Ile Ile Glu Asn Ser 
1185 - # 1190 - # 1195 
- - CAC GTG AAG AAA GAT GAC ATC TGG CCC TCA GG - #G GGC CAA ATG ACT GTC 
3768 
His Val Lys Lys Asp Asp Ile Trp Pro Ser Gl - #y Gly Gln Met Thr Val 
1200 - # 1205 - # 1210 
- - AAA GAT CTC ACA GCA AAA TAC ACA GAA GGT GG - #A AAT GCC ATA TTA GAG 
3816 
Lys Asp Leu Thr Ala Lys Tyr Thr Glu Gly Gl - #y Asn Ala Ile Leu Glu 
1215 - # 1220 - # 1225 
- - AAC ATT TCC TTC TCA ATA AGT CCT GGC CAG AG - #G GTG GGC CTC TTG GGA 
3864 
Asn Ile Ser Phe Ser Ile Ser Pro Gly Gln Ar - #g Val Gly Leu Leu Gly 
1230 - # 1235 - # 1240 
- - AGA ACT GGA TCA GGG AAG AGT ACT TTG TTA TC - #A GCT TTT TTG AGA CTA 
3912 
Arg Thr Gly Ser Gly Lys Ser Thr Leu Leu Se - #r Ala Phe Leu Arg Leu 
1245 1250 - # 1255 - # 1260 
- - CTG AAC ACT GAA GGA GAA ATC CAG ATC GAT GG - #T GTG TCT TGG GAT TCA 
3960 
Leu Asn Thr Glu Gly Glu Ile Gln Ile Asp Gl - #y Val Ser Trp Asp Ser 
1265 - # 1270 - # 1275 
- - ATA ACT TTG CAA CAG TGG AGG AAA GCC TTT GG - #A GTG ATA CCA CAG AAA 
4008 
Ile Thr Leu Gln Gln Trp Arg Lys Ala Phe Gl - #y Val Ile Pro Gln Lys 
1280 - # 1285 - # 1290 
- - GTA TTT ATT TTT TCT GGA ACA TTT AGA AAA AA - #C TTG GAT CCC TAT GAA 
4056 
Val Phe Ile Phe Ser Gly Thr Phe Arg Lys As - #n Leu Asp Pro Tyr Glu 
1295 - # 1300 - # 1305 
- - CAG TGG AGT GAT CAA GAA ATA TGG AAA GTT GC - #A GAT GAG GTT GGG CTC 
4104 
Gln Trp Ser Asp Gln Glu Ile Trp Lys Val Al - #a Asp Glu Val Gly Leu 
1310 - # 1315 - # 1320 
- - AGA TCT GTG ATA GAA CAG TTT CCT GGG AAG CT - #T GAC TTT GTC CTT GTG 
4152 
Arg Ser Val Ile Glu Gln Phe Pro Gly Lys Le - #u Asp Phe Val Leu Val 
1325 1330 - # 1335 - # 1340 
- - GAT GGG GGC TGT GTC CTA AGC CAT GGC CAC AA - #G CAG TTG ATG TGC TTG 
4200 
Asp Gly Gly Cys Val Leu Ser His Gly His Ly - #s Gln Leu Met Cys Leu 
1345 - # 1350 - # 1355 
- - GCT AGA TCT GTT CTC AGT AAG GCG AAG ATC TT - #G CTG CTT GAT GAA CCC 
4248 
Ala Arg Ser Val Leu Ser Lys Ala Lys Ile Le - #u Leu Leu Asp Glu Pro 
1360 - # 1365 - # 1370 
- - AGT GCT CAT TTG GAT CCA GTA ACA TAC CAA AT - #A ATT AGA AGA ACT CTA 
4296 
Ser Ala His Leu Asp Pro Val Thr Tyr Gln Il - #e Ile Arg Arg Thr Leu 
1375 - # 1380 - # 1385 
- - AAA CAA GCA TTT GCT GAT TGC ACA GTA ATT CT - #C TGT GAA CAC AGG ATA 
4344 
Lys Gln Ala Phe Ala Asp Cys Thr Val Ile Le - #u Cys Glu His Arg Ile 
1390 - # 1395 - # 1400 
- - GAA GCA ATG CTG GAA TGC CAA CAA TTT TTG GT - #C ATA GAA GAG AAC AAA 
4392 
Glu Ala Met Leu Glu Cys Gln Gln Phe Leu Va - #l Ile Glu Glu Asn Lys 
1405 1410 - # 1415 - # 1420 
- - GTG CGG CAG TAC GAT TCC ATC CAG AAA CTG CT - #G AAC GAG AGG AGC CTC 
4440 
Val Arg Gln Tyr Asp Ser Ile Gln Lys Leu Le - #u Asn Glu Arg Ser Leu 
1425 - # 1430 - # 1435 
- - TTC CGG CAA GCC ATC AGC CCC TCC GAC AGG GT - #G AAG CTC TTT CCC CAC 
4488 
Phe Arg Gln Ala Ile Ser Pro Ser Asp Arg Va - #l Lys Leu Phe Pro His 
1440 - # 1445 - # 1450 
- - CGG AAC TCA AGC AAG TGC AAG TCT AAG CCC CA - #G ATT GCT GCT CTG AAA 
4536 
Arg Asn Ser Ser Lys Cys Lys Ser Lys Pro Gl - #n Ile Ala Ala Leu Lys 
1455 - # 1460 - # 1465 
- - GAG GAG ACA GAA GAA GAG GTG CAA GAT ACA AG - #G CTT TAGAGAGCAG 
4582 
Glu Glu Thr Glu Glu Glu Val Gln Asp Thr Ar - #g Leu 
1470 - # 1475 - # 1480 
- - CATAAATGTT GACATGGGAC ATTTGCTCAT GGAATTGGAG CTCGTGGGAC AG - 
#TCACCTCA 4642 
- - TGGAATTGGA GCTCGTGGAA CAGTTACCTC TGCCTCAGAA AACAAGGATG AA - 
#TTAAGTTT 4702 
- - TTTTTTAAAA AAGAAACATT TGGTAAGGGG AATTGAGGAC ACTGATATGG GT - 
#CTTGATAA 4762 
- - ATGGCTTCCT GGCAATAGTC AAATTGTGTG AAAGGTACTT CAAATCCTTG AA - 
#GATTTACC 4822 
- - ACTTGTGTTT TGCAAGCCAG ATTTTCCTGA AAACCCTTGC CATGTGCTAG TA - 
#ATTGGAAA 4882 
- - GGCAGCTCTA AATGTCAATC AGCCTAGTTG ATCAGCTTAT TGTCTAGTGA AA - 
#CTCGTTAA 4942 
- - TTTGTAGTGT TGGAGAAGAA CTGAAATCAT ACTTCTTAGG GTTATGATTA AG - 
#TAATGATA 5002 
- - ACTGGAAACT TCAGCGGTTT ATATAAGCTT GTATTCCTTT TTCTCTCCTC TC - 
#CCCATGAT 5062 
- - GTTTAGAAAC ACAACTATAT TGTTTGCTAA GCATTCCAAC TATCTCATTT CC - 
#AAGCAAGT 5122 
- - ATTAGAATAC CACAGGAACC ACAAGACTGC ACATCAAAAT ATGCCCCATT CA - 
#ACATCTAG 5182 
- - TGAGCAGTCA GGAAAGAGAA CTTCCAGATC CTGGAAATCA GGGTTAGTAT TG - 
#TCCAGGTC 5242 
- - TACCAAAAAT CTCAATATTT CAGATAATCA CAATACATCC CTTACCTGGG AA - 
#AGGGCTGT 5302 
- - TATAATCTTT CACAGGGGAC AGGATGGTTC CCTTGATGAA GAAGTTGATA TG - 
#CCTTTTCC 5362 
- - CAACTCCAGA AAGTGACAAG CTCACAGACC TTTGAACTAG AGTTTAGCTG GA - 
#AAAGTATG 5422 
- - TTAGTGCAAA TTGTCACAGG ACAGCCCTTC TTTCCACAGA AGCTCCAGGT AG - 
#AGGGTGTG 5482 
- - TAAGTAGATA GGCCATGGGC ACTGTGGGTA GACACACATG AAGTCCAAGC AT - 
#TTAGATGT 5542 
- - ATAGGTTGAT GGTGGTATGT TTTCAGGCTA GATGTATGTA CTTCATGCTG TC - 
#TACACTAA 5602 
- - GAGAGAATGA GAGACACACT GAAGAAGCAC CAATCATGAA TTAGTTTTAT AT - 
#GCTTCTGT 5662 
- - TTTATAATTT TGTGAAGCAA AATTTTTTCT CTAGGAAATA TTTATTTTAA TA - 
#ATGTTTCA 5722 
- - AACATATATT ACAATGCTGT ATTTTAAAAG AATGATTATG AATTACATTT GT - 
#ATAAAATA 5782 
- - ATTTTTATAT TTGAAATATT GACTTTTTAT GGCACTAGTA TTTTTATGAA AT - 
#ATTATGTT 5842 
- - AAAACTGGGA CAGGGGAGAA CCTAGGGTGA TATTAACCAG GGGCCATGAA TC - 
#ACCTTTTG 5902 
- - GTCTGGAGGG AAGCCTTGGG GCTGATCGAG TTGTTGCCCA CAGCTGTATG AT - 
#TCCCAGCC 5962 
- - AGACACAGCC TCTTAGATGC AGTTCTGAAG AAGATGGTAC CACCAGTCTG AC - 
#TGTTTCCA 6022 
- - TCAAGGGTAC ACTGCCTTCT CAACTCCAAA CTGACTCTTA AGAAGACTGC AT - 
#TATATTTA 6082 
- - TTACTGTAAG AAAATATCAC TTGTCAATAA AATCCATACA TTTGTGT - # 
6129 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1480 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Gln Arg Ser Pro Leu Glu Lys Ala Ser Va - #l Val Ser Lys Leu Phe 
1 5 - # 10 - # 15 
- - Phe Ser Trp Thr Arg Pro Ile Leu Arg Lys Gl - #y Tyr Arg Gln Arg Leu 
20 - # 25 - # 30 
- - Glu Leu Ser Asp Ile Tyr Gln Ile Pro Ser Va - #l Asp Ser Ala Asp Asn 
35 - # 40 - # 45 
- - Leu Ser Glu Lys Leu Glu Arg Glu Trp Asp Ar - #g Glu Leu Ala Ser Lys 
50 - # 55 - # 60 
- - Lys Asn Pro Lys Leu Ile Asn Ala Leu Arg Ar - #g Cys Phe Phe Trp Arg 
65 - # 70 - # 75 - # 80 
- - Phe Met Phe Tyr Gly Ile Phe Leu Tyr Leu Gl - #y Glu Val Thr Lys Ala 
85 - # 90 - # 95 
- - Val Gln Pro Leu Leu Leu Gly Arg Ile Ile Al - #a Ser Tyr Asp Pro Asp 
100 - # 105 - # 110 
- - Asn Lys Glu Glu Arg Ser Ile Ala Ile Tyr Le - #u Gly Ile Gly Leu Cys 
115 - # 120 - # 125 
- - Leu Leu Phe Ile Val Arg Thr Leu Leu Leu Hi - #s Pro Ala Ile Phe Gly 
130 - # 135 - # 140 
- - Leu His His Ile Gly Met Gln Met Arg Ile Al - #a Met Phe Ser Leu Ile 
145 1 - #50 1 - #55 1 - 
#60 
- - Tyr Lys Lys Thr Leu Lys Leu Ser Ser Arg Va - #l Leu Asp Lys Ile 
Ser 
165 - # 170 - # 175 
- - Ile Gly Gln Leu Val Ser Leu Leu Ser Asn As - #n Leu Asn Lys Phe Asp 
180 - # 185 - # 190 
- - Glu Gly Leu Ala Leu Ala His Phe Val Trp Il - #e Ala Pro Leu Gln Val 
195 - # 200 - # 205 
- - Ala Leu Leu Met Gly Leu Ile Trp Glu Leu Le - #u Gln Ala Ser Ala Phe 
210 - # 215 - # 220 
- - Cys Gly Leu Gly Phe Leu Ile Val Leu Ala Le - #u Phe Gln Ala Gly Leu 
225 2 - #30 2 - #35 2 - 
#40 
- - Gly Arg Met Met Met Lys Tyr Arg Asp Gln Ar - #g Ala Gly Lys Ile 
Ser 
245 - # 250 - # 255 
- - Glu Arg Leu Val Ile Thr Ser Glu Met Ile Gl - #u Asn Ile Gln Ser Val 
260 - # 265 - # 270 
- - Lys Ala Tyr Cys Trp Glu Glu Ala Met Glu Ly - #s Met Ile Glu Asn Leu 
275 - # 280 - # 285 
- - Arg Gln Thr Glu Leu Lys Leu Thr Arg Lys Al - #a Ala Tyr Val Arg Tyr 
290 - # 295 - # 300 
- - Phe Asn Ser Ser Ala Phe Phe Phe Ser Gly Ph - #e Phe Val Val Phe Leu 
305 3 - #10 3 - #15 3 - 
#20 
- - Ser Val Leu Pro Tyr Ala Leu Ile Lys Gly Il - #e Ile Leu Arg Lys 
Ile 
325 - # 330 - # 335 
- - Phe Thr Thr Ile Ser Phe Cys Ile Val Leu Ar - #g Met Ala Val Thr Arg 
340 - # 345 - # 350 
- - Gln Phe Pro Trp Ala Val Gln Thr Trp Tyr As - #p Ser Leu Gly Ala Ile 
355 - # 360 - # 365 
- - Asn Lys Ile Gln Asp Phe Leu Gln Lys Gln Gl - #u Tyr Lys Thr Leu Glu 
370 - # 375 - # 380 
- - Tyr Asn Leu Thr Thr Thr Glu Val Val Met Gl - #u Asn Val Thr Ala Phe 
385 3 - #90 3 - #95 4 - 
#00 
- - Trp Glu Glu Gly Phe Gly Glu Leu Phe Glu Ly - #s Ala Lys Gln Asn 
Asn 
405 - # 410 - # 415 
- - Asn Asn Arg Lys Thr Ser Asn Gly Asp Asp Se - #r Leu Phe Phe Ser Asn 
420 - # 425 - # 430 
- - Phe Ser Leu Leu Gly Thr Pro Val Leu Lys As - #p Ile Asn Phe Lys Ile 
435 - # 440 - # 445 
- - Glu Arg Gly Gln Leu Leu Ala Val Ala Gly Se - #r Thr Gly Ala Gly Lys 
450 - # 455 - # 460 
- - Thr Ser Leu Leu Met Met Ile Met Gly Glu Le - #u Glu Pro Ser Glu Gly 
465 4 - #70 4 - #75 4 - 
#80 
- - Lys Ile Lys His Ser Gly Arg Ile Ser Phe Cy - #s Ser Gln Phe Ser 
Trp 
485 - # 490 - # 495 
- - Ile Met Pro Gly Thr Ile Lys Glu Asn Ile Il - #e Phe Gly Val Ser Tyr 
500 - # 505 - # 510 
- - Asp Glu Tyr Arg Tyr Arg Ser Val Ile Lys Al - #a Cys Gln Leu Glu Glu 
515 - # 520 - # 525 
- - Asp Ile Ser Lys Phe Ala Glu Lys Asp Asn Il - #e Val Leu Gly Glu Gly 
530 - # 535 - # 540 
- - Gly Ile Thr Leu Ser Gly Gly Gln Arg Ala Ar - #g Ile Ser Leu Ala Arg 
545 5 - #50 5 - #55 5 - 
#60 
- - Ala Val Tyr Lys Asp Ala Asp Leu Tyr Leu Le - #u Asp Ser Pro Phe 
Gly 
565 - # 570 - # 575 
- - Tyr Leu Asp Val Leu Thr Glu Lys Glu Ile Ph - #e Glu Ser Cys Val Cys 
580 - # 585 - # 590 
- - Lys Leu Met Ala Asn Lys Thr Arg Ile Leu Va - #l Thr Ser Lys Met Glu 
595 - # 600 - # 605 
- - His Leu Lys Lys Ala Asp Lys Ile Leu Ile Le - #u His Glu Gly Ser Ser 
610 - # 615 - # 620 
- - Tyr Phe Tyr Gly Thr Phe Ser Glu Leu Gln As - #n Leu Gln Pro Asp Phe 
625 6 - #30 6 - #35 6 - 
#40 
- - Ser Ser Lys Leu Met Gly Cys Asp Ser Phe As - #p Gln Phe Ser Ala 
Glu 
645 - # 650 - # 655 
- - Arg Arg Asn Ser Ile Leu Thr Glu Thr Leu Hi - #s Arg Phe Ser Leu Glu 
660 - # 665 - # 670 
- - Gly Asp Ala Pro Val Ser Trp Thr Glu Thr Ly - #s Lys Gln Ser Phe Lys 
675 - # 680 - # 685 
- - Gln Thr Gly Glu Phe Gly Glu Lys Arg Lys As - #n Ser Ile Leu Asn Pro 
690 - # 695 - # 700 
- - Ile Asn Ser Ile Arg Lys Phe Ser Ile Val Gl - #n Lys Thr Pro Leu Gln 
705 7 - #10 7 - #15 7 - 
#20 
- - Met Asn Gly Ile Glu Glu Asp Ser Asp Glu Pr - #o Leu Glu Arg Arg 
Leu 
725 - # 730 - # 735 
- - Ser Leu Val Pro Asp Ser Glu Gln Gly Glu Al - #a Ile Leu Pro Arg Ile 
740 - # 745 - # 750 
- - Ser Val Ile Ser Thr Gly Pro Thr Leu Gln Al - #a Arg Arg Arg Gln Ser 
755 - # 760 - # 765 
- - Val Leu Asn Leu Met Thr His Ser Val Asn Gl - #n Gly Gln Asn Ile His 
770 - # 775 - # 780 
- - Arg Lys Thr Thr Ala Ser Thr Arg Lys Val Se - #r Leu Ala Pro Gln Ala 
785 7 - #90 7 - #95 8 - 
#00 
- - Asn Leu Thr Glu Leu Asp Ile Tyr Ser Arg Ar - #g Leu Ser Gln Glu 
Thr 
805 - # 810 - # 815 
- - Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu Gl - #u Asp Leu Lys Glu Cys 
820 - # 825 - # 830 
- - Leu Phe Asp Asp Met Glu Ser Ile Pro Ala Va - #l Thr Thr Trp Asn Thr 
835 - # 840 - # 845 
- - Tyr Leu Arg Tyr Ile Thr Val His Lys Ser Le - #u Ile Phe Val Leu Ile 
850 - # 855 - # 860 
- - Trp Cys Leu Val Ile Phe Leu Ala Glu Val Al - #a Ala Ser Leu Val Val 
865 8 - #70 8 - #75 8 - 
#80 
- - Leu Trp Leu Leu Gly Asn Thr Pro Leu Gln As - #p Lys Gly Asn Ser 
Thr 
885 - # 890 - # 895 
- - His Ser Arg Asn Asn Ser Tyr Ala Val Ile Il - #e Thr Ser Thr Ser Ser 
900 - # 905 - # 910 
- - Tyr Tyr Val Phe Tyr Ile Tyr Val Gly Val Al - #a Asp Thr Leu Leu Ala 
915 - # 920 - # 925 
- - Met Gly Phe Phe Arg Gly Leu Pro Leu Val Hi - #s Thr Leu Ile Thr Val 
930 - # 935 - # 940 
- - Ser Lys Ile Leu His His Lys Met Leu His Se - #r Val Leu Gln Ala Pro 
945 9 - #50 9 - #55 9 - 
#60 
- - Met Ser Thr Leu Asn Thr Leu Lys Ala Gly Gl - #y Ile Leu Asn Arg 
Phe 
965 - # 970 - # 975 
- - Ser Lys Asp Ile Ala Ile Leu Asp Asp Leu Le - #u Pro Leu Thr Ile Phe 
980 - # 985 - # 990 
- - Asp Phe Ile Gln Leu Leu Leu Ile Val Ile Gl - #y Ala Ile Ala Val Val 
995 - # 1000 - # 1005 
- - Ala Val Leu Gln Pro Tyr Ile Phe Val Ala Th - #r Val Pro Val Ile Val 
1010 - # 1015 - # 1020 
- - Ala Phe Ile Met Leu Arg Ala Tyr Phe Leu Gl - #n Thr Ser Gln Gln Leu 
1025 1030 - # 1035 - # 1040 
- - Lys Gln Leu Glu Ser Glu Gly Arg Ser Pro Il - #e Phe Thr His Leu Val 
1045 - # 1050 - # 1055 
- - Thr Ser Leu Lys Gly Leu Trp Thr Leu Arg Al - #a Phe Gly Arg Gln Pro 
1060 - # 1065 - # 1070 
- - Tyr Phe Glu Thr Leu Phe His Lys Ala Leu As - #n Leu His Thr Ala Asn 
1075 - # 1080 - # 1085 
- - Trp Phe Leu Tyr Leu Ser Thr Leu Arg Trp Ph - #e Gln Met Arg Ile Glu 
1090 - # 1095 - # 1100 
- - Met Ile Phe Val Ile Phe Phe Ile Ala Val Th - #r Phe Ile Ser Ile Leu 
1105 1110 - # 1115 - # 1120 
- - Thr Thr Gly Glu Gly Glu Gly Arg Val Gly Il - #e Ile Leu Thr Leu Ala 
1125 - # 1130 - # 1135 
- - Met Asn Ile Met Ser Thr Leu Gln Trp Ala Va - #l Asn Ser Ser Ile Asp 
1140 - # 1145 - # 1150 
- - Val Asp Ser Leu Met Arg Ser Val Ser Arg Va - #l Phe Lys Phe Ile Asp 
1155 - # 1160 - # 1165 
- - Met Pro Thr Glu Gly Lys Pro Thr Lys Ser Th - #r Lys Pro Tyr Lys Asn 
1170 - # 1175 - # 1180 
- - Gly Gln Leu Ser Lys Val Met Ile Ile Glu As - #n Ser His Val Lys Lys 
1185 1190 - # 1195 - # 1200 
- - Asp Asp Ile Trp Pro Ser Gly Gly Gln Met Th - #r Val Lys Asp Leu Thr 
1205 - # 1210 - # 1215 
- - Ala Lys Tyr Thr Glu Gly Gly Asn Ala Ile Le - #u Glu Asn Ile Ser Phe 
1220 - # 1225 - # 1230 
- - Ser Ile Ser Pro Gly Gln Arg Val Gly Leu Le - #u Gly Arg Thr Gly Ser 
1235 - # 1240 - # 1245 
- - Gly Lys Ser Thr Leu Leu Ser Ala Phe Leu Ar - #g Leu Leu Asn Thr Glu 
1250 - # 1255 - # 1260 
- - Gly Glu Ile Gln Ile Asp Gly Val Ser Trp As - #p Ser Ile Thr Leu Gln 
1265 1270 - # 1275 - # 1280 
- - Gln Trp Arg Lys Ala Phe Gly Val Ile Pro Gl - #n Lys Val Phe Ile Phe 
1285 - # 1290 - # 1295 
- - Ser Gly Thr Phe Arg Lys Asn Leu Asp Pro Ty - #r Glu Gln Trp Ser Asp 
1300 - # 1305 - # 1310 
- - Gln Glu Ile Trp Lys Val Ala Asp Glu Val Gl - #y Leu Arg Ser Val Ile 
1315 - # 1320 - # 1325 
- - Glu Gln Phe Pro Gly Lys Leu Asp Phe Val Le - #u Val Asp Gly Gly Cys 
1330 - # 1335 - # 1340 
- - Val Leu Ser His Gly His Lys Gln Leu Met Cy - #s Leu Ala Arg Ser Val 
1345 1350 - # 1355 - # 1360 
- - Leu Ser Lys Ala Lys Ile Leu Leu Leu Asp Gl - #u Pro Ser Ala His Leu 
1365 - # 1370 - # 1375 
- - Asp Pro Val Thr Tyr Gln Ile Ile Arg Arg Th - #r Leu Lys Gln Ala Phe 
1380 - # 1385 - # 1390 
- - Ala Asp Cys Thr Val Ile Leu Cys Glu His Ar - #g Ile Glu Ala Met Leu 
1395 - # 1400 - # 1405 
- - Glu Cys Gln Gln Phe Leu Val Ile Glu Glu As - #n Lys Val Arg Gln Tyr 
1410 - # 1415 - # 1420 
- - Asp Ser Ile Gln Lys Leu Leu Asn Glu Arg Se - #r Leu Phe Arg Gln Ala 
1425 1430 - # 1435 - # 1440 
- - Ile Ser Pro Ser Asp Arg Val Lys Leu Phe Pr - #o His Arg Asn Ser Ser 
1445 - # 1450 - # 1455 
- - Lys Cys Lys Ser Lys Pro Gln Ile Ala Ala Le - #u Lys Glu Glu Thr Glu 
1460 - # 1465 - # 1470 
- - Glu Glu Val Gln Asp Thr Arg Leu 
1475 - # 1480 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5635 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - CATCATCAAT AATATACCTT ATTTTGGATT GAAGCCAATA TGATAATGAG GG - 
#GGTGGAGT 60 
- - TTGTGACGTG GCGCGGGGCG TGGGAACGGG GCGGGTGACG TAGTAGTGTG GC - 
#GGAAGTGT 120 
- - GATGTTGCAA GTGTGGCGGA ACACATGTAA GCGCCGGATG TGGTAAAAGT GA - 
#CGTTTTTG 180 
- - GTGTGCGCCG GTGTATACGG GAAGTGACAA TTTTCGCGCG GTTTTAGGCG GA - 
#TGTTGTAG 240 
- - TAAATTTGGG CGTAACCAAG TAATGTTTGG CCATTTTCGC GGGAAAACTG AA - 
#TAAGAGGA 300 
- - AGTGAAATCT GAATAATTCT GTGTTACTCA TAGCGCGTAA TATTTGTCTA GG - 
#GCCGCGGG 360 
- - GACTTTGACC GTTTACGTGG AGACTCGCCC AGGTGTTTTT CTCAGGTGTT TT - 
#CCGCGTTC 420 
- - CGGGTCAAAG TTGGCGTTTT ATTATTATAG TCAGCTGACG CGCAGTGTAT TT - 
#ATACCCGG 480 
- - TGAGTTCCTC AAGAGGCCAC TCTTGAGTGC CAGCGAGTAG AGTTTTCTCC TC - 
#CGAGCCGC 540 
- - TCCGAGCTAG TAACGGCCGC CAGTGTGCTG CAGATATCAA AGTCGACGGT AC - 
#CCGAGAGA 600 
- - CCATGCAGAG GTCGCCTCTG GAAAAGGCCA GCGTTGTCTC CAAACTTTTT TT - 
#CAGCTGGA 660 
- - CCAGACCAAT TTTGAGGAAA GGATACAGAC AGCGCCTGGA ATTGTCAGAC AT - 
#ATACCAAA 720 
- - TCCCTTCTGT TGATTCTGCT GACAATCTAT CTGAAAAATT GGAAAGAGAA TG - 
#GGATAGAG 780 
- - AGCTGGCTTC AAAGAAAAAT CCTAAACTCA TTAATGCCCT TCGGCGATGT TT - 
#TTTCTGGA 840 
- - GATTTATGTT CTATGGAATC TTTTTATATT TAGGGGAAGT CACCAAAGCA GT - 
#ACAGCCTC 900 
- - TCTTACTGGG AAGAATCATA GCTTCCTATG ACCCGGATAA CAAGGAGGAA CG - 
#CTCTATCG 960 
- - CGATTTATCT AGGCATAGGC TTATGCCTTC TCTTTATTGT GAGGACACTG CT - 
#CCTACACC 1020 
- - CAGCCATTTT TGGCCTTCAT CACATTGGAA TGCAGATGAG AATAGCTATG TT - 
#TAGTTTGA 1080 
- - TTTATAAGAA GACTTTAAAG CTGTCAAGCC GTGTTCTAGA TAAAATAAGT AT - 
#TGGACAAC 1140 
- - TTGTTAGTCT CCTTTCCAAC AACCTGAACA AATTTGATGA AGGACTTGCA TT - 
#GGCACATT 1200 
- - TCGTGTGGAT CGCTCCTTTG CAAGTGGCAC TCCTCATGGG GCTAATCTGG GA - 
#GTTGTTAC 1260 
- - AGGCGTCTGC CTTCTGTGGA CTTGGTTTCC TGATAGTCCT TGCCCTTTTT CA - 
#GGCTGGGC 1320 
- - TAGGGAGAAT GATGATGAAG TACAGAGATC AGAGAGCTGG GAAGATCAGT GA - 
#AAGACTTG 1380 
- - TGATTACCTC AGAAATGATT GAAAACATCC AATCTGTTAA GGCATACTGC TG - 
#GGAAGAAG 1440 
- - CAATGGAAAA AATGATTGAA AACTTAAGAC AAACAGAACT GAAACTGACT CG - 
#GAAGGCAG 1500 
- - CCTATGTGAG ATACTTCAAT AGCTCAGCCT TCTTCTTCTC AGGGTTCTTT GT - 
#GGTGTTTT 1560 
- - TATCTGTGCT TCCCTATGCA CTAATCAAAG GAATCATCCT CCGGAAAATA TT - 
#CACCACCA 1620 
- - TCTCATTCTG CATTGTTCTG CGCATGGCGG TCACTCGGCA ATTTCCCTGG GC - 
#TGTACAAA 1680 
- - CATGGTATGA CTCTCTTGGA GCAATAAACA AAATACAGGA TTTCTTACAA AA - 
#GCAAGAAT 1740 
- - ATAAGACATT GGAATATAAC TTAACGACTA CAGAAGTAGT GATGGAGAAT GT - 
#AACAGCCT 1800 
- - TCTGGGAGGA GGGATTTGGG GAATTATTTG AGAAAGCAAA ACAAAACAAT AA - 
#CAATAGAA 1860 
- - AAACTTCTAA TGGTGATGAC AGCCTCTTCT TCAGTAATTT CTCACTTCTT GG - 
#TACTCCTG 1920 
- - TCCTGAAAGA TATTAATTTC AAGATAGAAA GAGGACAGTT GTTGGCGGTT GC - 
#TGGATCCA 1980 
- - CTGGAGCAGG CAAGACTTCA CTTCTAATGA TGATTATGGG AGAACTGGAG CC - 
#TTCAGAGG 2040 
- - GTAAAATTAA GCACAGTGGA AGAATTTCAT TCTGTTCTCA GTTTTCCTGG AT - 
#TATGCCTG 2100 
- - GCACCATTAA AGAAAATATC ATCTTTGGTG TTTCCTATGA TGAATATAGA TA - 
#CAGAAGCG 2160 
- - TCATCAAAGC ATGCCAACTA GAAGAGGACA TCTCCAAGTT TGCAGAGAAA GA - 
#CAATATAG 2220 
- - TTCTTGGAGA AGGTGGAATC ACACTGAGTG GAGGTCAACG AGCAAGAATT TC - 
#TTTAGCAA 2280 
- - GAGCAGTATA CAAAGATGCT GATTTGTATT TATTAGACTC TCCTTTTGGA TA - 
#CCTAGATG 2340 
- - TTTTAACAGA AAAAGAAATA TTTGAAAGCT GTGTCTGTAA ACTGATGGCT AA - 
#CAAAACTA 2400 
- - GGATTTTGGT CACTTCTAAA ATGGAACATT TAAAGAAAGC TGACAAAATA TT - 
#AATTTTGC 2460 
- - ATGAAGGTAG CAGCTATTTT TATGGGACAT TTTCAGAACT CCAAAATCTA CA - 
#GCCAGACT 2520 
- - TTAGCTCAAA ACTCATGGGA TGTGATTCTT TCGACCAATT TAGTGCAGAA AG - 
#AAGAAATT 2580 
- - CAATCCTAAC TGAGACCTTA CACCGTTTCT CATTAGAAGG AGATGCTCCT GT - 
#CTCCTGGA 2640 
- - CAGAAACAAA AAAACAATCT TTTAAACAGA CTGGAGAGTT TGGGGAAAAA AG - 
#GAAGAATT 2700 
- - CTATTCTCAA TCCAATCAAC TCTATACGAA AATTTTCCAT TGTGCAAAAG AC - 
#TCCCTTAC 2760 
- - AAATGAATGG CATCGAAGAG GATTCTGATG AGCCTTTAGA GAGAAGGCTG TC - 
#CTTAGTAC 2820 
- - CAGATTCTGA GCAGGGAGAG GCGATACTGC CTCGCATCAG CGTGATCAGC AC - 
#TGGCCCCA 2880 
- - CGCTTCAGGC ACGAAGGAGG CAGTCTGTCC TGAACCTGAT GACACACTCA GT - 
#TAACCAAG 2940 
- - GTCAGAACAT TCACCGAAAG ACAACAGCAT CCACACGAAA AGTGTCACTG GC - 
#CCCTCAGG 3000 
- - CAAACTTGAC TGAACTGGAT ATATATTCAA GAAGGTTATC TCAAGAAACT GG - 
#CTTGGAAA 3060 
- - TAAGTGAAGA AATTAACGAA GAAGACTTAA AGGAGTGCCT TTTTGATGAT AT - 
#GGAGAGCA 3120 
- - TACCAGCAGT GACTACATGG AACACATACC TTCGATATAT TACTGTCCAC AA - 
#GAGCTTAA 3180 
- - TTTTTGTGCT AATTTGGTGC TTAGTAATTT TTCTGGCAGA GGTGGCTGCT TC - 
#TTTGGTTG 3240 
- - TGCTGTGGCT CCTTGGAAAC ACTCCTCTTC AAGACAAAGG GAATAGTACT CA - 
#TAGTAGAA 3300 
- - ATAACAGCTA TGCAGTGATT ATCACCAGCA CCAGTTCGTA TTATGTGTTT TA - 
#CATTTACG 3360 
- - TGGGAGTAGC CGACACTTTG CTTGCTATGG GATTCTTCAG AGGTCTACCA CT - 
#GGTGCATA 3420 
- - CTCTAATCAC AGTGTCGAAA ATTTTACACC ACAAAATGTT ACATTCTGTT CT - 
#TCAAGCAC 3480 
- - CTATGTCAAC CCTCAACACG TTGAAAGCAG GTGGGATTCT TAATAGATTC TC - 
#CAAAGATA 3540 
- - TAGCAATTTT GGATGACCTT CTGCCTCTTA CCATATTTGA CTTCATCCAG TT - 
#GTTATTAA 3600 
- - TTGTGATTGG AGCTATAGCA GTTGTCGCAG TTTTACAACC CTACATCTTT GT - 
#TGCAACAG 3660 
- - TGCCAGTGAT AGTGGCTTTT ATTATGTTGA GAGCATATTT CCTCCAAACC TC - 
#ACAGCAAC 3720 
- - TCAAACAACT GGAATCTGAA GGCAGGAGTC CAATTTTCAC TCATCTTGTT AC - 
#AAGCTTAA 3780 
- - AAGGACTATG GACACTTCGT GCCTTCGGAC GGCAGCCTTA CTTTGAAACT CT - 
#GTTCCACA 3840 
- - AAGCTCTGAA TTTACATACT GCCAACTGGT TCTTGTACCT GTCAACACTG CG - 
#CTGGTTCC 3900 
- - AAATGAGAAT AGAAATGATT TTTGTCATCT TCTTCATTGC TGTTACCTTC AT - 
#TTCCATTT 3960 
- - TAACAACAGG AGAAGGAGAA GGAAGAGTTG GTATTATCCT GACTTTAGCC AT - 
#GAATATCA 4020 
- - TGAGTACATT GCAGTGGGCT GTAAACTCCA GCATAGATGT GGATAGCTTG AT - 
#GCGATCTG 4080 
- - TGAGCCGAGT CTTTAAGTTC ATTGACATGC CAACAGAAGG TAAACCTACC AA - 
#GTCAACCA 4140 
- - AACCATACAA GAATGGCCAA CTCTCGAAAG TTATGATTAT TGAGAATTCA CA - 
#CGTGAAGA 4200 
- - AAGATGACAT CTGGCCCTCA GGGGGCCAAA TGACTGTCAA AGATCTCACA GC - 
#AAAATACA 4260 
- - CAGAAGGTGG AAATGCCATA TTAGAGAACA TTTCCTTCTC AATAAGTCCT GG - 
#CCAGAGGG 4320 
- - TGGGCCTCTT GGGAAGAACT GGATCAGGGA AGAGTACTTT GTTATCAGCT TT - 
#TTTGAGAC 4380 
- - TACTGAACAC TGAAGGAGAA ATCCAGATCG ATGGTGTGTC TTGGGATTCA AT - 
#AACTTTGC 4440 
- - AACAGTGGAG GAAAGCCTTT GGAGTGATAC CACAGAAAGT ATTTATTTTT TC - 
#TGGAACAT 4500 
- - TTAGAAAAAA CTTGGATCCC TATGAACAGT GGAGTGATCA AGAAATATGG AA - 
#AGTTGCAG 4560 
- - ATGAGGTTGG GCTCAGATCT GTGATAGAAC AGTTTCCTGG GAAGCTTGAC TT - 
#TGTCCTTG 4620 
- - TGGATGGGGG CTGTGTCCTA AGCCATGGCC ACAAGCAGTT GATGTGCTTG GC - 
#TAGATCTG 4680 
- - TTCTCAGTAA GGCGAAGATC TTGCTGCTTG ATGAACCCAG TGCTCATTTG GA - 
#TCCAGTAA 4740 
- - CATACCAAAT AATTAGAAGA ACTCTAAAAC AAGCATTTGC TGATTGCACA GT - 
#AATTCTCT 4800 
- - GTGAACACAG GATAGAAGCA ATGCTGGAAT GCCAACAATT TTTGGTCATA GA - 
#AGAGAACA 4860 
- - AAGTGCGGCA GTACGATTCC ATCCAGAAAC TGCTGAACGA GAGGAGCCTC TT - 
#CCGGCAAG 4920 
- - CCATCAGCCC CTCCGACAGG GTGAAGCTCT TTCCCCACCG GAACTCAAGC AA - 
#GTGCAAGT 4980 
- - CTAAGCCCCA GATTGCTGCT CTGAAAGAGG AGACAGAAGA AGAGGTGCAA GA - 
#TACAAGGC 5040 
- - TTTAGAGAGC AGCATAAATG TTGACATGGG ACATTTGCTC ATGGAATTGG AG - 
#GTAGCGGA 5100 
- - TTGAGGTACT GAAATGTGTG GGCGTGGCTT AAGGGTGGGA AAGAATATAT AA - 
#GGTGGGGG 5160 
- - TCTCATGTAG TTTTGTATCT GTTTTGCAGC AGCCGCCGCC ATGAGCGCCA AC - 
#TCGTTTGA 5220 
- - TGGAAGCATT GTGAGCTCAT ATTTGACAAC GCGCATGCCC CCATGGGCCG GG - 
#GTGCGTCA 5280 
- - GAATGTGATG GGCTCCAGCA TTGATGGTCG CCCCGTCCTG CCCGCAAACT CT - 
#ACTACCTT 5340 
- - GACCTACGAG ACCGTGTCTG GAACGCCGTT GGAGACTGCA GCCTCCGCCG CC - 
#GCTTCAGC 5400 
- - CGCTGCAGCC ACCGCCCGCG GGATTGTGAC TGACTTTGCT TTCCTGAGCC CG - 
#CTTGCAAG 5460 
- - CAGTGCAGCT TCCCGTTCAT CCGCCCGCGA TGACAAGTTG ACGGCTCTTT TG - 
#GCACAATT 5520 
- - GGATTCTTTG ACCCGGGAAC TTAATGTCGT TTCTCAGCAG CTGTTGGATC TG - 
#CGCCAGCA 5580 
- - GGTTTCTGCC CTGAAGGCTT CCTCCCCTCC CAATGCGGTT TAAAACATAA AT - #AAA 
5635 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - ACTCTTGAGT GCCAGCGAGT AGAGTTTTCT CCTCCG - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - GCAAAGGAGC GATCCACACG AAATGTGCC - # - # 
29 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - CTCCTCCGAG CCGCTCCGAG CTAG - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - CCAAAAATGG CTGGGTGTAG GAGCAGTGTC C - # - # 
31 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - CGGATCCTTT ATTATAGGGG AAGTCCACGC CTAC - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - CGGGATCCAT CGATGAAATA TGACTACGTC CG - # - # 
32 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 170 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - GTACGGTTGA TCTTCTCCAT TCCCCGAGTG GTCAAGTTTT AGACTTCACC TC - 
#TGTCCTGG 60 
- - ACTCCACTGT TACTGTAGAT GAGACTGTAA GAGAGGAGTC CTGTAGAGGT TC - 
#AAACGTCT 120 
- - CTTTCTGTTA TATCAAGAAC CTCTTCCACC TTAGTGTGAC TCACCTCCAG - # 
170 
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