U.S. patent application number 09/568756 was filed with the patent office on 2002-11-07 for adenovirus vectors for gene therapy.
Invention is credited to Armentano, Donna, Couture, Larry A., Gregory, Richard J., Smith, Alan E..
Application Number | 20020164782 09/568756 |
Document ID | / |
Family ID | 22937349 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164782 |
Kind Code |
A1 |
Gregory, Richard J. ; et
al. |
November 7, 2002 |
Adenovirus vectors for gene therapy
Abstract
Gene Therapy vectors, which are especially useful for cystic
fibrosis, and methods for using the vectors are disclosed.
Inventors: |
Gregory, Richard J.;
(Westford, MA) ; Armentano, Donna; (Belmont,
MA) ; Couture, Larry A.; (Louisville, CO) ;
Smith, Alan E.; (Dover, MA) |
Correspondence
Address: |
GENZYME CORPORATION
LEGAL DEPARTMENT
15 PLEASANT ST CONNECTOR
FRAMINGHAM
MA
01701-9322
US
|
Family ID: |
22937349 |
Appl. No.: |
09/568756 |
Filed: |
May 11, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09568756 |
May 11, 2000 |
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09248026 |
Feb 10, 1999 |
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6093567 |
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Current U.S.
Class: |
435/320.1 ;
424/93.2; 435/325 |
Current CPC
Class: |
C12N 2710/10343
20130101; C12N 15/86 20130101; A61K 48/00 20130101 |
Class at
Publication: |
435/320.1 ;
435/325; 424/93.2 |
International
Class: |
A61K 048/00; C12N
015/74; C12N 005/06 |
Claims
1. An adenovirus-based gene therapy vector comprising the genome of
an adenovirus 2 serotype 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.
2. The adenovirus-based gene therapy vector of claim 1, wherein the
genetic material of interest is DNA encoding cystic fibrosis
transmembrane conductance regulator
3. The adenovirus-based gene therapy vector of claim 1 further
comprising PGK promoter operably linked to the genetic material of
interest.
4. The adenovirus-based gene therapy vector of claim 2 having
substantially the same nucleotide sequence as shown in Table II
(SEQ ID NO:3).
5. An adenovirus-based gene therapy vector comprising adenovirus
inverted terminal repeat nucleotide sequences and the minimal
nucleotide sequences necessary for efficient replication and
packaging and genetic material of interest.
6. The adenovirus-based gene therapy vector of claim 5 having the
adenovirus 2 sequences shown in FIG. 17.
7. The adenovirus-based gene therapy vector of claim 5 further
comprising PGK promoter operably linked to the genetic material of
interest.
8. The adenovirus-based gene therapy vector of claim 5 in which the
genetic material of interest is selected from the group consisting
of DNA encoding: cystic fibrosis transmembrane conductance
regulator, Factor VIII, and Factor IX.
9. An adenovirus-based gene therapy vector comprising an adenovirus
genome which has been deleted for all E4 open reading frames,
except open reading fine 6, and additionally comprising genetic
material of interest.
10. The adenovirus-based gene therapy vector of claim 9 further
comprising PGK promoter operably linked to the genetic material of
interest.
11. The adenovirus-based gene therapy vector of claim 9 in which
the E1a and E1b regions of the genome, which are involved in early
stages of viral replication, have been deleted.
12. The adenovirus-based gene therapy vector of claim 9 in which
the E3 region has been deleted.
13. An adenovirus-based gene therapy vector comprising an
adenovirus genome which has been deleted for all E4 open reading
frames, except open reading frame 3, and additionally comprising
genetic material of interest.
14. The adenovirus-based gene therapy vector of claim 13 in which
the E1a and E1b regions of the genome, which are involved in early
stages of viral replication, have been deleted.
15. The adenovirus-based gene therapy vector of claim 13 further
comprising PGK promoter operably linked to the genetic material of
interest.
16. The adenovirus-based gene therapy vector of claim 13 in which
the E3 region has been deleted.
17. A method for treating or preventing cystic fibrosis in a
patient comprising administering to the pulmonary airways of the
patient, a gene therapy vector comprising DNA encoding cystic
fibrosis transmembrane conductance regulator.
18. The method of claim 17 wherein the gene therapy vector is an
adenovirus-based gene therapy vector comprising the genome of an
adenovirus 2 serotype 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 DNA encoding cystic fibrosis
transmembrane conductance regulator.
19. The method of claim 17 wherein the gene therapy vector further
comprises PGK promoter operably linked to the DNA encoding cystic
fibrosis transmembrane conductance regulator.
20. The method of claim 17 wherein the gene therapy vector is an
adenovirus-based gene therapy vector comprising adenovirus inverted
terminal repeats and the minimal sequences necessary for efficient
replication and packaging and DNA encoding cystic fibrosis
tranmembrane conductance regulator.
21. The method of claim 20 wherein the gene therapy vector further
comprises PGK promoter operably linked to the DNA encoding cystic
fibrosis transmembrane conductance regulator.
22 The method of claim 17 wherein the gene therapy vector is an
adenovirus-based gene therapy vector comprising an adenovirus
genome which has been deleted for all E4 open reading frames,
except open reading frame 6, and additionally comprising DNA
encoding cystic fibrosis transmembrane conductance regulator.
23. The method of claim 22 wherein the gene therapy vector further
comprises PGK promoter operably linked to the DNA encoding cystic
fibrosis transmembrane conductance regulator.
24. The method of claim 17 wherein the gene therapy vector is an
adenovirus-based gene therapy vector comprising an adenovirus
genome which has been deleted for all E4 open reading frames,
except open reading frame 6, and has been deleted for the E1a and
E1b regions of the genome, which are involved in early stages of
viral replication, and additionally comprising DNA encoding cystic
fibrosis tranmembrane conductance regulator.
25. The method of claim 24 wherein the gene therapy vector further
comprises PGK promoter operably linked to the DNA encoding cystic
fibrosis transmembrane conductance regulator.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
U.S. Ser. No. 07/985,478, filed Dec. 2, 1992, which is a
continuation-in-part of application U.S. Ser. No. 07/613,592, filed
Nov. 15, 1990, which is in turn a continuation-in-part application
of U.S. Ser. No. 07/589,295, filed Sep. 27, 1990, which is itself a
continuation-in-part application of U.S. Ser. No. 07/488,307, filed
Mar. 5, 1990. The contents of all of the above co-pending patent
applications are incorporated herein by reference. Definitions of
language or terms not provided in the present application are the
same as those set forth in the copending applications. Any reagents
or materials used in the examples of the present application whose
source is not expressly identified also is the same as those
described in the copending application, e.g., A F508 CFTR gene and
CFTR antibodies.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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 freuent 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.
[0004] 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; Rominens. 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.
[0005] 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
multidrug 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.
[0006] 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).
[0007] Sequence analysis of the CFTR gene of CF chromosomes has
revealed a variety of mutations (Cutting, G. R. et al. (1990)
Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863-870; and
Kerem, B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S. et al.
(1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). Population studies
have indicated that the most common CF mutation, a deletion of the
3 nucleotides that encode phenylalanine at position 508 of the CFTR
amino acid sequence (AF508), is associated with approximately 70%
of the cases of cystic fibrosis. This mutation results in the
failure of an epithelial cell chloride channel to respond to cAMP
(Frizell 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.
[0008] Studies on the biosynthesis (Cheng, S. H. et al. (1990) Cell
63:827-834; Gregory, R. J. et al. (1991) Mol. Cell Biol.
11:3886-3893) and localization (Denning, G. M. et al. (1992) J.
Cell Biol. 118:551-559) of CFTR AF508, as well as other CFTR
mutants, indicate that many CFTR mutant proteins are not processed
correctly and, as a result, are not delivered to the 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 AF508 (Rich, D. P. et al. (1990)
Nature 347:358-363; Anderson, M. P. et al. (1991) Science
251:679-682).
[0009] 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).
[0010] 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. Natl. Sci. Acad USA 87:9188). Preliminary reports suggest
that aerosolized enzyme may be effective in reducing the viscosity
of mucus. This could be helpful in clearing the airways of
obstruction and perhaps in reducing infections. In an attempt to
limit damage caused by anexcess of neutrophil derived elastase,
protease inhibitors have been tested. For example,
alpha-1-antitrsin 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.
[0011] Using a different rationale, other investigators have
attempted to use pharmacological agents to reverse the abnormally
decreased chloride secretion and increased sodium absorption in CF
airways. Defective electrolyte transport by airway epithelia is
thought to alter the composition of the respiratory secretions and
mucus (Boat, T. F. et al. in The Metabolic Basis of Inherited
Diseases (Scriver, C. R. et al. eds., McGraw-Hill, New York (1989);
Quinton, P. M. (1990) 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.
[0012] 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.
[0013] 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
[0014] 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.
[0015] In one embodiment, the adenovirus-based gene therapy vector
comprises an adenovirus 2 serotpe 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).
[0016] In another embodiment, the adenovirus-based therapy vector
is a pseudo-adenovirus (PAV). PAVs contain no potentially harmful
viral genes, have a theoretical capacity for foreign material of
nearly 36 kb, may be produced in reasonably high titers and
maintain the tropism of the parent adenovirus for dividing and
non-dividing human target cell types. PAVs comprise adenovirus
inverted terminal repeats and the minimal sequences of a 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.
[0017] 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.
[0018] The invention also relates to methods of gene therapy using
the disclosed vectors and genetically engineered cells produced by
the method.
BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS
[0019] Further understanding of the invention may be had by
reference to the tables and figures wherein:
[0020] Table I shows CFTR mutants wherein the known association
with CF (Y, yes or N, no), exon localization, domain location and
presence (+) or absence (-) of bands A, B, and C of mutant CFTR
species is shown. TM6, indicates transmembrane domain 6; NBD
nucleotide binding domain; ECD, extracellular domain and Term,
termination at 21 codons pastresidue 1337;
[0021] Table II shows the nucleotide sequence of Ad2/CFTR-1;
[0022] Table III depicts a nucleotide analysis of
Ad2ORF6/PGK-CFTR;
[0023] The convention for naming mutants is first the amino acid
normally found at the particular residue, the residue number
(Riordan, T. R. et al. (1989) Science 245:1066-1073). and the amino
acid to which the residue was converted. The single letter amino
acid code is used: D, aspartic acid; F, phenylalanine; G, glycine;
I, isoleucine; K, lysine; M, methionine; N, asparagine; Q,
glutamine; R, arginine; S, serine; W, tryptophan. Thus G551D is a
mutant in which glycine 551 is converted to aspartic acid;
[0024] FIG. 1 shows alignment of CFTR partial cDNA clones used in
construction of cDNA containing complete coding sequence of the
CFTR, only restriction sites relevant to the DNA constructions
described below are shown;
[0025] FIG. 2 depicts plasmid construction of the CFTR cDNA clone
pKK-CFTR1;
[0026] FIG. 3 depicts plasmid construction of the CFTR cDNA clone
pKK-CFTR2;
[0027] FIG. 4 depicts plasmid construction of the CFTR cDNA clone
pSC-CFTR2;
[0028] FIG. 5 shows a plasmid map of the CFTR cDNA clone
pSC-CFTR2;
[0029] FIG. 6 shows the DNA sequence of synthetic DNAs used for
insertion of an intron into the CFTR cDNA sequence, with the
relevant restriction endonuclease sites and nucleotide positions
noted;
[0030] FIGS. 7A and 7B depict plasmid construction of the CFTR cDNA
clone pKK-CFTR3;
[0031] FIG. 8 shows a plasmid map of the CFTR cDNA pKK-CFTR3
containing an intron between nucleotides 1716 and 1717;
[0032] FIG. 9 shows treatment of CFTR with glycosidases;
[0033] FIGS. 10A and 10B show an analysis of CFTR expressed from
COS-7 transfected cells;
[0034] FIGS. 11A and, 11B show pulse-chase labeling of wild type
and AF508 mutant CFTR in COS-7 transfected cells;
[0035] FIGS. 12A-12D show immunolocalization of wild type and AF508
mutant CFTR; and COS-7 cells transfected with pMT-CFTR or
pMT-CFTR-.DELTA.F508;
[0036] FIG. 13 shows an analysis of mutant forms of CFTR;
[0037] FIG. 14 shows a map of the first generation adenovirus based
vector encoding CFTR (Ad2/CFTR-1);
[0038] FIG. 15 shows the plasmid construction of the Ad2/CFTR-1
vector;
[0039] FIG. 16 shows a map of the second generation adenovirus
based vector, PAV;
[0040] FIG. 17 shows; the plasmid construction of a second
generation adenoviral vector 6 (Ad E4 ORF6).
[0041] FIG. 18 shows an example of UV fluorescence from an agarose
gel electrophoresis of products of nested RT-PCR from lung
homogenates of cotton rats which received Ad2/CFTR-1 stained with
ethidium bromide. The gel demonstrates that the homogenates were
positive for virally-encoded CFTR mRNA;
[0042] FIG. 19 depicts cotton rat bronchial epithelium from control
and infected rats stained with monoclonal antibodies for CFTR. CFTR
was detected in the epithelium from the rats infected with
Ad2/CFTR-1, but not in the epithelium of the control rats;
[0043] FIGS. 20A and 20B show differential cell analyses of
bronchoalveolar lavage specimens from control and infected rats.
These data demonstrate that none of the rats treated with
Ad2/CFTR-1 had a change in the total or differential white blood
cell count 4, 10, and 14 days after infection (FIG. 20A) and 3, 7,
14 days after infection (FIG. 20B);
[0044] FIG. 21 shows an example of UV fluorescence from an agarose
gel electrophoresis of products of nested RT-PCR from organ
homogenates of cotton rats stained with ethidium bromide. The gel
demonstrates that all organs of the infected rats were negative for
Ad2/CFTR-1 with the exception of the small bowel;
[0045] FIG. 22 shows hematoxilyn and eosin stained sections of
cotton rat tracheas from both treated and control rats sacrificed
at different time points after infection with Ad2/CFTR-1. The
sections demonstrate that there were no observable differences
between the treated and control rats;
[0046] FIGS. 23A and 23B show examples of UV fluorescence from an
agarose gel electrophoresis, stained with ethidium bromide, of
products of RT-PCR from nasal bushings of Rhesus monkeys after
application of Ad2/CFTR-1 or Ad2/0-Gal;
[0047] FIG. 24 shows lights microscopy and immunocytochemistry from
monkey nasal brushings. The microscopy revealed that there was a
positive reaction when nasal epithelial cells from monkeys exposed
to Ad2/CFTR-1 were stained with antibodies to CFTR;
[0048] FIG. 25 shows immunocytochemistry of monkey nasal turbinate
biopsies. This microscopy reveals increased immunofluorescence at
the apical membrane of the surface epithelium from biopsies
obtained from monkeys treated with Ad2/CFTR-1 over that seen at the
apical membrane of the surface epithelium from biopsies obtained
from control monkeys;
[0049] FIGS. 26A-26D show serum antibody titers in Rhesus monkeys
after three vector administrations. These graphs demonstrate that
all three monkeys treated with Ad2/CFTR-1 developed antibodies
against adenovirus;
[0050] FIG. 27 shows hematoxilyn and eosin stained sections from
monkey medial turbinate biopsies. These sections demonstrate that
turbinate biopsy specimens from control monkeys could not be
differentiated from those from monkeys treated with Ad2/CFTR-1 when
reviewed by an independent pathologist;
[0051] FIGS. 28A-28I show photomicrographs of human nasal mucosa
immediately before, during, and after Ad2/CFTR-1 application. These
photomicrographs demonstrate that inspection of the nasal mucosa
showed mild to moderate erythema, edema, and exudate in patients
treated with Ad2/CFTR-1 (FIGS. 28A-28C) and in control patients
(FIGS. 28G-28I). These changes were probably due to local
anesthesia and vasocontriction because when an additional patient
was exposed to Ad2/CFTR in a method which did not require the use
of local anesthesia or vasoconstriction, there were no symptoms and
the nasal mucosa appeared normal (FIGS. 28D-28F);
[0052] FIG. 29 shows a photomicrograph of a hematoxilyn and eosin
stained biopsy of human nasal mucosa obtained from the third
patient three days after Ad2/CFTR-1 administration. This section
shows a morphology consistent with CF, i.e., a thickened basement
membrane and occasional morphonuclear cells in the submucosa, but
no abnormalities that could be attributed to the adenovirus
vector;
[0053] FIG. 30 shows transepithelial voltage (V.sub.t) across the
nasal epithelium of a normal human subject. Amiloride (.mu.M) and
terbutaline (.mu.M) were perfused onto the mucosal surface
beginning at the times indicated. Under basal conditions V.sub.t
was electrically negative. Perfusion of amiloride onto the mucosal
surface inhibited V.sub.t by blocking apical Na.sup.+ channels;
[0054] FIGS. 31A and 31B show transepithelial voltage (V.sub.t
across the nasal epithelium of normal human subjects (FIG. 31A) and
patients with CF (FIG. 31B). Values were obtained under basal
conditions, during perfusion with amiloride (.mu.M), and during
perfusion of amiloride plus terbutaline (.mu.M) onto the mucosal
surface. Data are from seven normal subjects and nine patients with
CF. In patients with CF, V.sub.t was more electrically negative
than in normal subjects (FIG. 31B). Amiloride inhibited V.sub.t in
CF patients, as it did in normal subjects. However, V.sub.t failed
to hyperpolarize when terbutaline was perfused onto the epithelium
in the presence of amiloride. Instead, V.sub.t either did not
change or became less negative, a result very different from that
observed in normal subjects;
[0055] FIGS. 32A and 32B show transepithelial voltage (V.sub.t)
across the nasal epithelium of a third patient before (FIG. 32A)
and after (FIG. 32B) administration of approximately 25 MOI of
Ad2/CFTR-1 Amiloride and terbutaline were perfused onto the mucosal
surface beginning at the times indicated.
[0056] FIG. 32A shows an example from the third patient before
treatment.
[0057] FIG. 32B shows that in contrast to the response before
Ad2/CFTR-1 was applied, after virus replication, in the presence of
amiloride, terbutaline stimulated V.sub.t;
[0058] FIG. 33 shows the time of course changes in transepithelial
electrical properties before and after administration of
Ad2/CFTR-1.
[0059] FIGS. 33A and 33B are from the first patient who received
approximately 1 MOI;
[0060] FIGS. 33C and 33D are from the second patient who received
approximately 3 MOI; and
[0061] FIGS. 33E and 33F are from the third patient who received
approximately 25 MOI. FIGS. 33A, 33C, and 33E show values of basal
transeptithelial voltage (V.sub.t) and FIGS. 33B, 33D, and 33F show
the change in transepithelial voltage (.DELTA.V.sub.t) following
perfusion of terbutaline in the presence of amiloride. Day zero
indicates the day of Ad2/CFTR-1 administration. FIGS. 33A, 33C, and
33E show the time course of changes in basal V.sub.t for all three
patients. The decrease in basal V.sub.t suggests that application
of Ad2/CFTR-1 corrected the CF electrolyte transport defect in
nasal epithelium of all three patients. Additional evidence came
from an examination of the response to terbutaline. FIGS. 33B, 33D,
and 33F show the time course of the response. These data indicate
that Ad2/CFTR-1 corrected the CF defect in Cl.sup.- transport;
and
[0062] FIG. 34 shows the time course of changes in transepithelial
electrical properties before and after administration of saline
instead of Ad2/CFTR-1 to CF patients. Day zero indicates the time
of mock administration. The top graph shows basal transepithelial
voltage (V.sub.t) and the bottom graph shows the change in
transepithelial voltage following perfusion with terbutaline in the
presence of amiloride (.DELTA.V.sub.t). Closed symbols are data
from two patients that received local anesthetic/vasoconstriction
and placement of the applicator for thirty minutes. Open symbol is
data from a patient that received local
anesthetic/vasoconstriction, but not placement of the applicator.
Symptomatic changes and physical findings were the same as those
observed in CF patients treated with a similar administration
procedure and Ad2/CFTR-1.
[0063] FIG. 35 is a schematic of Ad2-ORF6/PGK-CFTR which differs
from Ad2/CFTR 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.
[0064] FIG. 36 shows short-circuit currents from CF nasal polyp
epithelial cells infected with Ad2-ORF6/PGK-CFTR at multiplicities
of 0.3, 3, and 50. 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-2carboxyla- te were added to the mucosal
solution.
[0065] FIG. 37 shows immunocytochemistry of nasal brushings by
laser scanning microscopy of the Rhesus monkey C, before infection
(A) and on 7 days (B); 24 (C); and 38 (E) after the first infection
with Ad2-ORF6/PGK-CFTR;
[0066] FIG. 38 shows immunocytochemistry of nasal brushings by
laser scanning microscopy of Rhesus monkey D, before infection (A)
and on days 7 (B); 24 (C); and 48 (E) after the first infection
with Ad2ORF6/PGK-CFTR;
[0067] FIG. 39 shows immunocytochemistry of nasal brushings by
laser scanning microscopy of the Rhesus monkey E, before infection
(A) and on days 7 (B); 24 (C); and 48 (E) after the first infection
with Ad2-ORF6/PGK-CFTR;
[0068] FIG. 40 shows summaries the clinical signs (or lack thereof)
of infection with Ad2-ORF6/PGK-CFTR;
[0069] FIGS. 41A-41C shows a summary of blood counts, sedimentation
rate, and clinical chemistries after infection with
Ad2-ORF6/PGK-CFTRfor monkeys C, D, and E. There was no evidence of
a systemic inflammatory response or other abnormalities of the
clinical chemistries;
[0070] FIG. 42 shows summaries of white blood cells counts in
monkeys C, D, and E after infection with Ad2-ORF61PGK-CFTR. These
date indictate that the administration of Ad2-ORF6/PGK-CFTR caused
no change in the distribution and number of inflammatory cells at
any of the time points following viral administration;
[0071] FIG. 43 shows histology of submucosal biopsy performed on
Rhesus monkey C on day 4 after the second viral instillation of
Ad2-ORF6/PGK-CFTR. Hematoxylin and eosin stain revealed no evidence
of inflammation or cytopathic changes;
[0072] FIG. 44 shows histology of submucosal biopsy performed on
Rhesus monkey D on day 11 after the second viral instillation of
Ad2ORF6/PGK-CFTR. Hematoxylin and eosin stain revealed no evidence
of inflammation or cytopathic changes;
[0073] FIG. 45 shows histology of submucosal biopsy performed on
Rhesus monkey E on day 18 after the second viral instillation of
Ad2-ORF6/PGK-CFTR. Hematoxylin and eosin stain revealed no evidence
of inflammation or cytopatic changes; and
[0074] FIG. 46 shows antibody titers to adenovirus prior to and
after the first and second administrations of Ad2-ORF6/PGK-CFTR.
Prior to administration of Ad2-ORF6/PGK-CFTR, the monkeys had
received instillations of Ad2/CFTR-1. Antibody titers measured by
ELISA rose within one week after the first and second
administrations of Ad2-ORF6/PGK-CFTR. Serum neutralizing antibodies
also rose within a week after viral administration and peaked at
day 24. No antiadenoviral antibodies were detected by ELISA or
neutralizing assay in nasal washings of any of the monkeys.
DETAILED DESCRIPTION AND BEST MODE
[0075] Gene Therapy
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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 retrovinises
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.
[0082] Even if efficient DNA integration could be achieved using
viruses, the human genome contains elements involved in the
regulation of cellular growth only a small fraction of which 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.
[0083] 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.
[0084] Other issues associated with the use of viruses are the
possibility of recombination with related viruses naturally
infecting the treated patient, complementation of the viral defects
by simultaneous expression of wild type virus proteins and
containment of aerosols of the engineered viruses.
[0085] 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 amy
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 efficacy of treatment.
[0086] CF Gene Therapy Vectors--Possible Options
[0087] 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.
[0088] 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 nondividing 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.
[0089] Plasmid DNA--Naked plasmid can be introduced into muscle
cells by injection into the tissue. Expression can extend over many
months but the number of positive cells is low (Wolff, J. et al.
(1989) Science 247:1465). Cationic lipids aid introduction of DNA
into some cells in culture (Felgner, P. and Ringold, G. M. (1989)
Nature 337:387). Injection of cationic lipid plasmid DNA complexes
into the circulation of mice has been shown to result in expression
of the DNA in lung (Brigham, K. et al. (1989) Am. J. Med. Sci.
298:278). 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.
[0090] 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.
[0091] 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 retrovirses. 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 252:431434; 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).
[0092] The following properties would be desirable in the design of
an adenovirus vector to transfer the gene for CFTR to the airway
cells of 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.
[0093] 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.
[0094] 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 endogenous 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.
[0095] The E3 region of the Ad2/CFTR-1 encodes a variety of
proteins. One of these proteins, gp19, is believed to interact with
and prevent 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.
[0096] 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.
[0097] 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.
[0098] 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, human HeLa, human CF PAC cells (see Example 8) and airway
epithelial cells from CF patients (Rich, O. et al. (1990) Nature
347:358).
[0099] 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.
[0100] Second Generation Adenoviral Vectors
[0101] 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.
[0102] Pseudo-Adenovirus Vector (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.
[0103] 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.
[0104] 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 11. By not containing
most native adenoviral DNA, the PAVs described herein are less
likely to produce a patient immune reponse or to replicate in a
host.
[0105] 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-antitypsin)
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.
[0106] Ad2-E4/ORE6 Adenovirus Vectors
[0107] 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
fires 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.
[0108] 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.
[0109] 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.
[0110] 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 ORF6/7 would prevent the potential activation of transcription
factor E2F in non-dividing primary cells and thereby reduce the
potential for viral DNA replication.
[0111] Target Tissue
[0112] 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 (Deining, 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.
[0113] Correction of the genetic defect in the airway epithelial
cells is likely to reverse the CF pulmonary phenotype. The identity
of the specific cells in the airway epithelium that express CFTR
cannot be accurately determined by immunocytochemical means,
because of the low abundance of protein. However, functional
studies suggest that the ciliated epithelial cells and perhaps
nonciliated cells of the surface epithelium are among the main cell
types involved in electrolyte transport. Thus, in practical terms,
the present preferred target cell for gene therapy would appear to
be the mature cells that line the pulmonary airways. These are 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.
[0114] 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.
[0115] Efficiency of Gene Delivery Required to Correct the Genetic
Defect
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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 immunoprecipitation or
immunoblotting and has been exceedingly difficult to detect with
immunocytochemical techniques (Denning, G. M. et al. (1992) J. Cell
Biol. 118:551). Although CFTR has been detected in some cases using
laser-scanning confocal microscopy, the signal is at the limits of
detection and cannot be detected above background in every case.
Despite that minimal levels of CFTR, this small amount is
sufficient to generate substantial cAMP-stimulated chloride
secretion. The reason that a very small number of CFTR chloride
channels can support a large chloride secretory rate is that a
large number of ions can pass through a single channel
(10.sup.6-10.sup.7 ions/sec) (Hille, B. (1984) Sinauer Assoc. Inc.,
Sunderland, Mass. 420426).
[0122] f. Previous studies using quantitative PCR have reported
that the airway epithelial cells contain at most one to two
transcripts per cell (Trapnell, B. C. et al. (1991) Proc. Natl.
Acad Sci. USA 88:6565).
[0123] 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-potassiumchloride cotransporter and potassium channel. serve
as important regulators of transeptihelial secretion (Welsh, M. J.
(1987) Physiol. Rev. 67:1143-1184).
[0124] 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) BiolTechnology 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).
[0125] 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
[0126] Nearly all of the commonly used DNA cloning vectors are
based on plasmids containing modified pMB1 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 fill
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 postulation, 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 most 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 plasmids constructed from it to replicate at 15-20
copies per host E. coli cell (Sambrook et al. Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press 1989). The
resultant plasmid clone was called pkk-4.5.
[0127] Partial CFTR clone T11 was cleaved with Eco R1 and Hinc 11
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 plasmid
Bluescript Sk- (Stratagene, catalogue number 212206), such that the
CFTR sequences were now flanked on the upstream (5') side by a 11
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-CFTR1 contains exons 1
through 24 of the CFTR cDNA. It was discovered that this plasmid is
stably maintained in E. coli cells and confers no measureably
disadvantageous growth characteristics upon host cells.
[0128] pkk-CFTR1 contains, between nucleotides 1716 and 1717, the
119 bp insert DNA derived from partial cDNA clone T164.5 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.
[0129] 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 1. 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-CFTR2Alternatively, pKK-CFTR2
could have been constructed using corresponding restriction
fragments from the partial CFTR cDNA clone C1-1/5. pKK-CFTR2
contains the uninterrupted CFTR protein coding sequence and
conferred slow growth upon E. coli host cells in which it was
inserted, whereas pKK-CFTR1 did not. The origin of replication of
pKK-CFTR2 is derived from pMB1 and confers a plasmid copy number of
15-20 copies per host cell.
Example 2
Improving Host Cell Viability
[0130] 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 Pyu 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 at
al.).
[0131] 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
fragment which was retained within the column. The column eluted
fractions from the pKK-CFTR2 digest and the pSC-3Z digest were
mixed and ligated to produce pSC-CFTR2. A map of this plasmid is
presented in FIG. 5. Host cells containing CFTR cDNAs at this and
similar gene dosages grow well and have stably maintained the
recombinant plasmid with the fill length CFTR coding sequence. In
addition, this plamid contains a bacteriophage T7 RNA polymerase
promoter adjacent to the CFTR coding sequence and is therefore
convenient for in vitro transcription/translation of the CFTR
protein. The nucleotide sequence of CFTR coding region from
pSC-CFTR2 plasmid is presented in Sequence Listing 1 as SEQ ID
NO:1. Significantly, this sequence differs from the previously
published (Riordan, J. R. et al. (1989) Science 245:1066-1073) CFTR
sequence at position 1990 where there is C in place of the reported
A. See Gregory, R. 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 Cells Viability
[0132] 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). 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.
[0133] T16-intron was then digested with Eco RI and Sma I and the
large fragment was isolated by agarose gel purification. T164.5 was
digested with Eco RI and Sca I and 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.
[0134] T-16-intron-2 was then cleaved with Xba I and Hpa I and the
1800 bp fragment was isolated by agarose gel purification.
pKK-CFTR1 was digested with Xba I and Hpa I and the large fragment
was also isolated by agarose gel purification and ligated with the
fragment derived from T16-intron-2 to yield pKK-CFTR3, shown in
FIG. 8. The CFTR cDNA within pKK-CFTR3 is identical to that within
pSC-CFTR2 and pKK-CFTR2 except for the insertion of the 83 bp
intron between nucleotides 1716 and 1717. The insertion of this
intron resulted in improved growth characteristics for cells
harboring pKK-CFTR3 relative to cells containing the unmodified
CFTR cDNA in pKK-CFTR2.
Example 4
In vitro Transcription/Translation
[0135] In addition to sequence analysis, the integrity of the CFTR
cDNA open reading frame was verified by in vitro
transcription/translation. This method also provided the initial
CFTR protein for identification purposes. 5 micrograms of pSC-CFTR2
plasmid DNA were linearized with Sal I and used to direct the
synthesis of CFTR RNA transcripts with T7 RNA polymerase as
described by the supplier (Stratagene). This transcript was
extracted with phenol and chloroform and precipitated with ethanol.
The transcript was resuspended in 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.35S-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.35S labelled proteins were detected by
fluorgraphy. A band of approximately 180 kD was detected,
consistent with translation of the fill length CFTR insert.
Example 5
Elimination of Crytic Regulatory Signals
[0136] 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 transcription and translation could also be advantageously
altered and/or deleted by the same method.
[0137] Futher analysis has identified a sequence extending from
nucleotide 908 to 936 which functions efficiently as a
transcriptional promoter element in E. coli (Gregory, R. J. et al.
(1990) Nature 347:382-386). Mutation at position 936 is capable of
inactivating this promoter and allowing the CFTR cDNA to be stably
maintained as a plasmid in E. coli (Cheng, S. H. et al. (1990) Cell
63:827-834). Specifically position 936 has been altered from a C to
a T residue without the amino acid sequence encoded by the cDNA
being altered. Other mutations within this regulatory element
described in Gregory, R. J. et al. (1990) 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 System
[0138] 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)
[0139] 1. DNA Preparation
[0140] Construction of the recombinant Ad/CFTR-1 virus (shown in
Table II and as SEQ ID NO:3) was accomplished as follows: The CFTR
cDNA was excised from the plasmid pCMV-CFTR-936C using restriction
enzymes 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.
[0141] 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 coning 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.
[0142] 2. Virus Preparation from DNA
[0143] To generate the recombinant Ad2/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
plasmid 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
supernatant was then used to infect 293 cells and upon delvelopment
of CPE, expression of CFTR was assayed by the protein kinase A
(PKA) immunoprecipitation assay (Gregory, R. J. et al. (1990)
Nature 347:382). Following these verification procedures, the virus
was further purified by two rounds of plaque purification.
[0144] 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.
[0145] 3. Virus Host Cell
[0146] 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.
[0147] 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.
[0148] 4. Growth of Production Lots of Virus
[0149] 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 or 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.
[0150] 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
(CE) 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.
[0151] 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:
[0152] 5. Structure and Purity of Virus
[0153] SDS polyacrylamide gel electrophoresis of purified virions
reveals a number of polypeptides, many of which have been
characterized. When preparations of virus were subjected to one or
two additional rounds of CsCl centrifigation, the protein profile
obtained was indistinguishable. This indicates that additional
equilibrium centrifigation 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.
[0154] 6. Contaminating Materials
[0155] The material to be administered to patients will 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 moleuclar
mass for adenovirus of 150.times.10.sup.6.
[0156] The origin of the materials from which a production lot of
the purified Ad2/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.
[0157] 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 AdCFTR-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 serum albumin (BSA), transferrin and IgG
heavy and light chain derived from the serum added to the growth
medium, Tests using such reagents to examine research batches of
Ad2/CFTR-1 by both ELISA and Western blots are in progress.
[0158] 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.
[0159] Nucleic acids and proteins would be expected to be separated
from purified virus preparations upon equilibrium density
centrifiugation. 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
[0160] a. Hamster Studies
[0161] 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.
[0162] 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
epithelium was observed at any time point or virus dose level.
Second, a time- and dose-dependent mild inflammatory response was
observed, being maximal at 48 hr in the high virus dose animals. By
seven days, the inflammatory response had completely resolved, such
that the lungs from animals in all treatment groups were
indistinguishable.
[0163] 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.
[0164] 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.
[0165] b. Primate Studies.
[0166] 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.
[0167] 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 e 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.
[0168] In the first pilot study the right nostril of Monkey A was
infected with Ad-o-Gal (.about.1 ml). This viral preparation was
purified by CsCl gradient centrifigation 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-p-Gal viruses were .about.2.times.10.sup.10
pfu/ml and >1.times.10.sup.13 pfu/ml, respectively, and both
preparations produced detectable .beta.-galactosidase activity in
293 cells.
[0169] 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.
[0170] 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 (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 (1.5 ml) and Monkey B received the crude virus
(6 ml). (note that this was the second exposure of Monkey A to the
recombinant adenovirus).
[0171] 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.
[0172] 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 cells/slide using a Cytospin
3 (Shandon, Pa.)) for subsequent staining (see below).
[0173] To determine viral efficacy, nasal, pharyngeal, and tracheal
cells were stained for .beta.-galactosidase using X-gal (5
bromo-4-chloro-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.
[0174] RT-PCR (reverse transcriptase-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 mRNA
from endogenous mRNA. PCR was also used to detect the presence of
the recombinant adenovirus DNA. Cytospin preparations was used to
assess for the presence of virally produced .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.
[0175] 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.
[0176] 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).
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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 night 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.
[0181] RT-PCR on day 3 post infection revealed a band of the
correct size that hybridized with a R-Gal probe, consistent with
(3-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.
[0182] 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.
[0183] c. Human Explant Studies
[0184] In a second type of experiment, epithelial cells from a
nasal polyp of a CF patient were cultured on permeable filter
supports. These cells form an electrically tight epithelial
monolayer after several days in culture. Eight days after seeding,
the cells were exposed to the Ad/CFTR virus for 6 hours. Three days
later, the short-circuit current (lsc) 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 pfu/cell) express functional CFTR; cAMP agonists
stimulated lsc, 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.
[0185] The experiments using primary cultures of human airway
epithelial cells indicate that the Ad2/CFTR virus is able to enter
CF airway epithelial cells and express sufficient CFTR to correct
the defect in chloride transport.
Example 9
In vivo Delivery to and Expression of CFTR in Cotton Rat and Rhesus
Monkey Epithelium
[0186] Materials and Methods
[0187] Adenovirus Vector.
[0188] Ad2/CFTR-1 was prepared as described in Example 7. The DNA
construct comprises a full length copy of the Ad2 genome of
approximately 37.5 kb from which the early region 1 genes
(nucleotides 546 to 3497) have been replaced by cDNA for CFTR
(nucleotides 123 to 4622 of the published CFTR sequence with 53
additional linker nucleotides). The viral 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.
[0189] Animals.
[0190] 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.
[0191] 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.
[0192] 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/CFTR-1 14 days later. Groups of one control rat and three
experimental rats were sacrificed at 3, 7, or 14 days after the
second administration of virus. Before necropsy, the trachea was
cannulated and brochoaveolar lavage (BAL) was performed with 3 ml
aliquots of phosphate-buffered saline. A median stemotomy was
performed and the right ventricle cannulated for blood collection.
The right lung and trachea were fixed in 4% formaldehyde and the
left lung was frozen in liquid nitrogen and kept at -70.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).
[0193] Monkeys. Three female Rhesus monkeys were used for this
study; a fourth female monkey was kept in the same room, and was
used as control. For application of the virus, the monkeys were
anesthetized by intramuscular injection of ketamine (15 mg/kg). The
entire epithelium of one nasal cavity in each monkey was used for
virus application. A foley 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.
[0194] The animals were evaluated I 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.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] Serology
[0199] 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
{fraction (1/50)}. The samples were incubated for 1 hour, the
plates washed, and a goat anti-human IgG HRP conjugate (Jackson
ImmunoResearch Laboratories, West Grove, Pa.) was added and
incubated for 1 hour. The plates were washed and O-Phenylenediamine
(Sigma Chemical Corp., St. Louis, Mo.) was added for 30 min. at
room temperature. The assay was stopped with 4.5 M H.sub.2SO.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>0.100.
[0200] 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
>95% cytopathic effect. The titer was calculated as the product
of the reciprocal of the initial dilution times the reciprocal of
the dilution in the last well showing >95% cytopathic
effect.
[0201] Bronchoalveolar Lavage and Nasal Brushings for Cytology.
[0202] 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/mil. 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.
[0203] Histology of Lung Sections and Nasal Biopsies.
[0204] 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.
[0205] Immunocytochemistry
[0206] 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, M14) (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.
[0207] PCR.
[0208] PCR was performed on pieces of small bowel, brain, heart,
kidney, liver, ovaries, and spleen from cotton rats. Approximately
I 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.
[0209] 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:
1 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)
[0210] The nested primer set amplifies a 506 bp fragment and is
shown below:
2 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)
[0211] 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 DNTP,
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.
[0212] 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.
[0213] RT-PCR.
[0214] 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%
sarcosvl. 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.
[0215] Southern Analysis.
[0216] 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.32P]-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.
[0217] Culture of Ad2/CFTR-1
[0218] Viral cultures were performed on the permissive 293 cell
line. For culture of virus from lung tissue, 1 g of lung was
frozen/thawed 3-6 times and then mechanically disrupted in 200
.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 p 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.
[0219] Results
[0220] Efficacy of Ad2/CFTR-1 in the Lungs of Cotton Rats
[0221] 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.l 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. FIG. 18 shows that the lungs of
animals which received Ad2/CFTR-1 were positive for virally-encoded
CFTR mRNA. The lungs of all control rats were negative.
[0222] To detect the protein, lung sections were immunostained with
antibodies specific to CFTR. FIG. 19 shows that 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).
[0223] 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.
[0224] Safety of Ad2/CFTR-1 in Cotton Rats.
[0225] Because the E1 region of Ad2 is deleted in the Ad2CFFR-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 4:461476). 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.
[0226] 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 inflammatory
response. To assess the pulmonary inflammatory response more
directly, bronchoalveolar lavage was performed on each of the rats.
FIG. 20Ashows that there was no change in the total number of cells
recovered from the lavage or in the differential cell count.
[0227] 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.
[0228] It seemed possible that the recombinant adenovirus could
escape from the lung into other tissues. To test for this
possibility, other organs from the rats were evaluated using nested
PCR to detect viral DNA. All organs tested from infected rats were
negative, with the exception of small bowel which was positive in 3
of 7 rats. FIG. 21 shows the results of 2 infected rats and one
control sacrificed on day 4 after infection. 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.
[0229] Repeat Administration of Ad2/CFTR-1 to Cotton Rats.
[0230] 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.
[0231] 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.
Examples of some sections are shown in FIG. 22. 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.
[0232] 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.
[0233] Evidence that Ad2/CFTR-1 Expresses CFTR in Primate Airway
Epithelum.
[0234] 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.
[0235] To assess gene transfer, RT-PCR was used as described above
for the cotton rats. RT-PCR was positive on cells brushed from the
right nostril of all three monkeys, although it was only detectable
for 18 days after virus administration. An example of the results
are shown in FIG. 23A. 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.
[0236] 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-detected CFTR transcript.
[0237] 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.
[0238] To assess the presence of CFTR proteins directly, cells
obtained by brushing were plated onto slides by cytospin and
stained with antibodies to CFTR. FIG. 24 shows an example of the
immunocytochemistry of the brushed cells. A positive reaction is
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.
[0239] 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 (FIG. 25). 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
immunostraining 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).
[0240] Safety of Ad2/CFTR-1 Administered to Monkeys.
[0241] FIG. 26 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. 26).
[0242] 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
administration, 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.
[0243] 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.
[0244] 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.
[0245] 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. (FIG.
27).
[0246] 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 of
humans with cystic fibrosis.
[0247] 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 consderations: 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).
[0248] This study also provides the first comprehensive data on the
safety of adenovirus vectors for gene transfer 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.
[0249] 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
[0250] Experimental Procedures
[0251] Adenovirus Vector.
[0252] The recombinant adenovirus AdCFTR-1 was used to deliver CFTR
cDNA. The construction and preparation of AdCFTR-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 transcripts. The E3
region of the virus was conserved.
[0253] Patients.
[0254] 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 >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 twice by FEV1, cytology of nasal mucosa, visual inspection,
and measurement of V.sub.t before treatment. Prior to treatment, a
coronal computed tomographic scan of the paranasal sinuses and a
chest X-ray were obtained.
[0255] 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.
[0256] Transepithelial Voltage.
[0257] 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.2PO.sub.2, K.sub.2HPO.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 (Keithley Instuments 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 V.sub.t was
greater than .+-.4 mV. The rubber catheter was introduced into the
nostril under telescopic guidance (Hoplons 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 V.sub.t was recorded until no changes in V.sub.t 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 V.sub.t 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 V.sub.t were recorded.
[0258] Measurements of basal V.sub.t 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 V.sub.t
(.DELTA.V.sub.t) ranged from 0 mV to +4 mV; hyperpolarization of
V.sub.t was never observed. In contrast, in 7 normal subjects
.DELTA.V.sub.t ranged from -1 mV to -5 mV; hyperpolarization was
always observed.
[0259] Ad2/CFTR-1 Application and Cell Acquisition.
[0260] 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. 28A-28I). 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 vital 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 V.sub.t
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.
[0261] 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 {fraction (1/100,000)}
epinephrine. The area of virus application on the inferior
turbinate was removed. The specimen was fixed in 4% formaldehyde
and stained.
[0262] Results
[0263] 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.
[0264] 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.
28A-28C). 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. 28G-28I).
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/vasoconstrict- ors
used before virus administration), but there were no abnormalities
suggestive of virus-mediated damage.
[0265] 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. 28E). 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. 28D-28F). 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. 29), 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.
[0266] 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 witht the normal course of disease in
each individual.
[0267] 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 (V.sub.t) 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. 30 shows an example from a normal subject. Under
basal conditions, V.sub.t was electrically negative (lumen
referenced to the submucosal surface). Perfusion of amiloride (100
.mu.M) onto the mucosal surface inhibited V.sub.t by blocking
apical Na.sup.+ channels (Knowles, M. et al (1981) N. Eng. J. Med.
305:1489-1495; Quinton, P. M. (1190) FASFB J. 4:2709-2717: Welsh,
M. T. (1992) Neuron 8:821-929). Subsequent perfusion of with
teroutaline (10 .mu.M) a .beta.-adrenergic agonist, hyperpolarized
V.sub.t 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. 31A shows results from seven normal subjects:
basal V.sub.t was -10.5.+-.1.0 mV, and in the presence of
amiloride, terbutaline hyperpolarized V.sub.t by -2.3.+-.0.5
mV.
[0268] In patients with CF, V.sub.t was more electrically negative
than in normal subjects (FIG. 31B), as has been previously reported
(Knowles, M. et al. (1981) N. Eng. J. Med. 305:1489-1495). Basal
V.sub.t was -37.0.+-.2.4 mV, much more negative than values in
normal subjects (P<0.001). (Note the difference in scale in FIG.
31A and FIG. 31B). Amiloride inhibited V.sub.t, as it did in normal
subjects. However, V.sub.t failed to hyperpolarize when terbutaline
was perfused onto the epithelium in the presence of amiloride.
Instead, V.sub.t either did not change or became less negative: on
average V.sub.t depolarized by +1.8.+-.0.6 mV, a result very
different from that observed in normal subjects. (P<0.001).
[0269] After Ad2/CFTR-1 was applied, basal V.sub.t became less
negative in all three CF patients: FIG. 32A shows an example from
the third patient before (FIG. 32A) and after (FIG. 32B) treatment
and FIGS. 33A, 33C, and 33E show the time course of changes in
basal V.sub.t for all three patients The decrease in basal V.sub.t
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. 32B shows that in contrast to the response before
Ad2/CFTR-1 was applied, after virus replication, in the presence of
amiloride, terbutaline stimulated V.sub.t. FIGS. 33B, 33D, and 33F
show the time course of the response. These data indicate that
Ad2/CFTR-1 corrected the CF defect in Cl transport. Correction of
the Cl 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. 34).
Moreover, the effects of the anesthesia were generalized on the
nasal mucosa, but basal V.sub.t decreased only in the area of virus
administration. Finally, similar changes were observed in the left
nasal mucosa of the third patient (FIGS. 33E and 33F), which had no
symptomatic or physical response after the modified application
procedure.
[0270] Unsuccessful attempts were made to detect CFTR transcripts
by reverse transciptase-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 AdCFTR-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
delete 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.
[0271] With time, the electrical changes that indicate correction
of the CF defect reverted toward pretreatment values. However, the
basal V.sub.t appeared to revert more slowly than did the change in
V.sub.t 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 V.sub.t to zero for at
least two days afterwards, thus preventing an accurate assessment
of duration of the effect of Ad2/CFTR-1.
[0272] Efficacy of Adenovirus-mediated Gene Transfer.
[0273] 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 transport that is characteristic
of CF epithelia.
[0274] 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.
[0275] 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 4:461476 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.
[0276] 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).
[0277] 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 transcibe CFTR, the inability to detect
CFTR protein and CFTR mRNA 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 transciptase reaction may have been suboptimal. This could
have occurred because of factors in the tissue that inhibit the
reverse transcriptase. Although there is little doubt that the
changes in electrolyte transport measured here result from
expression of CFTR, it remains to be seen whether this will lead to
measurable clinical changes in lung function.
[0278] Safety Considerations.
[0279] 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.
[0280] 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 Immunological 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)
[0281] With reference to FIG. 17, 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.
[0282] 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 (UP) and tripartite
leader (TPL) DNA and an SV40 T-antigen nuclear localization signal
(NLS) and polyadenylation signal (SVpA). As can be seen in FIG. 17,
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.
[0283] Generation of PAV infectious particles was performed by
excision of PAV from the plasmid with the Aga I and k II
restriction endonucleases and co-transfection into 293 cells (an
E1a/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.
[0284] 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.
[0285] 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.
[0286] 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 fill-length Ad genome but not genomes <95%
required the presence of the adenoviral gene designated pIX.
[0287] 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 fill 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 full 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 capstu
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
[0288] 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. 16, 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 Pac I and ligated to Ad2 DNA digested with Pac
I. This Pac I 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 Same was ORF6.
[0289] 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
[0290] An adenoviral vector is prepared as described in Example 7
while substituting the PGK promoter for the E1a promoter.
Example 14
[0291] 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-ORE6/PGK-CFTR
[0292] This protocol uses a second generation adenovirus vector
named Ad2-ORF6/PGK-CFTR. This virus lacks E1 and in its place
contains a modified transcription 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.
[0293] 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 fame 6 (ORF6) of the
Ad2 E4 region. The Adenovirus vector is referred to as
AD2-ORF6/PGK-CFTR and is illustrated schematically in FIG. 35. 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.
[0294] 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, 4:461467; and
Zabner et al. (1993) Nature Genetics, In Press).
[0295] 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. 35.
[0296] 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. 35. 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. See Table III for the sequence of
Ad2-ORF6/PGK-CFTR.
[0297] 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, 32:409417) at nucleotides
828, 829 and 837 of the recombinant vector (Singer-Sam et al.
(1984) Gene, 32:409417). 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.
[0298] 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.
[0299] Regulatory Characteristics of the Elements of the
AD2-ORF6/PGK-CFTR
[0300] 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.
[0301] PGK Promoter
[0302] 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 "shutdown" 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).
[0303] Polyadenylation Signal
[0304] 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 Ad2/CMV .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.
[0305] Alterations of the E4 Region
[0306] 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.
[0307] 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.
[0308] 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).
[0309] Replication Origin
[0310] 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.
[0311] Steps Used to Derive the DNA Construct
[0312] 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.
[0313] 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 1234622 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.
[0314] 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.
[0315] In vitro Studies With Ad2-ORF6/PGK-CFTR
[0316] 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
immunoprecipitation and by functional assays of halide efflux.
Gregory, R. J. et al. (1990) Nature 347:382-386; Cheng, S. H. et
al. (1990) Cell 63:827-834. 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 IBM (100 .mu.m) increased SPQ
fluorescence indicating the presence of functional chloride
channels produced by the vector.
[0317] 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. 36 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 addied to the mucosal
solution.
[0318] In vivo Studies With Ad2-ORF6/PGK-CFTR
[0319] Virus Preparation
[0320] 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.
[0321] Animals
[0322] 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 fee 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.
[0323] Virus Administration
[0324] 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.
[0325] 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.
[0326] Timing of Evaluations.
[0327] 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.
[0328] 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.
[0329] Animal Evaluation.
[0330] 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.
[0331] Hematology and Serum Chemistry
[0332] 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.
[0333] Serology
[0334] 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, Ca) 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 {fraction (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.2SO.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>0.100.
Nasal washings from the monkeys were obtained and antiadenoviral
antibody titers were measured by ELISA, starting at a dilution of
1/4.
[0335] Nasal Washings.
[0336] 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.
[0337] Cytology
[0338] 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 106 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.
[0339] Culture for Ad2-ORF6/PFK-CFTRB
[0340] 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 (Chericon,
Light Diagnostics, Temecuca, Calif.) for 30 min. Positive nuclear
immunofluorescence was interpreted as positive culture.
[0341] Immunocytochemistry for the Detection of CFTR.
[0342] Cells were obtained by brushing. Eighty ill 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, M14, and M64) (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 microscope.
[0343] Biopsies
[0344] 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.
[0345] Results
[0346] Studies of Efficacy.
[0347] 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. FIGS. 37-39
show examples from each monkey.
[0348] Studies of Safety
[0349] 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 FIG. 40.
[0350] The presence of live virus was tested in the supernatant of
cell suspensions from swabs and brushes from each nostril and the
pharynx. Each supernatant was used to infect the virus-sensitive
293 cell line. Live virus was never detected at any of the time
points The rapid loss of live virus suggests that there was no
viral replication.
[0351] The results of complete blood counts, sedimentation rate,
and clinical chemistries are shown in FIGS. 42A-41C. There was no
evidence of a systemic inflammatory response or other abnormalities
of the clinical chemistries.
[0352] 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 FIG. 42. 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-ORF6/PGK-CFTR
administration were reviewed by an independent pathologist, who
found no evidence of inflammation or any other cytopathic effects.
FIGS. 43 to 45 show an example from each monkey.
[0353] FIG. 46 shows 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.
[0354] These results combined with demonstrate the ability of a
recombinant adenovirus encoding CFTR (Ad2-ORF6/PGK-CFTR) to express
CFTR cDNA in the airway epithelium of monkeys. These monkeys have
been followed clinically for 12 months after the first viral
administration and no complications have been observed.
[0355] 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.
[0356] 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.
[0357] Equivalents
[0358] 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 CWU 1
1
* * * * *