Note: Descriptions are shown in the official language in which they were submitted.
CA 02271826 1999-OS-11
BACKGROUND OF THE INVENTION
I. Field of the Invention
The present invention relates to the fields of virology, cellular and
molecular biology.
More particularly, the invention relates to the development of a method for
increasing the
susceptibility of epithelial cells to infection by viruses and viral vectors,
including viral vectors
used in the gene therapy. Thus, the invention also relates to the delivery of
therapeutic genes to
diseased tissues.
II. Related Art
Numerous diseases exist which are the result of congenital and acquired
genetic defects.
Diseases resulting from congenital inherited defects include cystic fibrosis
(CF) and various
other genetic deficiencies. CF is a common, recessive disease characterized by
decreased
chloride ion permeability in epithelial tissues (Quinton, 1990). While several
tissues are affected
by the disease, it is chronic lung disease that causes 95% of the mortality
associated with CF
(Welsh et al., 1995). The CF gene product has been identified (Tsui et al.,
1989) and is called
cystic fibrosis transmembrane conductance regulator (CFTR). Over 700 different
mutations of
CFTR have been associated with clinical disease. Studies have established that
transfer of the
wild-type CFTR cDNA into CF epithelia corrects the characteristic CF defect in
chloride ion
secretion (Rich et al., 1990; Drumm et al., 1990).
Another important genetic disease is cancer. Cancer is usually the result of
an
accumulation of genetic damage, most of which is acquired, but some of which
may be the result
of congenital genetic defects. As described by Foulds (1958), cancer is
usually the product of a
multistep biological process, which is presently known to occur by the
accumulation of genetic
damage. On a molecular level, the multistep process of tumorigenesis involves
the disruption of
both positive and negative regulatory effectors (Weinberg, 1989). The
molecular basis for
human colon carcinomas has been postulated by Vogelstein and coworkers (1990)
to involve a
number of oncogenes, tumor suppressor genes and repair genes. Similarly,
defects leading to the
development of retinoblastoma have been linked to another tumor suppressor
gene (Lee et al.,
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CA 02271826 1999-OS-11
1987). Still other oncogenes and tumor suppressors have been identified in a
variety of other
malignancies.
The development of effective gene therapies therefore is critical to the
treatment of
chronic and progressive diseases resulting from genetic defects. Gene transfer
to epithelial cells
in particular would be required for treatment of numerous diseases caused by
genetic defects
effecting epithelial tissue. Examples of such diseases include lung cancer,
tracheal cancer,
asthma, surfactant protein B deficiency, alpha-1-antitrypsin deficiency and
cystic fibrosis.
However, transfer of foreign DNAs into human cells in vivo has proved to be a
challenging undertaking. Various viral vectors have been designed for use in
gene therapy in
order to deliver foreign DNA to human tissues, including retrovirus (both
marine virus and
lenitvirus), adenovirus, papillorna virus, herpesvirus, parvovirus and
poxivirus. All of these
vectors have been successful, but there remain various obstacles that limit
the efficacy of these
vectors. One of the most serious obstacles to be overcome in gene therapy is
low cellular viral
infection rates, and therefore low gene transfer efficiency, particularly in
non-dividing cells.
Thus, there remains a need to improve the efficiency of infection of target
cells, in the
context of gene therapy, by various viral vectors. With the current interest
in gene therapy, the
need for improving the existing gene therapy vectors is greater than ever.
SUMMARY OF THE INVENTION
Therefore, it is an objective of the present invention to provide a method
increasing gene
transfer efficiency to epithelial tissue when using viruses and viral vectors.
It is also an objective
to provide a means of targeting gene transfer to all the cells in the
epithelial sheet, including
basal cells. It also is an objective to provides compositions for use in these
methods.
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CA 02271826 1999-OS-11
r n
In accordance with the foregoing objectives, there is provided, in one
embodiment, a
method of increasing the susceptibility of epithelial cells to viral infection
by increasing the
transepithelial permeability. T'he epithelial cells may of any epithelial
tissue type but, in
particular embodiments is airway epithelial (issue, most particularly airway
epithelial tissue
selected from the group of tracheal, bronchial, bronchiolar and alveolar
tissue.
In another embodiment the susceptibility of epithelial cells to viral
infection by
increasing the transepithelial permeability may be further modified by
increasing the
proliferation of the epithelial cells by eorrtactiag them with a proliferative
factor. Any
proliferative factor may be used, but in a particular embodiment the
proliferative factor is a
growth factor. In further embodiments, the proliferative factor may be
delivered as an aerosol or
as a topical solution.
In a further embodiment method of increasing the susceptibility of epithelial
cells to viral
infection by increasing the transepithelial permeability of epithelial tissue,
the increase in
transepithelial permeability is achieved by contacting the epithelial tissue
with a tissue
permeabilizing agent. Any tissue permeabilizing agent may be used, but in
specific
embodiments, the tissue permeabilizing agent is selected from a group
including hypotonic
solutions, ion chelators, cationic peptides, occludin peptides, cytoskeletal
disruption agents,
ether, neurotransmitters, FCCP, oxidants, and mediators of inflammation. In
further specific
embodiments, the ion chelator may be EGTA, BAPTA or EDTA; the cationic peptide
may be
poly-L-lysine; the c~-toskeletal disruption agent may be cytochalasin B or
colchicine; the
neurotransmitter may be capsianoside; the oxidant may be hydrogen peroxide or
ozone; and the
mediator of inflammation may be TNFa. Finally, in yet another embodiment, the
tissue
permeabilizing agent may be delivered as an aerosol or as a topical solution.
Yet another embodiment provides a method of increasing the susceptibility of
epithelial
cells to viral infection by increasing the transepithelial permeability,
further comprising infecting
the epithelial tissue with a virus vector selected from the group including
virus from the virus
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CA 02271826 1999-OS-11
families retrovirus, adenovirus, parvovirus, papovavirus and paramyxovirus,
from the virus
genera lentivirus and adeno-associated virus, and the vaccinia virus. This
embodiment is further
modified in still further embodiments wherein the viral vector contains a non-
viral gene under
the control of a promoter active in eukaryotic cells. Any non-viral gene may
be used, but in a
particular embodiment the non-viral gene is a human gene, and in yet another
embodiment the
human gene encodes a polypeptide selected from the group consisting of a tumor
suppressor, a
cytokine, an enzyme, a toxin, a growth factor, a membrane channel, an inducer
of apoptosis, a
transcription factor, a hormone and a single chain antibody. In another
embodiment the virus
vector may be a replication-defective virus, and in a further embodiment the
replication-defective
virus is a retroviral vector.
In still another embodiment there is provided a method of increasing the
susceptibility of
epithelial cells to viral infection by increasing the transepithelial
permeability wherein the
epithelial tissue is diseased. In a fiu~ther embodiment the disease of the
epithelial tissue may be
lung cancer, tracheal cancer, asthma, surfactant protein B deficiency, alpha-1-
antitrypsin
deficiency or cystic fibrosis.
As a further embodiment, the invention provides a composition comprising both
a tissue
permeabilizing agent and a cell proliferative factor suitable for aerosol
application, and in
another embodiment, suitable for topical application. Any tissue
permeabilizing agent may be
used in either composition, but in a further embodiment the tissue
permeabilizing agent of the
composition is selected from the group of a hypotonic solution, a cytokine, a
cationic peptide, a
cytoskeletal disruptor, a mediator of inflammation, an oxidant, a
neurotransmitter or an ion
chelator. It is understood that any proliferative factor can be used in the
aforementioned
compositions. An additional embodiment of the compositions further comprises a
packaged viral
vector. The packaged viral vector in other embodiments comprises a non-viral
gene or is a
retroviral vector.
The invention also provides a method for redistributing the viral receptors or
enhancing
accessibility of viral receptors on epithelial cells of an epithelial tissue
by increasing the
CA 02271826 1999-OS-11
transepithelial permeability of the epithelial tissue. Any viral receptor may
be redistributed, but
in a another embodiment the viral receptor is a retroviral receptor.
The invention provides a further embodiment which is a method for expressing a
polypeptide in cells of an epithelial tissue comprising the steps of (a)
providing a packaged viral
vector comprising a polynucleotide encoding said polypeptide; (b) increasing
the permeability of
said epithelial tissue; and (c) contacting cells of the epithelial tissue with
the packaged viral
vector under conditions permitting the uptake of the packaged viral vector by
the cells and
expression of said polypeptide therein. Other embodiments of this method
further comprises
increasing the proliferation of cells in the epithelial tissue or further
comprises a viral vector
which is a retroviral vector.
Also, the invention provides a method for treating epithelial tissue disease
comprising the
steps of (a) providing a packaged viral vector comprising a polynucleotide
encoding the
therapeutic polypeptide; (b) increasing the permeability of the diseased
epithelial tissue; and (c)
contacting cells of the epithelial tissue with the packaged viral vector under
conditions permitting
the uptake of the packaged viral vector by the cells and expression of the
therapeutic polypeptide
therein, whereby expression of the therapeutic polypeptide treats the disease.
A further
embodiment of the method comprises increasing the proliferation of the cells
of the diseased
epithelial tissue. Any means of increasing the proliferation of the cells of
the diseased epithelial
tissue may be used but a in further embodiment the means of increasing the
proliferation is
contacting the epithelial cells with a proliferative agent. Another further
embodiment of the
method comprises a method in which the diseased epithelial tissue being
treated is airway tissue.
Any airway tissue may be treated, but a further embodiment treats airway
tissue selected from
the group of alveolar tissue, brochiolar tissue, bronchial tissue and tracheal
tissue. A fiuther
embodiment of the method is one which comprises the treatment of epithelial
tissue disease
wherein the disease is cancer. Any cancer may be treated but further
embodiments are directed
to the treatment of lung cancer or tracheal cancer.
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CA 02271826 1999-OS-11
In still another embodiment of the method, the epithelial tissue disease being
treated is an
inherited genetic defect. The invention is directed to any inherited genetic
defect, but further
embodiments are specifically directed to the inherited genetic defects
surfactant protein B
deficiency, alpha-1-antitrypsin deficiency or cystic fibrosis. The method for
treating an epithelial
tissue disease has a further embodiment wherein the method further comprises
the use of a
therapeutic polypeptide selected from the group consisting of a tumor
suppressor, a cytokine, an
enzyme, a toxin, a growth factor, a membrane channel, an inducer of apoptosis,
a transcription
factor, a hormone and a single chain antibody. Another embodiment of the
method for treating
epithelial tissue disease is one in which the step of increasing the
permeability of the diseased
epithelial tissue comprises contacting cells of said diseased epithelial
tissue with a tissue
permeabilizing agent. Finally, the method of treating an epithelial tissue
disease has an
embodiment in which the viral vector used is specifically a retroviral vector.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The development of gene therapy methods has long been a goal of medical
research.
Since 1990, numerous gene therapy trials have been attempted in humans and
have shown that
gene therapy is a safe and potentially efficacious treatment for genetic
disorders. Initially, there
were numerous difficulties to overcome in the development of effective gene
therapy methods.
These difficulties included the identification of the genetic defects
responsible for various
illnesses, the isolation of functional copies of these genes and the design
and delivery of vehicles
to transport functional copies of the defective genes into diseased tissues.
Currently the genetic
defects responsible for or contributing to many illnesses are known and
functional copies of the
genes have been isolated.
Genetically engineered viruses have been designed that are capable of
delivering
therapeutic genes to various target tissues. One ongoing difficulty is the
efficient delivery of
therapeutic genes to target tissues. While some vectors may have high levels
of infectivity in
vitro, conditions in vivo may be altered such that the vectors have much lower
rates of infectivity.
For many disease states, it is important that high levels of transgene
expression, be achieved.
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CA 02271826 1999-OS-11
Even where highly active promoters are used, and transgene product turnover is
low, the inability
to infect target cells with high efficiency is highly limiting.
The present invention is designed to overcome these deficiencies by providing
methods
for increasing the susceptibility of epithelial cells to viral infection
comprising increasing the
transepithelial permeability of epithelial tissue. Treatment with tissue
permeablizing agents such
as hypotonic shock or EGTA increases transepithelial permeability and enhances
gene transfer
by viral vectors applied to the mucosal surface of epithelial tissue. Using
this approach, cells
throughout the epithelial sheet, including basal cells, are targeted. It was
shown that, using this
approach, it was possible to correct the Cl- transport defect in
differentiated CF airway epithelia
in vitro.
An additional enhancement of gene transfer efficiency in the invention can be
achieved
by stimulating division of epithelial cells by increasing the proliferation of
said epithelial cells by
contacting the cells with a cell proliferative factor. Recent studies by the
inventors and by others
have identified epithelial specific growth factors which stimulate
proliferation in vivo without
prior injury. Keratinocyte growth factor (KGF) stimulates proliferation of
epithelia in multiple
organs including the bronchial and alveolar cells of the lung (Ulich, et al.,
1994; Housley et al.,
1994). Hepatocyte growth factor (HGF) also is a potent in vivo mitogen for
proliferation in
pulmonary epithelia (Mason et al., 1994; Ohmichi et al., 1996). In vivo, KGF
appears to
stimulate only 1 to 2 cycles of cell division and is not mutagenic (Ulich, et
al., 1994; Housley et
al., 1994). The inventors' in vivo data shows that KGF and HGF stimulate
epithelial
proliferation in the lungs and liver of rodents (Bosch et al., 1996; Bosch et
al., 1998).
A. TARGET TISSUES
The present invention is designed to increase the susceptibility of epithelial
cells to viral
infection. Epithelial tissue includes skin, the lining of the gastrointestinal
tract and the lining of
the airway and lungs. The airway and lungs include the nasal passages, the
oral cavity, the upper
part of the pharynx (throat), the larynx (voice box), the trachea (windpipe),
bronchi, bronchioles
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CA 02271826 1999-OS-11
uroepithelium of the kidneys and bladder, mammary epithelia, lining of brain
ventricles.
leptomenengis, and the alveoli of the lungs.
B. THERAPEUTIC GENES AND DISEASE STATES
Gene therapy has become an increasingly viable endeavor in the past decade
because for
the mere reason that genetic defects responsible for numerous genetic diseases
have been
identified. Such genes include cytokines, hormones, transporters, enzymes and
receptors.
Examples include the genes responsible for cystic fibrosis (CF), surfactant
protein B deficiency
and alpha-1-antitrypsin deficiency. Additionally, various antisense oncogene
constructs, tumor
suppressor genes, inducers of apoptosis, repair genes and toxins have been
identified as potential
therapeutics in various cancers. A list of potential therapeutic genes is set
forth below.
I. Tumor Suppressors
p53 currently is recognized as a tumor suppressor gene. High levels of mutant
p53 have
been found in many cells transformed by chemical carcinogenesis, ultraviolet
radiation, and
several viruses. The p53 gene is a frequent target of mutational inactivation
in a wide variety of
human tumors and is already documented to be the most frequently-mutated gene
in common
human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al.,
1991) and in a
wide spectrum of other tumors.
The p53 gene encodes a 393-amino acid phosphoprotein that can form complexes
with
host proteins such as SV40 large-T antigen and adenoviral E1B. The protein is
found in normal
tissues and cells, but at concentrations which are minute by comparison with
transformed cells or
tumor tissue. Interestingly, wild-type p53 appears to be important in
regulating cell growth and
division. Overexpression of wild-type p53 has been shown in some cases to be
anti-proliferative
in human tumor cell lines. Thus, p53 can act as a negative regulator of cell
growth (Weinberg,
1991 ) and may directly suppress uncontrolled cell growth or indirectly
activate genes that
suppress this growth. Thus, absence or inactivation of wild-type p53 may
contribute to
transformation. However, some studies indicate that the presence of mutant p53
may be
necessary for full expression of the transfonming potential of the gene.
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CA 02271826 1999-OS-11
Wild-type p53 is recognized as an important growth regulator in many cell
types.
Missense mutations are common for the p53 gene and are essential for the
transforming ability of
the oncogene. A single genetic change prompted by point mutations can create
carcinogenic
p53, in as much as mutations in p53 are known to abrogate the tumor suppressor
capability of
wild-type p53. Unlike other oncogenes, however, p53 point mutations are known
to occur in at
least 30 distinct codons, often creating dominant alleles that produce shifts
in cell phenotype
without a reduction to homozygosity. Additionally, many of these dominant
negative alleles
appear to be tolerated in the organism and passed on in the germ line. Various
mutant alleles
appear to range from minimally dysfunctional to strongly penetrant, dominant
negative alleles
(Weinberg, 1991).
Casey and colleagues have reported that transfection of DNA encoding wild-type
p53
into two human breast cancer cell lines restores growth suppression control in
such cells (Casey
et al., 1991). A similar effect has also been demonstrated on transfection of
wild-type, but not
mutant, p53 into human lung cancer cell lines (Takahasi et al., 1992). p53
appears dominant
over the mutant gene and will select against proliferation when transfected
into cells with the
mutant gene. Normal expression of the transfected p53 does not affect the
growth of normal or
non-malignant cells with endogenous p53. Thus, such constructs might be taken
up by normal
cells without adverse effects. It is thus proposed that the treatment of p53-
associated cancers
with wild-type p~3 will reduce the number of malignant cells or their growth
rate.
The major transitions of the eukaryotic cell cycle are triggered by cyclin-
dependent
kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4), regulates
progression through
the G,. The activity of this enzyme may be to phosphorylate Rb at late G,. The
activity of
CDK4 is controlled by an activating subunit, D-type cyclin, and by an
inhibitory subunit pl6~Ka.
The p 16~~' has been biochemically characterized as a protein that
specifically binds to and
inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al., 1993;
Serrano et al.,
1995). Since the p16~K4 protein is a CDK4 inhibitor (Serrano, 1993), deletion
of this gene may
CA 02271826 1999-OS-11
increase the activity of CDK4, resulting in hyperphosphorylation of the Rb
protein. p16 also is
known to regulate the function of CDK6.
p 16~''~'4 belongs to a newly described class of CDK-inhibitory proteins that
also includes
p15 ~~48, p2lW'~i, and p27~P1. The p16~K4 gene maps to 9p21, a chromosome
region
frequently deleted in many tumor types. Homozygous deletions and mutations of
the p16°~~'4
gene are frequent in human tumor cell lines. This evidence suggests that the
p16~K4 gene is a
tumor suppressor gene. This interpretation has been challenged, however, by
the observation
that the frequency of the p 164 gene alterations is much lower in primary
uncultured tumors
than in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;
Hussussian et al., 1994; Kamb
et al., 1994; Kamb et al., 1994; Mori et al., 1994; Okamoto et al., 1994;
Nobori et al., 1995;
Orlow et al., 1994; Arap et al., 1995). However, it was later shown that while
the p16 gene was
intact in many primary tumors, there were other mechanisms that prevented p 16
protein
expression in a large percentage of some tumor types. p16 promoter
hypermethylation is one of
these mechanisms (Merlo et al., 1995; Herman, 1995; Gonzalez-Zulueta, 1995).
Restoration of
wild-type p16~4 function by transfection with a plasmid expression vector
reduced colony
formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).
Delivery of p16 with
adenovirus vectors inhibits production of some human cancer lines and reduces
the growth of
human tumor xenografts
C-CAM is expressed in virtually all epithelial cells (Odin and Obrink, 1987).
C-CAM,
with an apparent molecular weight of 105 kD, was originally isolated from the
plasma membrane
of the rat hepatocyte by its reaction with specific antibodies that neutralize
cell aggregation
(Obrink, 1991 ). Recent studies indicate that, structurally, C-CAM belongs to
the
immunoglobulin (Ig) superfamily and its sequence is highly homologous to
carcinoembryonic
antigen (CEA) (Lin and Guidotti, 1989). Using a baculovirus expression system,
Cheung et al.
(1993) demonstrated that the first Ig domain of C-CAM is critical for cell
adhesive activity.
Cell adhesion molecules, or CAM's are known to be involved in a complex
network of
molecular interactions that regulate organ development and cell
differentiation (Edelman, 1985).
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CA 02271826 1999-OS-11
Recent data indicate that aberrant expression of CAM's maybe involved in the
tumorigenesis of
several neoplasms; for example, decreased expression of E-cadherin, which is
predominantly
expressed in epithelial cells, is associated with the progression of several
kinds of neoplasms
(Edelman and Crossin, 1991; Frixen et al., 1991; Bussemakers et al., 1992;
Matsura et al., 1992;
Umbas et al., 1992). Also, Giancotti and Ruoslahti (1990) demonstrated that
increasing
expression of as/3i integrin by gene transfer can reduce tumorigenicity of
Chinese hamster ovary
cells in vivo. C-CAM now has been shown to suppress tumor growth in vitro and
in vivo.
Other tumor suppressors that may be ee~phyed acEOrding to the present
invention include
p21, p15, BRCA1, BRCA2, IRF-1, PTEN, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I,
MEN-
II, zacl, p73, VHL, FCC and MCC.
II. Inducers ofApoptosis
Inducers of apoptosis, such as Bax, Bak, Bcl-Xs, Bad, Bim, Bik, Bid, Harakiri,
Ad E1B,
Bad and ICE-CED3 proteases, similarly could find use according to the present
invention,
particularly in the treatment of cancers.
111. Enrymes
Various enzyme genes are of interest according to the present invention. Such
enzymes
include human copper zinc superoxide dismutase (U.S. Patent No. 5,196,335),
cytosine
deaminase, adenosine deaminase, hypoxanthine-guanine
phosphoribosyltransferase, galactose-1-
phosphate uridyltransferase, phenylalanine hydroxylase, glucocerbrosidase,
sphingomyelinase,
a-L-iduronidase, glucose-6-phosphate dehydrogenase, (3-glucuronidase, HSV
thymidine kinase
and human thymidine kinase and extracellular proteins such as collagenase and
matrix
metalloprotease.
IY. Cytokines
Another class of genes that is contemplated to be inserted into the retroviral
vectors of the
present invention include interleukins and cytokines. Interleukin 1 (IL-1), IL-
2. IL-3, IL-4, IL-5,
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CA 02271826 1999-OS-11
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, (3-
interferon, a-interferon, y-
interferon, angiostatin, thrombospondin, endostatin, METH-I, METH-2, GM-CSF, G-
CSF, M-
CSF and tumor necrosis factor.
V. Toxins
Various toxins are also contemplated to be useful as part of the expression
vectors of the
present invention, these toxins include bacterial toxins such as ricin A-chain
(Burbage, 1997),
diphtheria toxin A (Massuda et al., 1997; Lidor, 1997), pertussis toxin A
subunit, E. coli
enterotoxin toxin A subunit, cholera toxin A subunit and Pseudomonas toxin C-
terminal.
Recently, it was demonstrated that transfection of a plasmid containing the
fusion protein
regulatable diphtheria toxin A chain gene was cytotoxic for cancer cells.
Thus, gene transfer of
regulated toxin genes might also be applied to the treatment of cancers
(Massuda et al., 1997).
YI. Antisense Constructs
Antisense methodology takes advantage of the fact that nucleic acids tend to
pair with
"complementary" sequences. By complementary, it is meant that polynucleotides
are those
which are capable of base-pairing according to the standard Watson-Crick
complementarity
rules. That is, the larger purines will base pair with the smaller pyrimidines
to form
combinations of guanine paired with cytosine (G:C) and adenine paired with
either thymine
(A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of
RNA. Inclusion of
less common bases such as inosine, 5-methylcytosine, 6-methyladenine,
hypoxanthine and others
in hybridizing sequences does not interfere with pairing. As part of the
present invention,
particular interest will be paid to the delivery of antisense oncogenes.
Particular oncogenes that
are targets for antisense constructs are ras, myc, neu, raf, erb, src, fms,
jun, trk, ret, hst, gsp, bcl-2
and abl.
Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix
formation;
targeting RNA will lead to double-helix formation. Antisense polynucleotides,
when introduced
into a target cell, specifically bind to their target polynucleotide and
interfere with transcription,
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CA 02271826 1999-OS-11
RNA processing, transport, translation and/or stability. Antisense RNA
constructs, or DNA
encoding such antisense RNA's, may be employed to inhibit gene transcription
or translation or
both within a host cell, either in vitro or in vivo, such as within a host
animal, including a human
subj ect.
Antisense constructs may be designed to bind to the promoter and other control
regions,
exons, introns or even exon-intron boundaries of a gene. It is contemplated
that the most
effective antisense constructs will include regions complementary to
intron/exon splice junctions.
Thus, it is proposed that a preferred embodiment includes an antisense
construct with
complementarity to regions within 50-200 bases of an intron-exon splice
junction. It has been
observed that some exon sequences can be included in the construct without
seriously affecting
the target selectivity thereof. The amount of exonic material included will
vary depending on the
particular exon and intron sequences used. One can readily test whether too
much exon DNA is
included simply by testing the constructs in vitro to determine whether normal
cellular function
is affected or whether the expression of related genes having complementary
sequences is
affected.
As stated above, "complementary" or "antisense" means polynucleotide sequences
that
are substantially complementary over their entire length and have very few
base mismatches.
For example, sequences of fifteen bases in length may be termed complementary
when they have
complementary nucleotides at thirteen or fourteen positions. Naturally,
sequences which are
completely complementary will be sequences which are entirely complementary
throughout their
entire length and have no base mismatches. Other sequences with lower degrees
of homology
also are contemplated. For example, an antisense construct which has limited
regions of high
homology, but also contains a non-homologous region (e.g., ribozyme; see
below) could be
designed. These molecules, though having less than 50% homology, would bind to
target
sequences under appropriate conditions.
It may be advantageous to combine portions of genomic DNA with cDNA or
synthetic
sequences to generate specific constructs. For example, where an intron is
desired in the ultimate
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CA 02271826 1999-OS-11
construct, a genomic clone will need to be used. The cDNA or a synthesized
polynucleotide may
provide more convenient restriction sites for the remaining portion of the
construct and,
therefore, would be used for the rest of the sequence.
VII. Ribozymes
Although proteins traditionally have been used for catalysis of nucleic acids,
another
class of macromolecules has emerged as useful in this endeavor. Ribozymes are
RNA-protein
complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have
specific catalytic
domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et
al., 1987; Forster
and Symons, 1987). For example, a large number of ribozymes accelerate
phosphoester transfer
reactions with a high degree of specificity, often cleaving only one of
several phosphoesters in an
oligonucleotide substrate (Cook et al., 1981; Michel and Westhof, 1990;
Reinhold-Hurek and
Shub, 1992). This specificity has been attributed to the requirement that the
substrate bind via
specific base-pairing interactions to the internal guide sequence ("IGS") of
the ribozyme prior to
chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific
cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al.,
1981). For
example, U.S. Patent 5,354,855 reports that certain ribozymes can act as
endonucleases with a
sequence specificity greater than that of known ribonucleases and approaching
that of the DNA
restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of
gene expression
may be particularly suited to therapeutic applications (Scanlon et al., 1991;
Sarver et al., 1990).
Recently, it was reported that ribozymes elicited genetic changes in some
cells lines to which
they were applied; the altered genes included the oncogenes H-ras, c-fos and
genes of HIV.
Most of this work involved the modification of a target mRNA, based on a
specific mutant codon
that is cleaved by a specific ribozyme. Targets for this embodiment will
include angiogenic
genes such as VEGFs and angiopoeiteins as well as the oncogenes (e.g., ras,
myc, neu, raf, erb,
src, fms, jun, trk, ret, hst, gsp, bcl-2, EGFR, grb2 and abl. Other constructs
will include
overexpression of antiapoptotic genes such as bcl-2.
CA 02271826 1999-OS-11
Vlll. Single Chain Antibodies
In yet another embodiment, one gene may comprise a single-chain antibody.
Methods for
the production of single-chain antibodies are well known to those of skill in
the art. The skilled
artisan is referred to U.S. Patent 5,359,046, (incorporated herein by
reference) for such methods.
A single chain antibody is created by fusing together the variable domains of
the heavy and light
chains using a short peptide linker, thereby reconstituting an antigen binding
site on a single
molecule.
Single-chain antibody variable fragments (scFvs) in which the C-terminus of
one variable
domain is tethered to the N-terminus of the other via a 15 to 25 amino acid
peptide or linker,
have been developed without significantly disrupting antigen binding or
specificity of the
binding (Bedzyk et al., 1990; Chaudhary et al., 1990). These Fvs lack the
constant regions (Fc)
present in the heavy and light chains of the native antibody.
Antibodies to a wide variety of molecules are contemplated, such as oncogenes,
growth
factors, hormones, enzymes, transcription factors or receptors. Also
contemplated are secreted
antibodies, targeted to serum, against angiogenic factors (VEGF/VSP; (3FGF;
aFGF) and
endothelial antigens necessary for angiogenesis (i.e. V3 integrin).
Specifically contemplated are
growth factors such as transforming growth factor and platelet derived growth
factor.
IX. Transcription Factors and Regulators
Another class of genes that can be applied in an advantageous combination are
transcription factors. Examples include C/EBPa, IxB, NficB and Par-4.
X. Cell Cycle Regulators
Cell cycle regulators provide possible advantages, when combined with other
genes.
Such cell cycle regulators include p27, p16, p21, p57, p18, p73, p19, p15, E2F-
l, E2F-2, E2F-3,
p 107, p 130 and E2F-4. Other cell cycle regulators include anti-angiogenic
proteins, such as
soluble Fltl (dominant negative soluble VEGF receptor), soluble Wnt receptors,
soluble
16
CA 02271826 1999-OS-11
Tie2/Tek receptor, soluble hemopexin domain of matrix metalloprotease 2 and
soluble receptors
of other angiogenic cytokines (e.g., VEGFR1/KDR, VEGFR3/Flt4, both VEGF
receptors).
XI. Chemokines
Genes that code for chemokines also may be used in the present invention.
Chemokines
generally act as chemoattractants to recruit immune effector cells to the site
of chemokine
expression. It may be advantageous to express a particular chemokine gene in
combination with,
for example, a cytokine gene, to enhance the recruitment of other immune
system components to
the site of treatment. Such chemokines include RANTES, MCAF, MIPl-alpha. MIP1-
Beta, and
IP-10. The skilled artisan will recognize that certain cytokines are also
known to have
chemoattractant effects and could also be classified under the term
chemokines.
XII. Combination Therapy
As described herein, it is contemplated that any one particular gene may be
combined
with any other particular gene in the form of a combined therapy. Other
combinations include
the use of a particular therapeutic gene with a more traditional
pharmaceutical therapy, such as
the combination of a tumor suppressor gene with chemo- or radiotherapy. For
example, the
herpes simplex-thymidine kinase (HS-tk) gene, when delivered to brain tumors
by a retroviral
vector system, successfully induced susceptibility to the antiviral agent
ganciclovir (Culver et al.,
1992). In the context of the present invention, it is contemplated that gene
therapy to induce a
therapeutic effect in for example a cancer cell could be used similarly in
conjunction with chemo-
or radiotherapeutic intervention. It also may prove effective to combine a
particular gene therapy
with immunotherapy.
In a cancer phenotype to kill cells, inhibit cell growth, inhibit metastasis,
inhibit
angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor
cells, using the
methods and compositions of the present invention, one would generally contact
a "target" cell with
an expression construct containing a particular gene. In CF it is contemplated
that the CFTR gene
is delivered and caused and achieves a correction, of Cl- transport or an
amelioration of the
detrimental effects of loss of Cl- transport seen in CF.
17
CA 02271826 1999-OS-11
As stated above, a gene therapy may be administered alone or in combination
with at least
one other agent. These compositions would be provided in a combined amount
effective to kill or
inhibit proliferation of a cancer cell or restore Cf transport function in CF.
This process may
involve contacting the cells with the expression construct and the agents) or
factors) at the same
time. This may be achieved by contacting the cell with a single composition or
pharmacological
formulation that includes both agents, or by contacting the cell with two
distinct compositions or
formulations, at the same time, wherein one composition includes the
expression construct and the
other includes the agent.
Alternatively, the gene therapy treatment may precede or follow the other
agent treatment
by intervals ranging from minutes to weeks. In embodiments where the other
agent and expression
construct are applied separately to the cell, one would generally ensure that
a significant period of
time did not expire between the time of each delivery, such that the agent and
expression construct
would still be able to exert an advantageously combined effect on the cell. In
such instances, it is
contemplated that one would contact the cell with both modalities within about
12-24 hours of each
other and, more preferably, within about 6-12 hours of each other, with a
delay time of only about
12 hours being most preferred. In some situations, it may be desirable to
extend the time period for
treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to
several wk (1, 2, 3, 4, 5,
6, 7 or 8) lapse bet~-een the respective administrations.
It also is conceivable that more than one administration of either the gene
therapy or the
other agent will be desired. Various combinations may be employed, where the
primary gene
therapy (e.g. CFTR in CF) is "A" and the other agent is "B", as exemplified
below:
AB/A B/AB BB/A A/AB B/A/A ABB BBBlA BBlAB
AlABB ABlAB A/BB/A BB/A/A B/AB/A B/A/AB BBBlA
A/A/AB B/A/AJA A/B/AlA A/AB/AABBB BlABB BBlAB
~8
CA 02271826 '1999-OS-11
Other combinations are contemplated. Again, to achieve a therapeutic outcome,
both agents
are delivered to a cell in a combined amount effective to restore a normal
state in the cell.
In a cancer therapy, agents or factors suitable for use in a combined therapy
are any
chemical compound or treatment method that induces DNA damage when applied to
a cell. Such
agents and factors include radiation and waves that induce DNA damage such as,
y-irradiation, X-
rays, LJV-irradiation, microwaves, electronic emissions, and the like. A
variety of chemical
compounds, also described as "chemotherapeutic agents," function to induce DNA
damage, all of
which are intended to be of use in the combined treatment methods disclosed
herein.
Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin,
5-fluorouracil (SFLn,
etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatzn (CDDP)
and even
hydrogen peroxide. The invention also encompasses the use of a combination of
one or more DNA
damaging agents, whether radiation-based or actual compounds, such as the use
of X-rays with
cisplatin or the use of cisplatin with etoposide.
In treating cancer according to the invention, one would contact the tumor
cells with an
agent in addition to the expression construct. This may be achieved by
irradiating the localized
tumor site with radiation such as X-rays, UV-light, y-rays or even microwaves.
Alternatively, the
tumor cells may be contacted with the agent by administering to the subject a
therapeutically
effective amount of a pharmaceutical composition comprising a compound such
as, adriamycin,
5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more
preferably,
cisplatin. The agent may be prepared and used as a combined therapeutic
composition, or kit, by
combining it with an expression construct, as described above.
Agents that directly cross-link nucleic acids, specifically DNA, are envisaged
to facilitate
DNA damage leading to a synergistic, antineoplastic combination with the gene
therapy. Agents
such as cisplatin, and other DNA alkylating agents may be used. Cisplatin has
been widely used to
treat cancer, with efficacious doses used in clinical applications of 20 mg/m2
for 5 days every three
19
CA 02271826 1999-OS-11 '
wk for a total of three courses. Cisplatin is not absorbed orally and must
therefore be delivered via
injection intravenously, subcutaneously, intratumorally or intraperitoneally.
Agents that damage DNA also include compounds that interfere with DNA
replication,
mitosis and chromosomal segregation. Such chemotherapeutic compounds include
adriamycin,
also known as doxorubicin, etoposide, verapamil, podophyllotoxin. and the
like. Widely used in a
clinical setting for the treatment of neoplasms, these compounds are
administered through bolus
injections intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals
for adriamycin, to
35-50 mg/m2 for etoposide intravenously or double the intravenous dose orally.
Agents that disrupt the synthesis and fidelity of nucleic acid precursors and
subunits also
lead to DNA damage. As such a number of nucleic acid precursors have been
developed.
Particularly useful are agents that have undergone extensive testing and are
readily available. As
such, agents such as 5-fluorouracil (5-FU), are preferentially used by
neoplastic tissue, making this
agent particularly useful for targeting to neoplastic cells. Although quite
toxic, 5-FU, is applicable
in a wide range of Garners, including topical, however intravenous
administration with doses
ranging from 3 to 15 mg/kg/day being commonly used.
Other factors that cause DNA damage and have been used extensively include
what are
commonly known as Y-rays, X-rays, and/or the directed delivery of
radioisotopes to tumor cells.
Other forms of DNA damaging factors also are contemplated such as microwaves
and UV-
irradiation. It is most likely that all of these factors effect a broad range
of damage DNA, on the
precursors of DNA, the replication and repair of DNA, and the assembly and
maintenance of
chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200
roentgens for
prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000
roentgens. Dosage ranges
for radioisotopes vary widely, and depend on the half life of the isotope, the
strength and type of
radiation emitted, and the uptake by the neoplastic cells.
CA 02271826 1999-OS-11 ''
The skilled artisan is directed to "Remington's Pharmaceutical Sciences" 1 Sth
Edition,
chapter 33, in particular pages 624-652. Some variation in dosage will
necessarily occur depending
on the condition of the subject being treated. The person responsible for
administration will, in any
event, determine the appropriate dose for the individual subject. Moreover,
for human
administration, preparations should meet sterility, pyrogenicity, general
safety and purity standards
as required by FDA Office of Biologics standards.
The inventors propose that the regional delivery of genetic expression
constructs to patients
will be a very efficient method for delivering a therapeutically effective
gene to counteract a clinical
disease. Similarly, the chemo- or radiotherapy may be directed to a
particular, affected region of
the subjects body. Alternatively, systemic delivery of expression construct
and/or the agent may be
appropriate in certain circumstances, for example, where extensive metastasis
has occurred.
In addition to combining specific gene therapies with chemo- and
radiotherapies, it also is
contemplated that combination with other gene therapies will be advantageous.
For example,
targeting of p53 and p16 mutations at the same time may produce an improved
anti-cancer
treatment. Any other tumor-related gene conceivably can be targeted in this
manner, for example,
p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1,
VHI.,
FCC, MCC, ras, myc, neu, raf, erb, src, fms, jun, trl~ ret, gsp, hst, bcl and
abl.
It also should be pointed out that any of the foregoing therapies may prove
useful by
themselves in treating a genetic abnormality. In this regard, reference to
chemotherapeutics and
non-gene therapy in combination should also be read as a contemplation that
these approaches may
be employed separately.
XIII. Disease States
Particular disease states that could be treated through gene replacement in
epithelial cells
include lung cancer, tracheal cancer, asthma, surfactant protein B deficiency,
alpha-1-antitrypsin
deficiency, breast cancer, bladder cancer and cystic fibrosis.
21
CA 02271826 1999-OS-11
C. VIRAL VECTORS
One aspect of the present invention is a virus that has been genetically
engineered to
deliver a therapeutic gene sequence to epithelial cells. These genetically
engineered viruses are
also referred to as viral vectors. Having identified and isolated functional
forms of the defective
genes responsible for various illnesses, gene therapy protocols require a
means of delivering the
functional gene to the diseased tissue. Researchers noted that viruses have
evolved to be able to
deliver their DNA to various host tissues despite the human body's various
defensive
mechanisms. For this reason, numerous viral vectors have been designed by
researchers seeking
to create vehicles for therapeutic gene delivery. Some of the types of viruses
that have been
engineered to create viral vectors for gene therapy are listed below.
I. Adenovirus
Knowledge of the genetic organization or adenovirus, a 36 kB, linear, double-
strained
DNA virus, allows substitution of large pieces of adenoviral DNA with foreign
sequences up to 7
kB (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the infection of
adenovira DNA in
host cells does not result in chromosomal integration because adenoviral DNA
can replicate in an
episomal manner. Also, adenoviruses are structurally stable, and no genome
rearrangement has
been detected after extensive amplification. Adenovirus can infect virtually
all epithelial cells
regardless of their cell cycle stage. This means that adenovirus can infect
non-dividing cells. So
far, adenoviral infection appears to be linked only to mild disease such as
acute respiratory
disease in humans. This group of viruses can be obtained in high titers, e.g.,
109-101
plaque-forming units per ml, and they are highly infective.
Adenovirus have been used in eukaryotic gene expression (Levrero et al., 1991;
Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992;
Graham and
Prevec, 1992). Animal studies have suggested that recombinant adenovirus could
be used for
gene therapy (Stratford-Perricaudet and Perncaudet, 1991; Stratford-
Perricaudet et al., 1990;
Rich et al., 1993). Studies in administering recombinant adenovirus to
different tissues include
tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et aL, 1992), muscle
injection (Ragot et
al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and
stereoctatic inoculation
22
CA 02271826 1999-OS-11
into the brain (Le Gal La Salle et al., 1993). Recently, phase I gene therapy
clinical trials have
begun in human volunteers where adenoviral vectors have been administered by
intradermal
injection and by intrabronchial infusion to determine what kind of
immunological response the
vectors elicit (Anderson, 1998).
II. Retroviruses
Particularly in the treatment of chronic illnesses, it may be preferable to
use DNA
expression vectors which will remain present in the treated tissue for long
periods of time
negating the need for frequent readministration of the gene therapy. One way
of achieving this is
through the use of integrating viral vectors. These viruses result in
integration of the transgene in
the host genome. The prototypical integrative virus is the retrovirus.
The retroviruses are a group of single-stranded RNA viruses characterized by
an ability to
convert their RNA to double-stranded DNA to infected cells by a process of
reverse-transcription
(Coffin, 1990). The resulting DNA then stably integrates into cellular
chromosomes as a
provirus and directs synthesis of viral proteins. The integration results in
the retention of the
viral gene sequences in the recipient cell and its descendants. The retroviral
genome contains
three genes, gag, pol, and env that code for capsid proteins, polymerase
enzyme, and envelope
components, respectively. A sequence found upstream from the gag gene, termed
yr, constitutes
the packaging signal for the virus. When a recombinant plasmid containing a
human cDNA,
together with the retroviral LTR and 4r seguences is introduced into this cell
line (by calcium
phosphate precipitation for example), the yr sequence allows the RNA
transcript of the
recombinant plasmid to be packaged into viral particles, which are then
secreted into the culture
media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The
media containing
the recombinant retroviruses is then collected, optionally concentrated, and
used for gene
transfer. Retroviral vectors are able to infect a broad variety of cell types.
However, integration
with MMLV-based retrovirus requires the division of host cells (Paskind et
al., 1975).
23
CA 02271826 1999-OS-11
The retrovirus family includes the subfamilies of the oncoviruses, the
lentiviruses and the
spumaviruses. Two oncoviruses are Moloney murine leukemia virus (MMLV) and
feline
leukemia virus (FeLV). The lentiviruses include human immunodeficiency virus
(HIV), simian
immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Among
the murine
viruses such as MMLV there is a further classification. Murine viruses may be
ecotropic,
xenotropic, polytropic or amphotropic. Each class of viruses target different
cell surface
receptors in order to initiate infection.
MMLV-based retroviruses have received extensive use in gene transfer studies
and are
approved for human trials. Further advances in retroviral vector design and
concentration
methods have allowed production of amphotropic and xenotropic viruses with
titers of 108 to 109
cfu/ml (Bowies et al., 1996; Irwin et al., 1994; Jolly, 1994; Kitten et al.,
1997).
One concern with the use of defective retrovirus vectors is the potential
appearance of
wild-type replication-competent virus in the packaging cells. This can result
from recombination
events in which the intact yr sequence from the recombinant virus inserts
upstream from the gag,
poi, env sequence integrated in the host cell genome. However, packaging cell
lines are now
available that should greatly decrease the likelihood of recombination
(Markowitz et al., 1988;
Hersdorffer et al., 1990). Another concern about retrovirus vectors is that
they usually integrate
into random sites in the cell genome. Theoretically, this can lead to
insertional mutagenesis
through the interruption of host genes or through the insertion of viral
regulatory sequences that
can interfere with the function of flanking genes (Varmus et al., 1981).
However, to date, the
only example of retroviral gene transfer producing cancer in large animals was
found to be due to
the integration of contaminant replication-competent virus and not due to the
retroviral vectors
themselves (Donahue et al., 1992; Anderson, 1998)
Two strategies have been proposed to increase the efficacy of retroviral
vectors. First,
one can pseudotype the virus and replace the envelope or to make an envelope
chimera by adding
a novel ligand. The second approach replaces a portion of the normal env
protein with a novel
24
CA 02271826 1999-OS-11
ligand that will interact with a more abundant cell membrane receptor or
component. Successful
examples include pseudotyping with the vesicular stomatitis G protein (VSV-G,
(Burns et al.,
1993)), and envelope chimeras containing heregulin (Han et al., 1995) and
erythropoetin
(Kasahara et al., 1994). The receptor for VSV-G has not been cloned but is
believed to be a
phosphoserine component of the plasma membrane (Schlegel et al., 1983).
MMLV-based retroviruses have received extensive use in gene transfer studies,
primarily
using ex vivo approaches, and have been approved for several human trials.
Replication
defective recombinant retroviruses are not acute pathogens in primates
(Chowdhury et al., 1991).
They have been successfully applied in cell culture systems to transfer the
CFTR gene and
generate cAMP-activated Cl- secretion in a variety of cell types including
human airway epithelia
(Drumm et al., 1990, Olsen et al., 1992; Anderson et al., 1991; Olsen et al.,
1993). While there
is evidence of immune responses to the viral gag and env proteins, this does
not prevent
successful readministration of vector (McCormack et al., 1997). Further, since
recombinant
retroviruses have no expressed gene products other than the transgene, the
risk of a host
inflammatory response due to viral protein expression is limited (McCormack et
al., 1997). As
for the concern about insertional mutagenesis, to date there are no examples
of insertional
mutagenesis arising from any human trial with recombinant retroviral vectors.
Until recently, one limitation to the use of retrovirus vectors in vivo was
the limited
ability to produce retroviral vector titers greater than 106 infections U/mL.
Titers 10- to
1,000-fold higher are necessary for many in vivo applications. Important
advances in viral
constructs and concentration methods have been made by Dr. Doug Jolly and
colleagues at
Chiron Technologies Center for Gene Therapy, resulting in titers of ampho- and
xenotropic
viruses in the 108 to 109 cfu/ml range (Jolly, 1994; Irwin et al., 1994).
Similar results have also
been reported by Woo et al. (Bowles et al., 1996) and Ferry and colleagues
(Kitten et al., 1997).
More recently, hybrid lentivirus vectors have been described combining
elements of
human immunodeficiency virus (HIV) (Naldini et al., 1996) or feline
immunodeficiency virus
CA 02271826 1999-OS-11
(FIV) (Poeschla et al., 1998) and MMLV. These vectors transduce nondividing
cells in the CNS
(Naldini et al., 1996; Blomer et al., 1997), liver (Kafri et al., 1997),
muscle (Kafri et al., 1997)
and retina (Miyoshi et al., 1997). However, a recent report in xenograft
models of human airway
epithelia suggests that in well-differentiated epithelia, gene transfer with
VSV-G pseudotyped
HIV-based lentivirus is inefficient (Goldman et al., 1997).
A recent report by Wilson and colleagues observed that primary cultures of
dividing
human airway epithelia expressed more transgene when infected with lentivirus
than confluent
epithelia (Goldman et al., 1997). This leads to the conclusion that HIV-based
lentivirus can
infect non-dividing, well-differentiated airway epithelia. Several other
recent studies confirm
that hybrid lentiviral vectors infect nondividing mammalian cells (Naldine et
al., 1996, Kafri et
al., 1997).
III. Adeno Associated Virus
Recently, adeno-associated virus (AAV) has emerged as a potential alternative
to the
more commonly used retroviral and adenoviral vectors. While studies with
retroviral and
adenoviral mediated gene transfer raise concerns over potential oncogenic
properties of the
former, and immunogenic problems associated with the latter, AAV has not been
associated with
any such pathological indications. This may be due to the fact that AAV
appears to integrate
preferentially into the short arm of human chromosome 19 (Anderson, 1998). AAV
vectors have
been shown to transduce brain, skeletal muscle and liver cells efficiently and
may be capable of
infecting non-dividing cells (Anderson, 1998). The sequence of AAV is provided
by Srivastava
et al. (1983). AAV is a member of the parvovirus family which includes the
genus parvovirus
and the genus dependovirus. AAV is classified as a dependovirus (Murphy and
Kingsbury,
1991). The use of AAV in gene transfer is described in U.S. Patents 5,139,941
and 5,252,479
(specifically incorporated herein by reference).
26
CA 02271826 1999-OS-11
IV. Vaccinia Virus
Vaccinia viruses are a genus of the poxvirus family. Vaccinia virus vectors
have been
used extensively because of the ease of their construction, relatively high
levels of expression
obtained, wide host range and large capacity for carrying DNA. Vaccinia
contains a linear,
double-stranded DNA genome of about 186 kB that exhibits a marked "A-T"
preference.
Inverted terminal repeats of about 10.5 kB flank the genome. The maj ority of
essential genes
appear to map within the central region, which is most highly conserved among
poxviruses.
Estimated open reading frames in vaccinia virus number from 150 to 200.
Although both strands
are coding, extensive overlap of reading firames is not common. U.S. Patent
5,656,465
(specifically incorporated by reference) describes in vivo gene delivery using
pox viruses.
V. Papovavirus
The papovavirus family includes the papillomaviruses and the polyomaviruses.
The
polyomaviruses include Simian Virus 40 (SV40), polyoma virus and the human
polyomaviruses
BKV and JCV. Papillomaviruses include the bovine and human papillomaviruses.
The genomes
of polyomaviruses are circular DNAs of a little more than 5000 bases. The
predominant gene
products are three virion proteins (VPl-3) and Large T and Small T antigens.
Some have an
additional structural protein, the agnoprotein, and others have a Middle T
antigen.
Papillomaviruses are somewhat larger, approaching 8 kB
Little is known about the cellular receptors for polyomaviruses, but polyoma
infection
can be blocked by treating with sialidase. SV40 W 11 still infect sialidase-
treated cells, but JCV
cannot hemagglutinate cells treated with sialidase. Because interaction of
polyoma VPl with the
cell surface activates c-myc and c fos, it has been hypothesized that the
virus receptor may have
some properties of a growth factor receptor. Papillomaviruses are specifically
tropic for
squamous epithelia, though the specific receptor has not been identified.
VI. Paramyxovirus
The paramyxovirus family is divided into three genera: paramyxovirus,
morbillivirus and
pneumovirus. The paramyxovirus genus includes the mumps virus and Sendai
virus, among
27
CA 02271826 1999-OS-11
others, while the morbilliviruses include the measles virus and the
pneumoviruses include
respiratory syncytial virus (RSV). Paramyxovirus genomes are RNA based and
contain a set of
six or more genes, covalently linked in tandem. The genome is something over 1
S kB in length.
The viral particle is 150-250 nm in diameter, with "fuzzy" projections or
spikes protruding
therefrom. These are viral glycoproteins that help mediate attachment and
entry of the virus into
host cells.
D. REGULATORY ELEMENTS
I. Promoters
Throughout this application, the term "expression construct" is meant to
include any type
of genetic construct containing a nucleic acid coding for gene products in
which part or all of the
nucleic acid encoding sequence is capable of being transcribed. The transcript
may be translated
into a protein, but it need not be. In certain embodiments, expression
includes both transcription
of a gene and translation of mRNA into a gene product. In other embodiments,
expression only
includes transcription of the nucleic acid encoding genes of interest.
The nucleic acid encoding a gene product is under transcriptional control of a
promoter.
A "promoter" refers to a DNA sequence recognized by the synthetic machinery of
the cell, or
introduced synthetic machinery, required to initiate the specific
transcription of a gene. The
phrase "under transcriptional control" means that the promoter is in the
correct location and
orientation in relation to the nucleic acid to control RNA polymerase
initiation and expression of
the gene.
The term promoter will be used here to refer to a group of transcriptional
control modules
and other sequences that initiate transcription. Exemplary are those sequences
clustered around
the initiation site for RNA polymerase II. Much of the thinking about how
promoters are
organized derives from analyses of several viral promoters, including those
for the HSV
thymidine kinase (tk) and SV40 early transcription units. These studies,
augmented by more
recent work, have shown that promoters are composed of discrete functional
modules, each
28
r
CA 02271826 1999-OS-11
consisting of approximately 7-20 by of DNA, and containing one or more
recognition sites for
transcriptional activator or repressor proteins.
At least one module in each promoter functions to position the start site for
RNA
synthesis. The best known example of this is the TATA box, but in some
promoters lacking a
TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl
transferase gene
and the promoter for the SV40 late genes, a discrete element overlying the
start site itself helps to
fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional
initiation.
Typically, these are located in the region 30-110 by upstream of the start
site, although a number
of promoters have recently been shown to contain functional elements
downstream of the start
site as well. The spacing between promoter elements frequently is flexible, so
that promoter
function is preserved when elements are inverted or moved relative to one
another. In the tk
promoter, the spacing between promoter elements can be increased to 50 by
apart before activity
begins to decline. Depending on the promoter, it appears that individual
elements can function
either co-operatively or independently to activate transcription.
The particular promoter employed to control the expression of a nucleic acid
sequence of
interest is not believed to be important, so long as it is capable of
directing the expression of the
nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is
preferable to position
the nucleic acid coding region adjacent to and under the control of a promoter
that is capable of
being expressed in a human cell. Generally speaking, such a promoter might
include either a
human or viral promoter.
In various embodiments, the human cytomegalovirus (CMV) immediate early gene
promoter, the SV40 early promoter, the Rous sarcoma virus long terminal
repeat, ~3-actin, rat
insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to
obtain high-
level expression of the coding sequence of interest. The use of other viral or
mammalian cellular
29
CA 02271826 1999-OS-11
or bacterial phage promoters which are well-known in the art to achieve
expression of a coding
sequence of interest is contemplated as well, provided that the levels of
expression are sufficient
for a given purpose. By employing a promoter with well-known properties, the
level and pattern
of expression of the protein of interest following transfection or
transformation can be optimized.
Selection of a promoter that is regulated in response to specific physiologic
or synthetic
signals can permit inducible expression of the gene product. For example in
the case where
expression of a transgene, or transgenes when a multicistronic vector is
utilized, is toxic to the
cells in which the vector is produced in, it may be desirable to prohibit or
reduce expression of
one or more of the transgenes. Examples of transgenes that may be toxic to the
producer cell line
are pro-apoptotic and cytokine genes. Several inducible promoter systems are
available for
production of viral vectors where the transgene product may be toxic.
The ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This system
is
designed to allow regulated expression of a gene of interest in mammalian
cells. It consists of a
tightly regulated expression mechanism that allows virtually no basal level
expression of the
transgene, but over 200-fold inducibility. The system is based on the
heterodimeric ecdysone
receptor of Drosophila, and when ecdysone or an analog such as muristerone A
binds to the
receptor, the receptor activates a promoter to turn on expression of the
downstream transgene
high levels of mRNA transcripts are attained. In this system, both monomers of
the
heterodimeric receptor are constitutively expressed from one vector, whereas
the ecdysone-
responsive promoter which drives expression of the gene of interest is on
another plasmid.
Engineering of this type of system into the gene transfer vector of interest
would therefore be
useful. Cotransfection of plasmids containing the gene of interest and the
receptor monomers in
the producer cell line would then allow for the production of the gene
transfer vector without
expression of a potentially toxic transgene. At the appropriate time,
expression of the transgene
could be activated with ecdysone or muristeron A.
Another inducible system that would be useful is the Tet-OffrM or Tet-OnTM
system
(Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen
and Bujard,
CA 02271826 1999-OS-11
1992; Gossen et al., 1995). This system also allows high levels of gene
expression to be
regulated in response to tetracycline or tetracycline derivatives such as
doxycycline. In the Tet-
OnTM system, gene expression is turned on in the presence of doxycycline,
whereas in the Tet-
OffrM system, gene expression is turned on in the absence of doxycycline.
These systems are
based on two regulatory elements derived from the tetracycline resistance
operon of E. coli. The
tetracycline operator sequence to which the tetracycline repressor binds, and
the tetracycline
repressor protein. The gene of interest is cloned into a plasmid behind a
promoter that has
tetracycline-responsive elements present in it. A second plasmid contains a
regulatory element
called the tetracycline-controlled transactivator, which is composed, in the
Tet-OffTM system, of
the VP16 domain from the herpes simplex virus and the wild-type tertracycline
repressor. Thus
in the absence of doxycycline, transcription is constitutively on. In the Tet-
OnTM system, the
tetracycline repressor is not wild type and in the presence of doxycycline
activates transcription.
For gene therapy vector production, the Tet-Offj'M system would be preferable
so that the
producer cells could be grown in the presence of tetracycline or doxycycline
and prevent
expression of a potentially toxic transgene, but when the vector is introduced
to the patient, the
gene expression would be constitutively on.
In some circumstances, it may be desirable to regulate expression of a
transgene in a gene
therapy vector. For example, different viral promoters with varying strengths
of activity may be
utilized depending on the level of expression desired. In mammalian cells, the
CMV immediate
early promoter if often used to provide strong transcriptional activation.
Modified versions of
the CMV promoter that are less potent have also been used when reduced levels
of expression of
the transgene are desired. When expression of a transgene in hematopoetic
cells is desired,
retroviral promoters such as the LTRs from MLV or MMTV are often used. Other
viral
promoters that may be used depending on the desired effect include SV40, RSV
LTR, HIV-1 and
HIV-2 LTR, adenovirus promoters such as from the ElA, E2A, or MLP region, AAV
LTR,
cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.
Similarly tissue specific promoters may be used to effect transcription in
specific tissues
or cells so as to reduce potential toxicity or undesirable effects to non-
targeted,tissues. For
31
CA 02271826 1999-OS-11
example, promoters that are selectively active in lung and other airway
tissues may be
particularly useful.
In certain indications, it may be desirable to activate transcription at
specific times after
administration of the gene therapy vector. This may be done with such
promoters as those that
are hormone or cytokine regulatable. For example in gene therapy applications
where the
indication is a gonadal tissue where specific steroids are produced or routed
to, use of androgen
or estrogen regulated promoters may be advantageous. Such promoters that are
hormone
regulatable include MMTV, MT-1, ecdysone and RuBisco. Other hormone regulated
promoters
such as those responsive to thyroid, pituitary and adrenal hormones are
expected to be useful in
the present invention. Cytokine and inflammatory protein responsive promoters
that could be
used include K and T Kininogen (Kageyama et al., 1987), c-fos, TNF-alpha, C-
reactive protein
(Arcone et al., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2,
C/EBP alpha, IL-1,
IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson et al., 1990), IL-8,
alpha-1 acid
glycoprotein (Drowse and Baumann, 1988), alpha-1 antitypsin, lipoprotein
lipase (Zechner et al.,
1988), angiotensinogen (Ron et al., 1991), fbrinogen, c-jun (inducible by
phorbol esters, TNF-
alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase
(induced by phorbol
esters and retinoic acid), metallothionein (heavy metal and glucocorticoid
inducible),
Stromelysin (inducible by phorbol ester, interleukin-l and EGF), alpha-2
macroglobulin and
alpha-1 antichymotrypsin.
It is envisioned that cell cycle regulatable promoters may be useful in the
present
invention. For example, in a bi-cistronic gene therapy vector, use of a strong
CMV promoter to
drive expression of a first gene such as p 16 that arrests cells in the G 1
phase could be followed
by expression of a second gene such as p53 under the control of a promoter
that is active in the
G1 phase of the cell cycle, thus providing a "second hit" that would push the
cell into apoptosis.
Other promoters such as those of various cyclins, PCNA, galectin-3, E2F1, p53
and BRCA1
could be used.
32
CA 02271826 1999-OS-11'
Tumor specific promoters such as osteocalcin, hypoxia-responsive element
(HRE),
MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase rnay also be used to
regulate gene
expression in tumor cells. Other promoters that could be used according to the
present invention
include Lac-regulatable, chemotherapy inducible (e.g., MDR), and heat
(hyperthermia) inducible
promoters, radiation-inducible (e.g., EGR (Joki et al., 1995)), Alpha-inhibin,
RNA pol III tRNA
met and other amino acid promoters, Ul snRNA (BartIett et al., 1996), MC-1,
PGK, (3-actin and
a-globin. Many other promoters that may be useful are listed in Walther and
Stein (1996).
It is envisioned that any of the above promoters alone or in combination with
another
may be useful according to the present invention depending on the action
desired. In addition,
this list of promoters is should not be construed to be exhaustive or
limiting, those of skill in the
art will know of other promoters that may be used in conjunction with the
promoters and
methods disclosed herein.
B. Enhancers
Enhancers are genetic elements that increase transcription from a promoter
located at a
distant position on the same molecule of DNA. Enhancers are organized much
like promoters.
That is, they are composed of many individual elements, each of which binds to
one or more
transcriptional proteins. The basic distinction between enhancers and
promoters is operational.
An enhancer region as a whole must be able to stimulate transcription at a
distance; this need not
be true of a promoter region or its component elements. On the other hand, a
promoter must
have one or more elements that direct initiation of RNA synthesis at a
particular site and in a
particular orientation, whereas enhancers lack these specificities. Promoters
and enhancers are
often overlapping and contiguous, often seeming to have a very similar modular
organization.
Any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base
EPDB)
could also be used to drive expression of the gene. Eukaryotic cells can
support cytoplasmic
transcription from certain bacterial promoters if the appropriate bacterial
polymerase is provided,
either as part of the delivery complex or as an additional genetic expression
construct.
33
CA 02271826 1999-OS-11 ~ -
In preferred embodiments of the invention, the expression construct comprises
a virus or
engineered construct derived from a viral genome. The ability of certain
viruses to enter cells via
receptor-mediated endocytosis and to integrate into host cell genome and
express viral genes
stably and efficiently have made them attractive candidates for the transfer
of foreign genes into
mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and
Sugden, 1986;
Temin, 1986).
III. Polyadenylation Signals
Where a cDNA insert is employed, one will typically desire to include a
polyadenylation
signal to effect proper polyadenylation of the gene transcript. The nature of
the polyadenylation
signal is not believed to be crucial to the successful practice of the
invention, and any such
sequence may be employed such as human or bovine growth hormone and SV40
polyadenylation
signals. Also contemplated as an element of the expression cassette is a
terminator. These
elements can serve to enhance message levels and to minimize read through from
the cassette
into other sequences.
IY. IRES
In certain embodiments of the invention, the use of internal ribosome entry
site (IRES)
elements is contemplated to create multigene, or polycistronic, messages. IRES
elements are
able to bypass the ribosome scanning model of 5' methylated Cap dependent
translation and
begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES
elements from two
members of the picornavirus family (poliovirus and encephalomyocarditis) have
been described
(Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message
(Macejak and
Samow, 1991). IRES elements can be linked to heterologous open reading frames.
Multiple
open reading frames can be transcribed together, each separated by an IRES,
creating
polycistronic messages. By virtue of the IRES element, each open reading frame
is accessible to
ribosomes for efficient translation. Multiple genes can be efficiently
expressed using a single
promoter/enhancer to transcribe a single message.
34
CA 02271826 1999-OS-11
Any heterologous open reading frame can be linked to IRES elements. This
includes
genes for secreted proteins, mufti-subunit proteins, encoded by independent
genes, intracellular
or membrane-bound proteins and selectable markers. In this way, expression of
several proteins
can be simultaneously engineered into a cell with a single construct and a
single selectable
marker.
E. VECTOR DELIVERY
1. Viral Receptor Expression
The inventors have identified one of the main barriers to viral uptake in
epithelial tissue
as the apparent lack of accessibility to viral receptors when viral particles
are applied to the
apical surface of polarized epithelial cells. The viral receptors appear to be
readily accessible
primarily from the basal side of the epithelia. In particular, the efficiency
of viral infection, as
exemplified here with retroviruses, is determined in part by the availability
of specific cellular
receptors that mediate virus entry (Miller, 1996; Weiss and Tailor, 1995).
Thus, the inventors'
observation that retroviral gene transfer to proliferating airway epithelia is
polar is very
important.
Little is known regarding the biology of retrovirus and its receptor
interactions in these
cells. Other than the observations that amphotropic (Olsen et al., 1993) or
GALV (Bayle et al.,
1993) enveloped vectors can infect airway epithelia, there has been little
work to characterize the
abundance, cellular location, or regulation of receptor expression in airway
epithelia. However,
there is considerable precedence in epithelia for viral infection to occur in
a polarized fashion.
Studies in high resistance MDCK cells conclusively showed that vesicular
stomatitis virus
infected at least 100 times more efficiently when applied to the basal side
than when applied to
the apical surface of these epithelia (Fuller et al., 1984).
Similarly, vaccina virus (Rodriguez et al., 1991) and canine parvovirus (Basak
and
Compans, 1989) preferentially infect the basolateral surface of epithelia,
while cytomegalovirus
CA 02271826 1999-OS-11
(Tugizov and Pereira, 1996), measles virus (Blare and Compares, 1995) and
simian virus 40
(Clayson and Compares, 1988) infects more efficiently from the apical surface.
Thus, other
vectors also show similar preferences for portions of polarized cells.
The efficiency of infection with retroviruses is determined in part by the
availability of
specific cellular receptors that mediate virus entry (Miller, 1996; Weiss and
Tailor, 1995). In the
case of amphotropic .enveloped (env) retrovirus, the receptor has been cloned
from rat and human
cells and is called Ram-1 (rodent) or GLVR-2 (human) or more recently Pitt. It
has been shown
to be a cell surface protein that functions as a sodium-dependent phosphate
transporter (Miller et
al., 1994; Miller and Miller, 1994). Several other specific MMLV receptors
exist and have been
cloned, but the receptor for the xenotropic envelope is currently unknown
(Miller, 1996).
Binding of the amphotropic envelope glycoprotein gp70 to Ram-1 initiates viral
infection and in
hematopoetic cells (Orlic et al., 1996) and hepatocytes (Hatzoglou et al.,
1995) levels of Ram-1
mRNA expression correlate directly with infection efficiencies. In some cases
receptor
abundance and infectivity is regulated by nutritional or hormonal conditions.
For example, there
is evidence for the regulation of Ram-1 mRNA expression by insulin,
dexamethasone (Wu et al.,
1994) or hypophosphatemia (Chien et al., 1997; Miller and Miller, 1994;
Richardson and Bank,
1996). The findings presented here suggest that, in addition to stimulating
cell proliferation,
growth factors also increase the expression of the Pit-2 amphotropic receptor
protein.
Il. Increasing Permeability
It was observed that procedures which increased transepithelial permeability
enhanced
gene transfer after vector was applied to the apical surface. The tight
junction, also known as
zonula occludens, is the apical-most component of the epithelial functional
complex (Anderson
and Itallie, 1995). A variety of tissue permeabilizing agents transiently
increase epithelial
permeability by disrupting tight junctions. Bhat and co-workers reported that
lowering infra- or
extracellular calcium levels or disrupting the cytoskeleton reversibly
increased permeability in
rabbit tracheal epithelium (Bhat et al.; 1993). Widdicombe and colleagues
found that hypotonic
shock from the application of water to the apical surface transiently
increased the permeability of
36
CA 02271826 1999-OS-11
cultured bovine or human tracheal epithelia (Widdicombe et al., 1996).
Hypotonic shock
reversibly increased both transcellular and paracellular permeability
(Widdicombe et al., 1996).
Intraepithelial permeability, according to the present invention, can
therefore be increased
by contacting the epithelial tissue with a tissue permeablizing agent
including those that lower
calcium levels, disrupt the cytoskeleton or cause hypotonic shock. Calcium
levels can be
lowered by introducing ion chelators such as EGTA, BAPTA or EDTA. Cytoskeletal
disruption
agents include cytochalasin B or colchicine. Hypotonic solutions are defined
relative to normal
osmolality, or normotonic solutions. Normotonic solutions are around 280-300
mosm/kg. The
hypotonic solutions according to the present invention are less than about 280
mosm/kg. One
particular buffer is about 105 mosm/kg, while others are about 25-SO mosm/kg.
Other tissue permeablizing agents include poly-L-lysine, occludin peptide,
ether,
neurotransmitters, FCCP, oxidants and mediators of inflammation.
Neurotransmitters that can be
used include capsianoside. Oxidants that can be used include ozone, and
mediators of
inflammation include TNFa.
Ill. Modes of Action
There are several possible reasons for the increase in gene transfer noted
under the
experimental conditions used herein. Perhaps the most simple interpretation is
that Pit-2
expression is polarized in human airway epithelia and primarily localized to
the basolateral
surface of all cells of the epithelial sheet. In support of this hypothesis,
techniques that are
known to transiently disrupt the integrity of epithelial tight junctions (i.e.
hypotonic shock, low
Ca2+) enhanced gene transfer efficiency with amphotropic or xenotropic vector
applied to the
mucosal surface. Disruption of tight junctions may also cause a transient loss
of cell polarity and
shifting of receptors to the apical pole. Since hypotonic conditions
transiently increase apical
membrane permeability to macromolecules (Widdicombe et al., 1996), it also is
conceivable that
vector may have entered cells via a receptor-independent fashion during
hypotonic conditions.
Finally . it also is possible that the apical treatment procedures removed
mucus or inhibitory
factors from the apical surface.
37
..
CA 02271826 1999-OS-11
Another potential advantage of delivering viral vectors via increased
transepithelial
permeability is the ability to target certain epithelial cells. There is
controversy regarding which
epithelial cells to target for gene therapy in chronic diseases such as CF.
Arguments can be made
in support of correcting cells of the surface epithelium, the submucosal
glands, or both (Yamaya,
1991; Engelhardt et al., 1992). A goal of gene transfer to the pulmonary
epithelium with
integrating vectors is to correct the genetic defect in a population of cells
which could pass the
corrected gene on to their progeny. There appear to be several epithelial cell
types in the lung
that are able to divide. Some of these cells may represent a pluripotent or
"stem cell" population.
Studies from several species and model systems suggest these populations
exist; basal cells and
non-ciliated columnar cells of the airways (Randell, 1992; Ford and Terzaghi-
Howe, 1992.);
Clara cells (Evans et al., 1976) and alveolar type II cells (Adamson and
Bowden. 1974; Evans, et
al., 1975) in the distal lung. The invention allows the pracnuoner w ~ar~m ~~~
~~ll~ l~l
infection by viral expression vectors. This in turn can enhance the duration
of transgenic
expression by targeting integrating vectors to epithelial cells with the
capacity to transmit genetic
material to daughter cells.
F. PHARMACEUTICALS AND ROUTES OF ADMINISTRATION
In clinical applications, it will be necessary to prepare the viral particles
of the present
invention as pharmaceutical compositions, i. e. in a form appropriate for in
vivo applications.
Generally, this will entail preparing compositions that are essentially free
of pyrogens, as well as
other impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render
viral vectors
compositions stable and allow for uptake by target cells. Aqueous compositions
of the present
invention comprise an effective amount of the viral vector, dissolved or
dispersed in a
pharmaceutically acceptable Garner or aqueous medium. Such compositions also
are referred to
as inocula. The phrase "pharmaceutically or pharmacologically acceptable"
refer to molecular
entities and compositions that do not produce adverse, allergic, or other
untoward reactions when
38
CA 02271826 1999-OS-11
administered to an animal or a human. As used herein, "pharmaceutically
acceptable carrier"
includes any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents,
isotonic and absorption delaying agents and the like. The use of such media
and agents for
pharmaceutically active substances is well know in the art. Except insofar as
any conventional
media or agent is incompatible with the vectors of the present invention, its
use in therapeutic
compositions is contemplated. Supplementary active ingredients also can be
incorporated into
the compositions.
The viral particles of the present invention include classic pharmaceutical
preparations.
Administration of these compositions according to the present invention will
be via any common
route so long as the target tissue is available via that route. This includes
oral, nasal, buccal,
rectal, vaginal or topical. Alternatively, administration rnay be by aerosol,
intraderrnal,
subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such
compositions would
normally be administered as pharmaceutically acceptable compositions,
described supra.
The particles may be administered via any suitable route, including
parenterally or by
injection. Solutions of the active compounds as free base or pharmacologically
acceptable salts
can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose.
Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof
and in oils. Under ordinary conditions of storage and use, these preparations
contain a
preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile
injectable solutions
or dispersions. In all cases the form must be sterile and must be fluid to the
extent that easy
syringability exists. It must be stable under the conditions of manufacture
and storage and must
be preserved against the contaminating action of microorganisms, such as
bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for example,
water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene glycol, and
the like), suitable
mixtures thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the
39
CA 02271826 1999-OS-11 -'
use of a coating, such as lecithin, by the maintenance of the required
particle size in the case of
dispersion and by the use of surfactants. The prevention of the action of
microorganisms can be
brought about by various antibacterial an antifungal agents, for example,
parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases,
it will be preferable
to include isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the
injectable compositions can be brought about by the use in the compositions of
agents delaying
absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the viral particles
in the required
amount in the appropriate solvent with various of the other ingredients
enumerated above, as
required, followed by filtered sterilization. Generally, dispersions are
prepared by incorporating
the various sterilized active ingredients into a sterile vehicle which
contains the basic dispersion
medium and the required other ingredients from those enumerated above. In the
case of sterile
powders for the preparation of sterile injectable solutions; the preferred
methods of preparation
are vacuum-drying and freeze-drying techniques which yield a powder of the
active ingredient
plus any additional desired ingredient from a previously sterile-filtered
solution thereof.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying
agents and the like. The use of such media and agents for pharmaceutical
active substances is
well known in the art. Except insofar as any conventional media or agent is
incompatible with
the active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary
active ingredients also can be incorporated into the compositions.
For oral administration the polypeptides of the present invention may be
incorporated
with excipients and used in the form of non-ingestible mouthwashes and
dentifrices. A
mouthwash may be prepared incorporating the active ingredient in the required
amount in an
appropriate solvent, such as a sodium borate solution (Dobell's Solution).
Alternatively, the
active ingredient may be incorporated into an antiseptic wash containing
sodium borate, glycerin
and potassium bicarbonate. The active ingredient may also be dispersed in
dentifrices, including:
CA 02271826 1999-OS-11
gels, pastes, powders and slurries. The active ingredient may be added in a
therapeutically
effective amount to a paste dentifrice that may include water, binders,
abrasives, flavoring
agents, foaming agents, and humectants.
The compositions of the present invention may be formulated in a neutral or
salt form.
Pharmaceutically-acceptable salts include the acid addition salts (formed with
the free amino
groups of the protein) and which are formed with inorganic acids such as, for
example,
hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and
the like. Salts formed with the free carboxyl groups also can be derived from
inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such
organic bases as isopropylamine, trimethylamine, histidine, procaine and the
like.
Upon formulation, solutions will be administered in a manner compatible with
the dosage
formulation and in such amount as is therapeutically effective. The
formulations are easily
administered in a variety of dosage forms such as injectable solutions, drug
release capsules and
the like. For parenteral administration in an aqueous solution, for example,
the solution should
be suitably buffered if necessary and the liquid diluent first rendered
isotonic with sufficient
saline or glucose. These particular aqueous solutions are especially suitable
for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
''Unit dose" is defined as a discrete amount of a therapeutic composition
dispersed in a
suitable carrier. For example, in accordance with the present methods, viral
doses include a
particular number of virus particles or plaque forming units (pfu). For
embodiments involving
adenovirus, particular unit doses include 103, 104, 105, 106, 10', 108, 109,
101°, 1011, 102, 1013 or
10'4 pfu. Particle doses may be somewhat higher (10 to 100-fold) due to the
presence of
infection defective particles.
In this connection, sterile aqueous media which can be employed will be known
to those
of skill in the art in light of the present disclosure. For example, a unit
dose could be dissolved
in 1 ml of isotonic NaCI solution and either added to 1000 ml of
hypodermoclysis fluid or
41
CA 02271826 1999-OS-11
injected at the proposed site of infusion, (see for example, "Remington's
Pharmaceutical
Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in
dosage will
necessarily occur depending on the condition of the subject being treated. The
person
responsible for administration will, in any event, determine the appropriate
dose for the
individual subject. Moreover, for human administration, preparations should
meet sterility,
pyrogenicity, general safety and purity standards as required by FDA Office of
Biologics
standards.
In one embodiment, the present invention is directed at the treatment of human
malignancies. A variety of different routes of administration are
contemplated. For example, a
classic and typical therapy will involve direct, intratumoral injection of a
discrete tumor mass.
The injections may be single or multiple; where multiple, injections are made
at about 1 cm
spacings across the accessible surface of the tumor. Alternatively, targeting
the tumor
vasculature by direct, local or regional infra-arterial injection are
contemplated. Also
contemplated are methods for aerosol delivery to the airway.
In another embodiment, the present invention is directed at the treatment of
diseases of
the airway, including the trachea and bronchial passages. An ideal delivery
method is via
aerosol. U.S. Patent 5,543,399 (incorporated by reference) describes methods
for the delivery of
compositions including the CFTR gene to airway. U.S. Patent 5,756,353 and U.S.
Patent
5,641,662 (both incorporated by reference) also describe delivery of genes to
the lung by aerosol.
Other airway delivery methods and compositions are described, for example, in
WO 93/12240,
WO 90/07469, WO 96/27393, WO 96/22765 and WO 96/32116, all of which are
incorporated by
reference.
G. EXAMPLES
Example 1
Materials and Methods. The present example details some of the methods
employed in
the present invention.
42
CA 02271826 1999-OS-11
I. Cell Culture Methods
Primary culture of human airway epithelia. Primary cultures of human airway
epithelia were prepared from trachea and bronchi by enzymatic dispersion as
previously
described (Konda et al., 1991; Yamaya et al., 1992; Zabner et al., 1996).
Briefly, epithelial cells
were dissociated and seeded onto collagen-coated, semipermeable membranes with
a 0.4 pm
pore size (Millicell-HA, surface area 0.6 cm2, Millipore Corp., Bedford, MA).
24 hours after
seeding, the mucosal media was removed and the cells were allowed to grow at
the air-liquid
interface as reported previously (Yamaya et al., 1992). The culture media
consisted of a 1:1
mixture of DMEM and Ham's F12 with 2°/ LJItroser G (Sepracor Inc.,
Marlborough, MA), 100
U/ml Penicillin and 100 pg/ml Streptomycin. Representative preparations from
all cultures were
scanned by EM and the presence of tight junctions confirmed by transepithelial
resistance
measurements (resistance >1000 Ohm x 2cm2). All preparations used in the study
were well
differentiated and only well differentiated cultures >2 wk old were used in
these studies.
Previous studies show that differentiated epithelia in this model are
multilayered and consist of
ciliated cells (cytokeratin 18 positive), secretory cells containing granules
that are reactive to
goblet and mucous cell specific antibodies, and basal cells positive for
cytokeratin 14 (Yamaya et
al., 1992; Zabner et al., 1996). This study was approved by the Institutional
Review Board of
the University of Iowa.
II. Reagents
Recombinant retrovirus and vector forrmrtat~iroa. High titer recombinant
amphotropic
and xenotropic retroviruses were prepared at Chiron Technologies-Center for
Gene Therapy, Inc.
(San Diego, CA) as described previously (Bosch et al., 1996; McCray et al.,
1997a). Reporter
viruses used included DA-(3ga1 ((3-galactosidase reporter, amphotropic
envelope) and DX-(3ga1
((3-galactosidase repoder, xenotropic envelope) (Irwin et al., 1994; Jolly,
1994). The
(3-galactosidase reporter gene was driven by retroviral LTR. The vector
formulation buffer was
19.5 mM trimethamine at pH 7.4, 37.5 mM NaCI, and 40 mg/ml lactose. The
osmolality of the
viral buffer was 105 mmol/kg as measured using a vapor pressure osmometer
(Model SS00,
43
~
CA 02271826 1999-OS-11
Wescor, Inc., Logan, UT). Polybrene was included in all infection mixtures at
a concentration of
8 p.g/ml.
A vector expressing human CFTR was prepared by cloning the human CFTR cDNA
(Rommens et al., 1989) into a retroviral vector plasmid with the viral LTR
promoter (Jolly,
1994). Producer clones were selected based on the ability of crude vector
stocks to confer
cAMP-activated Cl- transport to undifferentiated CF epithelia in vitro and a
stable producer cell
line was selected (Jolly, 1994; McCray et al., 1993). For gene transfer to
differentiated CF
airway epithelia, crude producer cell supernatants were concentrated by
centrifugation and
applied to epithelia. This may be performed with or without growth factor
stimulation. Epithelia
were tested for the presence of CFTR Cl- currents in Ussing chambers 3 to 10
days after gene
transfer as previously described (McCray et al., 1993).
In selected studies, transepithelial permeability was increased before or at
the time of
application of vector to the apical membrane of cultured epithelia. Treatment
conditions
included water or EGTA. For EGTA treatment, a solution of 1.5 mM EGTA in water
(osmolality 33 mmol/kg) was used to rinse the apical side of cells for 20 min.
An EGTA:virus
mixture was obtained by mixing the viral preparation and 3 mM EGTA in water at
a 1:1 ratio
(osmolality 48 mmol/kg). Gene transfer to the apical surface was performed by
applying vector
in 100 p,l volumes. For gene transfer to the basal side of the cell membrane,
the Millicell culture
insert was turned over and vector applied to the bottom of the membrane in a
100 ~l volume.
III. Assessment of Cell Proliferation
BrdU immunohistochemistry. BrdU labeling and immunostaining was performed
using
a kit from Zymed Laboratories Inc. (South San Francisco, CA). In studies that
investigate the
effects of growth factors, the cells may be treated with 50 - 100 ng/ml growth
factor for 36 h. A
1:100 dilution of the BrdU labeling reagent was added to the culture media and
cells labeled for
4 hours followed by fixation in 10% neutralized Formalin. BrdU histochemistry
was performed
following the methods of the Zymed BrdU kit. Labeled nuclei stained brown
under these
44
CA 02271826 1999-OS-11
conditions. Epithelial cell preparations were examined microscopically en face
or in cross
sections of paraffin embedded membranes and the percentage of brown staining
nuclei
determined. Hematoxylin or 4',6-diamidino-2-phenylindole, dihydrochloride
(DAPI) (Molecular
Probes, Eugene, OR) were used for counterstains.
(3-galactosidase expression. Epithelial cells were fixed with 2%
paraformaldehyde/PBS
solution for 20 min and rinsed with PBS t<vice for 5 min each. X-gal staining
solution was added
to the cells at 37°C for 4 hours to overnight as previously described
(McCray et al., 1995). Cell
membranes were examined microscopically en face or in cross section for (3-
galactosidase
expression. The percentage of ~i-gal positive cells was determined by counting
a minimum of
1000 cells from cross sections of each treated cell culture insert.
N. Identification of amphotropic retroviral receptor (Pit-2) in cultured human
airway epithelia.
Pit-2 antibody. Affinity purified polyclonal Pit-2 antisera were prepared by
immunizing
rabbits with a synthetic peptide (GLVR-2A), an extracellular domain sequence
that is conserved
in rat and human Pit-2 (Miller et al., 1994; Miller and Miller, 1994). The
peptide was coupled to
key hole limpet hemocyanin (KLH) and rabbits were then immunized. Different
post
immunization bleeds were tested using ELISA and immobilized 'free' peptide.
Resulting
anti-peptide antisera were pooled and affinity purified on columns of
immobilized GLVR-2A
peptides. These affinity purified antibodies were then used for all Pit-2
expression analyses.
Western blot. Airway epithelial cells were lysed in 10 mM Tris/HCl buffer (pH
7.4)
containing 0.5% Triton-X 100 and 1 mM PMSF. Cell lysates were collected and
protein
concentrations determined by the Lowry method. 35 p,g of protein in loading
buffer was
denatured at room temperature (not boiled) for 40 min and run on a 10%
polyacrylamide SDS
gel. Following electrophoresis, the proteins were transferred to a Nytran
membrane (Schleicher
and Schuell Inc., Keene, NH) by electroblotting and blocked with 10% skim milk
powder.
Immunoblotting was performed with the polyclonal antisera at a 1:10,000
dilution. Goat
anti-rabbit IgG conjugated with horseradish peroxidase was used for the
secondary antibody
CA 02271826 1999-OS-11
(Bio-Rad, Hercules, CA) and the proteins identified by autoradiography using
the ECL system
(Amersham, Arlington Heights, IL). The specificity of the antibody was
confirmed by
preincubating the antibody with 20 pM free synthetic peptide for 30 min in
PBS, 1% BSA at
room temperature prior to incubation with the blots.
Measurement of transepithelial resistance. Differentiated epithelial cells can
be treated
with SO ng/ml the desired growth factor for 24 h. Transepithelial resistance
was measured as
follows: Solutions of water or 1.5 mM EGTA in water are used to rinse the
apical side for
20 min. The solution was then replaced with viral formulation buffer alone, or
a 1:1 mixture of
viral formulation buffer plus 3 mM EGTA water solution. and incubated for 4 h.
Control cells
can receive growth factor treatment and PBS washes substituted for water or
EGTA washes.
Transepithelial resistance was monitored with an ohmmeter (EVOM; World
Precision
Instruments, Inc. Sarasota, FL) over 16-18 hours until resistance returned to
base line.
Example 2
Growth Factors Stimulate Proliferation of Differentiated Airway Epithelia In
Vitro
and In Vivo. To determine whether growth factors are mitogenic to human airway
epithelia,
primary cultures of cells grown at the air-liquid interface were utilized.
These cultures have ion
transport properties and morphology similar to the intact surface epithelium
(Smith et al., 1996;
Kondo et al., 1991) and after 14 days in culture the cells are well-
differentiated, ciliated, and
have ion transport properties similar to the intact airv~~ays (Zabner et al.,
1996; Yamaya et al.,
1992). The data show that 200 ng/ml of HGF stimulated cell division 3-fold as
measured by ['H]
thymidine incorporation. After 24 hours of growth factor treatment, growth
factor administration
doubles the monolayer cell number compared to control. HGF treatment (200
ng/ml) increased
cell labeling to 17-36%, while 5-10% of untreated controls were BrdU positive
(n = 3). EGF
(200 ng/ml) and heregulin (5 nM) also both increased BrdU incorporation over
control epithelia.
These results clearly document that differentiated, mitotically quiescent
human airway epithelia
proliferate in response to growth factors.
46
CA 02271826 1999-OS-11 ~ _
In order to investigate whether growth factors stimulate epithelial
proliferation in vivo,
the desired growth factor (e.g., 5 ~g/g) may be instilled into the tracheas of
3 wk old rats once
daily on two consecutive days. On days 3, 4, 5, and 7 following the first
instillation the animals
are given an injection of bromodeoxyuridine (BrdU) and sacrificed. The tissues
are formalin
fixed, paraffin embedded, and sections immunostained for BrdU and PCNA
(proliferating cell
nuclear antigen) (McCray et al., 1997b). Immunostaining with an antibody to
PCNA will serve
to identify the regions and cell types in the lung showing proliferation in
response to the growth
factor applied. It is predicted that it is likely that animals that receive
intratracheal growth
factors will develop a transient wave of epithelial cell proliferation that is
greatest in the alveolar
epithelium when compared to PBS treated control animals. Such a peak
proliferative response is
expected to occur within 72 hours after the second dose of growth factor for
both bronchiolar and
alveolar epithelia. Tissue sections are expected to demonstrate a "knobby"
epithelial
proliferation pattern in the alveolus suggestive of type II cell
proliferation. Morphologically
these findings would be similar to those reported in adult rats in response to
intratracheal KGF
(Ulich et al., 1994). Proliferating cells also may be noted in the bronchiolar
epithelium of
growth factor treated rats. These studies should conclusively demonstrate that
growth factors
stimulate airway epithelial proliferation both in vitro and in vivo.
Gene Transfer is Polar in Differentiated Human Airway Epithelia. Next, it was
of
interest to determine whether the levels of epithelial proliferation
stimulated by growth factors
supported gene transfer with high titer amphotropic enveloped MMLV expressing
(3-
galactosidase (DA-~igal). To address this question, airway epithelia are
stimulated with growth
factors for 24 hours followed by application of DA-(3ga1 amphotropic vector
(MOI ~20) to the
apical side of the membrane for 4 h. Three days after infection, X-gal
staining may be performed
to evaluate transgene expression. When vector is applied to the apical
membrane of quiescent or
growth factor -stimulated cells no gene transfer is seen. It was hypothesized
that the Pit-2
amphotropic receptors were not accessible from the apical surface. To test
this hypothesis, the
epithelial sheets were inverted and vector was applied to the basal surface
for 4 hours at an
estimated MOI of 20; 72 hours later numerous (3-gal expressing epithelia were
noted. Vector
47
..
CA 02271826 1999-OS-11
application to the basal side of growth factor-treated epithelia results in
improved gene transfer,
with significant numbers of cells expressing the transgene. The cells
expressing the transgene
are predominantly those with their basal membrane in contact with the membrane
support and
many had morphologic characteristics of basal cells. Cells that received no
growth factor also
show occasional X-gal positive cells when vector was applied to the basal
surface, in agreement
with the lower mitotic indices of cells grown under these conditions. Thus,
gene transfer with
MMLV is strikingly polar in differentiated human airway epithelia. It also was
found, using the
same experimental protocol, that gene transfer with MMLV vectors with the
xenotropic envelope
and VSV-G pseudotype show similar polarity of gene transfer. These results
were unexpected
and suggested that either the receptors for amphotropic and xenotropic viruses
were not present
or that they were inaccessible to virus applied to the apical surface. Next
studies were performed
to determine if the Pit-2 receptor was expressed on human airway epithelia and
if growth factor
application influenced Pit-2 expression.
The GLVR-2 amphotropic receptor is expressed in airway epithelia and
upregulated by growth factors. Retroviral transduction begins with the
interaction and binding
of viral envelope glycoproteins and cell surface receptors. In the case of
amphotropic enveloped
vectors, the receptor (Pit-2) has been cloned and identified as a sodium-
dependent phosphate
transporter (Miller et al., 1994; van Zeijl et al., 1994), a 656 amino acid
transmembrane protein.
Other than Northern blot data demonstrating that the amphotropic receptor mRNA
is present in
whole lung from rat (Miller et al., 1994), there are no data regarding the
abundance and
distribution of Pit-2 protein in the lung. Rabbit polyclonal antisera
generated against a synthetic
peptide sequence shared by rat and human Pit-2 was used in Western blots and
identified a
protein of ~62 kD in rat lung. The 62 kD band was competed off in the presence
of the synthetic
peptide. When rats are treated with intratracheal growth factors, the
abundance of the protein
will transiently increase, with the highest level of expression expected at 48
hours after the first
dose of growth factor. Similarly, quiescent human airway epithelial cells
express low levels of
Pit-2 protein and protein abundance can be increased following growth factor
treatment.
48
CA 02271826 1999-OS-11
Vector application from the mucosal and serosal surface targets different cell
populations. The marked polarity of gene transfer to differentiated epithelia
suggested that
access to receptors was extremely limited from the apical surface. Thus, it is
suggested that if
Pit-2 receptors are located on the basolateral membrane, transiently
disrupting epithelial tight
junctions might allow vector access to the receptor. This may be tested by
adding SO pl of water
or 3 mM EGTA in water to the apical surface of growth factor-stimulated human
monolayers for
20 min and then adding vector to the mucosal surface for 4 h. These treatments
should cause a
transient fall in transepithelial resistance that fully returns to baseline
over several hours. Such
interventions dramatically increase gene transfer efficiency. 3 ~ 0.5% of
epithelia from
preparations pretreated with water alone expressed 13-gal (mean ~ SE, n = 13
membranes from 3
preparations). Sequential treatment with water then EGTA in water for I 0 min
each, followed by
addition of vector result in 8 t 1.3 % cells positive. A further incremental
increase in expression
was seen in cells pretreated with a combination of water and EGTA for 20 min
followed by
addition of vector (20.3 ~ 2.5 % cells positive, mean t SE, n = 9 membranes
from 2
preparations). Finally, cells pretreated with water and EGTA for 20 min
followed by the
addition of vector containing EGTA showed a further increase in gene transfer
such that 34.3 ~
5.4 % of the cells were (3-gal positive 3 days following vector delivery (mean
~ SE, n = 9
membranes from 2 preparations). In control studies, it was shown that
application of H20 or
EGTA to epithelia had no effect on proliferation.
Different cell populations were targeted when vector was applied to the basal
surface or
when it was applied to the apical surface in the presence of EGTA. In contrast
to the results
obtained with vector applied to the basal surface of the epithelia, cells at
both the apical and basal
levels of the cell layer were transduced under these conditions (basal cells,
ciliated cells,
intermediate cells). To quantify the differences in cells targeted between
these two methods, (3-
gal expressing cells were identified in the epithelium and scored using
morphologic criteria as
basal cells (cuboidal cells in contact with the basement membrane whose apical
pole does not
reach the lumen), ciliated cells (columnar cells with cilia), or intermediate
cells (columnar cells
residing in the lower half of the surface epithelium with no lumenal contact
or secretory
49
CA 02271826 1999-OS-11
granules). Using these criteria, 200 (3-gal positive cells were counted in the
cell layers in which
vector was applied to basal side only and in cells in which vector applied to
apical surface with
EGTA in vector buffer. For the vector applied to the basal surface, the
results were: 80% basal,
13% intermediate, and 7% ciliated. In contrast, for the apical/EGTA
application, the results
were: 36% basal, 36% intermediate and 28% ciliated. Therefore, it was
concluded that vector
application to the basal surface targets predominantly basal cells while
apical application with
EGTA targets cells at all levels of the epithelial sheet.
Next, it was asked whether other growth factors that stimulate epithelial
proliferation
facilitated gene transfer with MMLV. Differentiated human airway epithelia
were treated with
HGF (200 ng/ml), EGF (200 ng/ml), or heregulin (5 nM) for 24 hours. Then high
titer
(1 x lOgcfu/ml) nuclear targeted (3-gal vector prepared in the Vector Core
(Manuel et al., 1997)
was applied to the apical surface (MOI ~10) in hypotonic buffer with EGTA.
Each growth factor
stimulated proliferation. Cells treated with HGF and heregulin showed similar
proliferative
responses. The finding of different levels of gene transfer following
equivalent proliferative
responses with HGF and heregulin suggests that in addition to cell division,
growth factors have
other effects that allow gene transfer with MMLV.
Gene transfer to proliferating differentiated CF airway epithelia corrects the
Cl-
transport defect. The results with MMLV (3-gal vectors in combination with
growth factors
suggest that it might be possible to correct the CF defect in epithelia using
such an approach.
However, it was not known if the cell types targeted using such an approach
would be su~cient
to correct Cl- transport. Therefore, a high titer amphotropic MMLV vector
expressing the human
CFTR cDNA (DA-CFTR) was generated. Then, the question was asked whether the
strategy
used above could also enhance gene transfer with a CFTR vector and correct the
Cl- transport
defect. Supernatants were collected from the DA-CFTR packaging cell line and
the vector was
concentrated. Differentiated human airway epithelia are treated with growth
factors for 24 hours
followed by application of DA-CFTR (estimated MOI ~1) to the mucosal surface
in the presence
of 3 mM EGTA. 10 days later epithelia with and without vector application were
assayed for
CA 02271826 1999-OS-llv'
cAMP activated Cf current in Ussing chambers. In control cells that received
no vector
application, correction of the CFTR transport defect was not detected. Only
cells receiving the
CFTR vector showed evidence of cAMP activated Cl- current. This is a novel
observation for
MMLV-based vectors as previous studies were only able to document correction
of the CF
transport defect if the retroviral vector was applied to poorly
differentiated, dividing cells
(Engelhardt et al., 1992).
Example 3
An integrating vector can target nondividing cells and produce persistent
expression
and correction of the CF defect. If an integrating vector can infect
nondividing cells it might
offer advantages for gene transfer to airway epithelia because the level of
proliferation in the
airways in vivo is normally very low. Several recent studies demonstrate that
hybrid lentiviral
vectors infect nondividing mammalian cells (Naldini et al., 1996; Kafri et
al., 1997). However,
the one report of gene transfer to human airway epithelia with HIV-based
lentivirus suggests that
the gene transfer efficiency is greater in cells that are proliferating
(Goldman et al., 1997). In a
preliminary study, HIV-based lentivirus was applied to the apical or basal
surface of
differentiated human airway in basal media in the absence of growth factors.
The vector
expresses E. coli (3-galactosidase and the envelope is pseudotyped with the
VSV-G protein.
Crude vector supernatants were prepared by transiently co-transfecting 293T
cells with 3
plasmids and the final concentration and purification of the vector was
completed. Similar to the
findings with MMLV-based vectors, when VSV-G lentivirus was applied to the
apical surface of
epithelia without pretreatment with growth factors (MOI ~ 1 ), no gene
transfer occurred. In
contrast, when vector was applied to the basal surface of quiescent epithelia,
f3-galactosidase
expressing cells were noted. Thus, the same polarity of gene transfer that was
noted with the
MMLV amphotropic envelope is noted for the VSV-G pseudotyped vector. When the
vector
was applied to the apical surface of quiescent cells in the presence of
EGTA/hypotonic buffer,
gene transfer was enhanced. From this study, it was concluded that HIV-based
lentivirus can
infect non-dividing well-differentiated airway epithelia. Similarly, a study
with FIV-based
sl
- CA 02271826 1999-OS-11 .
lentivirus gave similar results. Importantly, airway epithelia that are growth
arrested by
aphidicolin are susceptible to infection by HIV- and FIV-based lentiviruses.
Example 4
Integrating vectors can correct the CF defect in differentiated epithelia in
vivo Ca2+
chelation transiently disrupt epithelial tight junctions in vivo. Preliminary
studies were
performed in rats to test the feasibility of using hypotonic solutions with
EGTA to increase
transepithelial permeability in vivo. 3 wk old rats were tracheotomized and a
small caliber PE
catheter inserted into the left lobe of the lung. 100 pl of 3 or 12 mM EGTA in
water or PBS was
mixed with 100 nM fluorescent beads (a marker for viral particles) and
instilled into the lungs.
One hour later the animals were sacrificed and lung tissue sections examined
under fluorescence
microscopy to determine if the fluorescent beads penetrated the epithelial
layer. In the PBS
control animal, fluorescent particles were only noted in the airway lumen. In
contrast, in animals
receiving either 3 or 12 mM EGTA, beads were seen throughout the cell layer
and close to the
basement membrane. It also was asked if animals treated with EGTA/H20
developed changes in
lung morphology. Animals received PBS or 12, 60, or 120 mM EGTA/water into the
left lobe of
the lung and 1 wk later were sacrificed and H & E stained lung tissues
examined. The lungs of
all animals showed morphology similar to the control except for the animal
that was treated with
120 mM EGTA. In that animal thickening of the interstitial space was noted in
tissue sections.
These studies demonstrate the feasibility of using maneuvers that transiently
disrupt tight
junctions for in vivo gene transfer with integrating vectors.
Growth factors stimulate epithelial proliferation in a CF mouse model. The
OF508
CF mouse is a model for correcting the CFTR transport defect in vivo using the
nasal epithelium
as a model. In order to investigate whether the marine nasal epithelium
proliferates in response
to growth factors, adult mice are sedated and given growth factor compositions
via IV and
intranasally on 2 consecutive days. On day 3 the animals are given
intraperitoneal and intranasal
doses of BrdU and sacrificed 2 hours later. Based on BrdU histochemistry
animals treated with
52
CA 02271826 1999-OS-11
growth factor should show an increase in proliferating cells compared to
controls. Such results
provide confirmation that growth factors stimulate proliferation of nasal
epithelia.
Growth factors stimulate epithelial proliferation in human bronchial
xenografts.
The tracheal xenograft model closely resembles the CF human airways in terms
of morphology,
electrophysiologic defects and biochemical defects. Studies were performed to
verify whether
growth factors stimulate proliferation in tracheal xenografts populated with
human airway
epithelia. 100 ng/ml of the desired growth factor is instilled into the lumen
of mature
differentiated xenografts on 2 consecutive days. Simultaneously, animals are
given 5 pg/g
growth factor intravenously each day. PCNA histochemistry demonstrates that
growth factor-
treated grafts show an increase in the number of PCNA positive epithelia.
Example 5
In vivo Demonstration of Growth Factors Stimulation of Transient Epithelial
Proliferation. The present example provides a description of the type of
experiment to be
performed in order to evaluate whether growth factors stimulate transient
epithelial proliferation
in vivo. This example give details of the animals and procedures to be used in
suhc a study.
Animal procedures.
Sprague-Dawley rats (age 15-20 days, weight ~30 g) can be used in these
studies. Growth
factor and recombinant retrovirus may be delivered to the lung by direct
tracheal instillation.
Animals are then anesthetized with methoxyfluorane, gently restrained and the
larynx visualized.
A 22 gauge Teflon intravenous catheter is passed through the mouth and into
the trachea and the
growth factor or viral suspension instilled using a 1 ml tuberculin syringe
To stimulate epithelial proliferation in the lung, animals are given an
appropriate amount
of growth factor (e.g., 2.5 p,g/g body weight) intratracheally, twice daily on
consecutive days.
Control animals should receive PBS in equal volume. This growth factor dose
range has was
previously shown to stimulate proliferation in the alveolar and bronchiolar
epithelia of adult rats
53
CA 02271826 1999-OS-11
(Ulich et al., 1994). In the gene transfer studies, animals receive 80 p.l of
DA-(3gal
intratracheally on 3 consecutive days following growth factor administration
(total dose ~10~
cfu/animal). Control animals receive an equal volume of diluent.
Tissue histochemistry.
Cell proliferation by PCNA staining. Proliferating pulmonary epithelial cells
are
identified using antibodies against proliferating cell nuclear antigen (PCNA,
PC 10 clone, Dako)
as previously described (McCray et al., 1997; Schwarting, 1993). To determine
the percentage
of cells proliferating in response to a growth factor, groups of animals
receive the appropriate
amount of growth factor (e.g., 5 pg/g/day) or PBS on days 1 and 2. On days 3,
4 and 7 groups of
animals are killed and lungs are prepared for PCNA analysis.
Immunohistochemistry is performed on 5 micron thick sections of para~n-
embedded,
formalin fixed tissues. The PCNA antibody is applied at a 1:100 dilution
overnight at 4°C. This
monoclonal antibody recognizes the 36 kD polymerase delta accessory protein, a
DNA binding
protein expressed in cells in the Gl, S, M, and G2 phases of the cell cycle.
The labeled
strepavidin biotin peroxidase (LSAB Dako Corp., Santa Barbara, CA) detection
system can be
used for detection, after antigen retrieval (citrate buffer and microwave).
Positive cells will stain
brown with this method. Sections can be counterstained with hematoxylin. Human
tonsil may
be used as a positive control, while in the negative control the primary
antibody was omitted.
PCNA positive cells from random fields (40 X magnification) are counted from a
number
of non-adjacent fields for each section, with a minimum of 100 cells per field
counted per
animal. Brown staining nuclei is scored regionally as alveolar or bronchiolar.
Differences in
proliferation between growth factor-treated groups and PBS controls may be
analyzed. The
percentage of PCNA positive staining in the control (PBS) group is considered
background and
was subtracted from the growth factor groups in the analysis.
54
CA 02271826 1999-OS-11
X gal staining. To detect gene expression in animals treated with growth
factor, animals
are killed 5 days after the final dose of intratracheal retrovirus, lungs are
removed and perfused
with 2% paraformaldehyde in PBS and fixed overnight. Lungs are stained for 4 h
at 37°C with
40 mg/ml of X-gal from Gold Biotechnology Inc. (St. Louis, MO) using
previously described
techniques (McCray Jr. et al., 1995). After en bloc staining, tissues are
frozen in O.C.T. and 10
~m sections placed onto slides and counter-stained in nuclear fast red for
photomicroscopy.
Cells expressing (3-galactosidase stain blue with a cytoplasmic pattern using
this method.
Detection of amphotropic retrovirus receptor (Pit2) expression by western
blot. Lung
tissue is homogenized in lysis buffer (10 mM Tris/HCI, pH 7.4, 1 mM PMSF, 0.5%
Triton
X-100). The resultant cell lysate is collected and protein quantified by the
Lowry method. 35 pg
of total protein from each sample is then loaded in a 10% SDS-PAGE gel.
Following transfer to
a Nytran membrane (Midwest Scientific, St. Louis, MO), Pit2 protein is
identified by
immunoblotting with rabbit antisera. Affinity purified polyclonal Pitt
antisera may be prepared
by immunizing rabbits with a synthetic peptide (GLVR-2A), a Pit2 extracellular
domain
sequence that is conserved in rat and human (Miller et al., 1994; Miller and
Miller, 1994). The
peptide is coupled to key hole limpet hemocyanin (KLH) and rabbits are then
immunized.
Different post immunization bleeds may be tested using ELISA and immobilized
'free' peptide.
Resulting anti-peptide antisera is then pooled and affinity purified on
columns of immobilized
GLVR-2A peptides. The affinity purified antibodies are used for subsequent
Pit2 expression
analyses. Goat anti-rabbit serum conjugated with horseradish peroxidase may be
used as a
second antibody (Bio-Rad). Specific antigen and antibody reaction can be
detected by the ECL
system (Amersham).
In vitro gene transfer to rat airway epithelia by apical or basolateral
administration.
Primary cultures of rat airway epithelia can be prepared from trachea by
enzymatic dispersion
using methods similar to those described for human epithelia (Zabner et al.,
1996). Epithelial
cells can be dissociated and seeded at a density of 3 x 105 cells/cm2 onto rat
tail collagen-coated
permeable membranes with a 0.4 pm pore size (Millicell-HA, surface area 0.6
cm2, Millipore
CA 02271826 1999-OS-11
Corp., Bedford, MA). 24 h after seeding, the mucosal media is then removed and
the cells are
allowed to grow at the air-liquid interface as reported previously (Zabner et
al., 1996; Yamaya
et al., 1992). The cells are maintained at 37°C in a humidified
atmosphere of 7% C02 and air.
Preparations from all cultures are then examined by transepithelial resistance
measurements. It is
recommended that only well-differentiated airway cells which demonstrate tight
junction
formation and high transepithelial resistances (R~e >1000 Ohm x cm2) be used
in the study.
Nuclear targeted /3-gal retroviral vector is applied to growth factor-
stimulated differentiated
epithelia at an MOI of -20 on apical side or basolateral side and incubated
for 4 h. Three days
later, transgene expression can be assessed by X-gal histochemical staining.
Expected Results
Immunostaining with an antibody to PCNA will identify the regions and cell
types in the
lung which proliferate in response to the growth factor. It is expected that
those animals that
receive intratracheal growth factor over 2 consecutive days will likey develop
a transient wave of
epithelial cell proliferation that is greatest in the alveolar epithelium when
compared to PBS
treated control animals. Tissue sections may demonstrate a "knobby" epithelial
proliferation
pattern in the alveolus, suggestive of type II cell proliferation.
Proliferating cells also may be
present in the bronchioles.
Growth factor induced proliferation facilitates retroviral-mediated gene
transfer. In
order to determine if growth factor-induced epithelial proliferation
facilitates gene transfer with
high titer amphotropic enveloped retrovirus, 80 ~l of DA-(3ga1 retrovirus is
instilled
intratracheally for 3 consecutive days following pretreatment with growth
factor as described
above. The total delivered dose should be approximately 1 x 10' cfu/animal.
Five days
following the final dose of virus, animals are sacrificed and tissues fixed
and X-gal stained.
Tissue sections from animals that receive growth factor and retrovirus should
show epithelial
cells expressing cytoplasmic (3-galactosidase. (3-gal positive cells should be
most prevalent in the
alveolar epithelium with a more rare positive bronchiolar cells. In contrast,
rats that receive
retrovirus without growth factor pretreatment should show no ~i-gal expressing
cells.
56
CA 02271826 1999-OS-11 .
Expression of the amphotropic receptor (Pit2) in vivo. Amphotropic retroviral
infection
is mediated through the Pit2 receptor. A lack of expression or low abundance
of expression
might underlie the low efficiency of gene transfer in vivo. To test this
hypothesis Pit2 protein
expression may be measured by western blot in lung of animals with and without
growth factor
treatment. Pit2 protein should be detectable in both control samples and
growth factor treated
lungs. Using the same antibody, the pulmonary cell types expressing Pit2 can
be localized by
immunohistochemistry (Bosch et al., 1998).
Effects of epithelial polarity on gene transfer e~ciency. The airway
epithelium is a
polarized cell population. It was hypothesized that the apical surface, which
serves as a barrier
against infection in vivo, may impede gene transfer by retroviral vectors.
This may be tested in
vivo as follows. Primary cultures of differentiated rat tracheal epithelial
cells can be grown at the
air-liquid interface. This allows epithelia to differentiate into a tight
epithelial sheet which
closely mimics the in vivo airways. Growth factor is then added to the culture
media 24 h prior
to gene transfer to stimulate epithelial cell proliferation. After
stimulation, (3-gal retrovirus is
applied to the apical or basolateral surface at an MOI of ~20 and incubated
for 4 h at 37°C. For
basolateral transduction, the cell culture inserts are turned over and the
viral mixture is placed on
the underside of the insert for 1 h, then the insert are re-placed in the
upright position. Three
days later the (3-gal expression was evalutated with X-gal histochemistry. It
is expected that
growth factor will stimulate robust proliferation of cultured rat tracheal
epithelia.
57
..
CA 02271826 1999-OS-11
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