Note: Descriptions are shown in the official language in which they were submitted.
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Method of Inducing Vasodilation and Treating Pulmonary
Hypertension Using Adenoviral-Mediated Transfer
of the Nitric Oxide Synthase Gene
Background of the Invention
S Statement as to Rights to Inventions ll~ade Under
Federally Sponsored Researcl: and Development
Part of the work performed during development of this invention was
supported by U.S. Government funds. The U.S. Government may have certain
rights in this invention.
Field of the Invention
This invention relates to a gene therapy method for inducing pulmonary
vasodilation by transducing a nitric oxide synthase gene into lung tissue.
This
invention also relates to methods of treating pulmonary hypertension and
pharmaceutical compositions for treating pulmonary hypertension.
Related Art
Blood flow through the pulmonary circulation is highly regulated. For
example, the pulmonary endothelium regulates pulmonary blood flow and
maintains a low vascular resistance by releasing vasoactive substances, which
control vasomotor tone, vascular patency, and normal vessel wall architecture.
Vanhoutte, N. Engl. J. Med, 319:512-513 (1988).' Vasomotor tone relates to the
degree of active tension in the vessel wall and partially determines the
luminal
diameter of the vessel. Vascular patency refers to the condition of a blood
vessel
where the internal luminal diameter is normal and blood flow is unimpeded.
' This article and all other articles, patents, or other documents cited
or referred to in this application are specifically incorporated herein by
reference.
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Nitric oxide is one compound that plays an important role in regulating
pulmonary blood flow. However, it is a gas with no known storage mechanism,
which diffuses freely across membranes and is extremely labile. Nitric oxide
has
a biological half life on the order of seconds, and its production is tightly
regulated.
Nitric oxide is produced by two classes of nitric oxide synthases (NOS).
Nathan, FASEB J. 6:3051-3064 (1992}. The constitutively expressed nitric oxide
synthases, exist as two isoforms: the endothelial nitric oxide synthase
(ceNOS)
and the neuronal nitric oxide synthase (nNOS}. These isoforms are expressed in
vascular endothelial cells, platelets, and in neural tissues such as the
brain. This
class of nitric oxide synthase is calcium and calmodulin dependent. In blood
vessels ceNOS mediates endothelium dependent vasodilation in response to
acetylcholine, bradykinin, and other mediators. Nitric oxide levels increase
in
response to shear stress, i.e., forces on the blood vessels in the direction
of blood
flow, and the mediators of inflammation. Furchgott and Vanhoutte, FASEB J.
3:2007-2018 (1989); Ignarro, FASEB J. 3:31-36 (1989).
In the nervous system, the neuronal NOS isoform is localized to discrete
populations of neurons in the cerebellum, olfactory bulb, hippocampus, corpus
striatum, basal forebrain, and brain stem. Bredt et al., Nature 347:768-770
(1990). Neuronal NOS is also concentrated in the posterior pituitary gland, in
the
superoptic and paraventricular hypothalmic nuclei, and in discrete ganglion
cells
of the adrenal medulla. Id. The widespread cellular localization of the
neuronal
NOS isoform and the short half life and diffusion properties of nitric oxide
suggest that NOS plays a role in nervous system morphogenesis and synaptic
plasticity.
The second class, inducible nitric oxide synthase (iNOS), is expressed in
macrophages, hepatocytes, and tumor cells. Steuhr et al., Adv. Enzymol. Relat.
Areas Mol. Biol. 65:287-346 (1992); Lowenstein et al., Proc. Natl. Acad. Sci.
(USA) 89:6711-6715 (1992). The inducible form of NOS is not calcium
regulated, but its expression is induced by cytokines. This form of NOS
functions
as a cytotoxic agent, and NO produced by inducible NOS targets tumor cells and
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pathogens. Hibbs et al., Biochem. Biophys. Res. Comm. 157:87-94 (1988);
Nathan, FASEBJ. 6:3051-3064 (1992); Marietta, Trends Biochem. Sci. 14:488-
492 ( 1989).
All isoforms of NOS catalyze the conversion of L-arginine to L-citrulline
with production of NO. In vascular smooth muscle cells and in platelets, NO
activates soluble guanylate cyclase, which increases intracellular guanosine
3',5'-
cyclic monophosphate (cGMP), thereby inducing vasorelaxation and inhibiting
platelet aggregation. The anti-platelet effect of NO and its vasodilatory and
anti-
proliferative action on pulmonary vascular smooth muscle cells suggest that NO
may be an important modulator of pulmonary hypertension. Moncada et al.,
Pharmacol. Res. 43:109-142 (1991 ); Garg et al., J. Clin. Invest. 83:1774-1777
( 1989); Roberts et al., Circ. Res. 76:21 S-222 ( 1995); Heath, Eur. Respir.
Rev. 3:555-558 (1993); Radomski et al., Biochem. Biophys. Res. Commun.
148:1482-1489 (1987); Assender et al., J. Cardiovasc. Pharmacol. 17:104-107
(1991); de Graaf et al., Circulation 85:2284-2290 (1992).
The sequence of the various NOS isoforms have been published or are
available in Genbank under the following accession numbers:
Species:
Gene: Man Rat Mouse Cow
Neuronal U17327 X59949 D14552
D 16408
L02881
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Macrophage (iNOS) L09210 D14051 M87039 U18331
X85759-81 D83661 U43428 U14640
U18334 U26686 L23806
U31511 U16359 L09I26
U20141 D44591 M92649
U05810 X76881 M84373
X73029 U02534
L24553 L12562
Endothelial X76303-16 U18336 M89952
L26914 U28933 L27056
L23210 M95674
L10693 M99057
M95296 M89952
M93718
Each of these sequences are expressly incorporated herein by reference.
The different forms of NOS are about 50 to 60 percent homologous overall.
Several in vitro and in vivo results suggest that NO may play a role in the
pulmonary vascular response to hypoxia. For example, in perfused isolated
lungs,
hypoxia induces a significant reduction in contractile responses to
acetylcholine
and to inhibitors of NOS. Adnot et al., J. Clin. Invest. 87:155-162 {1991). In
isolated pulmonary vascular rings hypoxia suppresses basal and agonist-
stimulated release of NO. Johns et al., Circ. Res. 65:1508-1515 ( 1989); Shaul
et
al., J. Cardiovasc. Pharmacol. 22:819-827 (1993). In endothelial cells,
hypoxia
inhibits NO production by reducing ceNOS mRNA levels and ceNOS mRNA
stability. McQuillan et al. , Am. J. Physiol. 2G7:H 1921-H 1927 ( 1994).
Moreover,
downregulation of ceNOS mRNA and protein correlate inversely with the
severity of the plexogenic pulmonary arteriopathy in the lungs of patients
with
pulmonary hypertension. Giaid et al., N. Engl. J. Med. 333:214-221 (1995).
Therefore, hypoxia-induced hypertension may correlate with reduced NO
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generation from pulmonary endothelium affecting the balance between pulmonary
vasoconstrictive and vasodilatory stimuli.
In addition to hypoxia-induced pulmonary hypertension, there are other
forms of pulmonary hypertension. For example, pulmonary hypertension can
result from disease states such as interstitial lung diseases with fibrosis,
e.g.,
sarcoidosis and pneumoconioses, e.g., silicosis. Pulmonary hypertension can
also
result from emboli, from parasitic diseases such as schistosomiasis or
filariosis,
from multiple pulmonary artery thromboses associated with sickle cell disease,
and from cardiac disease, such as cor pulmonale, and from ischemic and
valvular
heart disease.
In addition to resulting from other disease, pulmonary hypertension can
also be a primary disease condition. Primary pulmonary hypertension is an
uncommon disease, which can only be diagnosed after a thorough search for the
usual causes of pulmonary hypertension. Ordinarily, the natural course of this
disease encompasses about five years, and it is normally fatal, with treatment
being palliative. While pharmacological vasodilator therapy for primary and
secondary pulmonary hypertension is known, these methods often have
undesirable systemic hypotensive side effects.
The use of gene therapy for the treatment of various diseases and disorders
has advanced significantly over the last several years. In contrast to
traditional
pharmaceuticals, gene therapy refers to the transfer and insertion of new
genetic
information into cells or the substitution of deficient genetic information
for the
therapeutic treatment of diseases or disorders. In some cases the gene is
expressed in the target cell, while in other cases expression is not required,
e.g.,
antisense technology. The foreign gene is normally transferred into a cell
that
proliferates to spread the new gene throughout the cell population. Often stem
cells or pluripotent progenitor cells are the target of gene transfer since
they
proliferate to various progeny lineages that may express the foreign gene.
High efficiency gene transfer systems for hematopoietic progenitor cell
transformation have been described. See Morrow, Arch. N. Y. Acad. Sci. 265:13
(1976); Salzar et al., In Organization and Expression of Globin Genes, A.R.
Liss,
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Inc., New York at 313; Bernstein, In Genetic Engineering: Principles and
Methods, Plenum Press, NY at 235; Dick et al., Trends in Genetics 2:165
(1986);
Kiem, Curr. Opin. Oncol. 7:107-114 (1995). Viral vector transfer systems, such
as retrovirus and adenovirus vectors, generally show a higher efficiency of
transformation than DNA-mediated gene transfer procedures, such as Ca3(P04)z
precipitation and DEAE dextran. Retroviral vector transfer systems also have
the
capacity to integrate transferred genes stably into a wide variety of cell
types.
However, retroviruses require proliferation of target cells for the expression
of the
newly transferred gene. Other non-viral methods of gene transfer include
microinjection, electroporation, liposomes, chromosome transfer, and
transfection
techniques. See Cline, Pharmacol. Ther. 29:69-92 (1985). However, these non-
viral vectors have a relatively Iow in vivo transduction efficiency.
Therefore, there exists a need in the art to develop gene therapy methods
to induce vasodilation in the pulmonary circulation and to treat pulmonary
hypertension.
Summary of the Invention
This invention satisfies these needs in the art by providing a method of
inducing pulmonary vasodilation comprising introducing a vector containing a
nitric oxide synthase gene operably linked to an expression control element
into
the Iungs of a patient in need of pulmonary vasodilation. The nitric oxide
synthase can be a constitutively expressed or an inducible nitric oxide
synthase
gene. In specific embodiments of this invention, the pulmonary vasodilation is
selective, the vector is an adenovirus vector, the nitric oxide synthase gene
is the
endothelial nitric oxide synthase gene, and this vector is transduced into
lung
tissue as an aerosol. In more specific embodiments, the resulting pulmonary
vasodilation does not significantly affect systemic blood pressure or cardiac
index.
This invention also relates to a method of treating pulmonary hypertension
comprising overexpressing nitric oxide synthase in the lungs of a patient in
need
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of treatment by introducing the nitric oxide synthase gene operably linked to
an
expression control element into the lungs of a patient in need of treatment.
In
specific embodiments, this method can be used to treat hypoxia-induced
. pulmonary hypertension, primary pulmonary hypertension, and pulmonary
hypertension secondary to pulmonary or cardiac disease states.
This invention further relates to a pharmaceutical composition comprising
the nitric oxide synthase gene operabIy linked to an expression control
element
and a means for transducing said gene into pulmonary tissue. In an exemplary
embodiment, the pharmaceutical composition comprises AdCMVceNOS in
admixture with a pharmaceutically acceptable carrier.
Further features, objects and' advantages of the present invention will
become more fully apparent to one of ordinary skill in the art from a detailed
consideration of the following description of the invention when taken
together
with the accompanying drawings.
Brief Description of the Figures
Figure 1. Figure 1 depicts the immunostaining for ceNOS in cultured rat
fetal lung fibroblasts infected with AdCMVceNOS (A, top) and AdCMVHirudin
(B, bottom). Abundant ceNOS immunoreactivity was observed in
AdCMVceNOS infected cells, but not in AdCMVHirudin infected cells. The
micrographs are at a 200-fold magnification.
Figure 2. Figure 2 shows the expression of ceNOS in rat lungs. Protein
extracts were obtained from the lungs of rats aerosolized with AdCMVceNOS
(lane 2) and AdCMV~3gal (lane 3). 70 pg samples of lung extracts were
fractionated using SDS-PAGE and were transferred to nitrocellulose membranes.
The presence of ceNOS was detected using a monoclonal antibody, and has an
apparent molecular weight of 135 kDa. An extract from human umbilical vein
endothelial cells was used as a positive control (lane 1 ). A monoclonal
antibody
directed against alpha-actin (42 kDa) was used to monitor the expression of
unrelated protein.
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_g_
Figure 3. Figure 3 depicts ceNOS and ~3-galactosidase gene expression
in the lungs of rats transduced with AdCMVceNOS and AdCMV~igal.
AdCMV(3ga1 transduced rat lungs show nuclear localized (3-galactosidase
staining
in basal airway epithelial cells, alveolar epithelial cells, and adventitial
cells of
the small pulmonary vessels (A, 200-fold magnification). AdCMVceNOS
transduced rat lungs show ceNOS staining in bronchial and alveolar epithelial
cells, and in the endothelium of medium-sized and small pulmonary vessels (B,
200-fold magnif cation; C, 400-fold magnification). AdCMV~3ga1 transduced rats
show no ceNOS staining {D, 200-fold magnification).
Figure 4. Figure 4 depicts cGMP production in RFL-6 cells. Cellular
cGMP content was measured under baseline conditions and after infection with
AdCMVceNOS in the absence and in the presence of L-NAME. Sodium
nitroprusside (SNP) was used as a positive control and AdCMVHirudin,
expressing the thrombin inhibitor, hirudin, was used as a second negative
control
for a virus containing an unrelated gene. The data depicted are means + SEM of
five determinations, except for AdCMVHirudin, which are means + SEM of three
determinations. * indicates P < 0.05 vs. control, AdCMVceNOS + L-NAME, and
AdCMVHirudin.
Figure 5. Figure 5 compares hypoxic pulmonary vasoreactivity in
AdCMVceNOS vs. AdCMV(igal transduced rats. Maximal changes in mean
pulmonary artery pressure (mPAP, left panel) and total pulmonary resistance
index (TPRI, right panel) are depicted for uninfected rats (open bars, n=8),
AdCMV~3ga1 transduced rats (dashed bars, n=8) and AdCMVceNOS transduced
rats {closed bars, n=8) during a 25 min. acute hypoxic challenge. Pressure and
resistance changes are expressed as a percentage of baseline. * indicates P <
0.05
vs. controls and AdCMV(~gal. Values are mean + SEM.
Figure 6. Figure 6 depicts the nucleotide sequence of the endothelial
isoform of the ceNOS gene.
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Detailed Description of the Preferred Embodiments
1. Definitions
In order to provide a clearer and more consistent understanding of the
invention, the following definitions are provided.
As used herein "vasodilation" refers to a physical change in a blood
vessel, which results in an increased blood flow capacity through the blood
vessel. Vasodilation can either be active vasodilation or passive
vasodilation.
Active vasodilation is caused by a decrease in the tonus of smooth muscle in
the
wall of the vessel. Passive vasodilation is caused by increased pressure in
the
lumen of the vessel.
As used herein "introduction" with reference to introducing nucleic acid
into a cell, tissue, or organ refers to the transfer of genetic material into
a cell
using a viral or non-viral vector. This term is meant to encompass
transduction,
transformation, and transfection.
As used herein "transduetion" refers to the transfer of genetic material
into a cell by viral infection. Transduction normally results in the
phenotypic
expression of the genetic material introduced into the recipient cell.
As used herein "pulmonary hypertension" refers to elevated blood
pressure in the pulmonary circulation. Pulmonary hypertension can be either
primary or secondary to pulmonary or cardiac disease. Typically, the pulmonary
blood pressure in humans suffering from pulmonary hypertension is greater than
mm Hg systolic and greater than 12 mm Hg diastolic, or a mean pulmonary
artery pressure in excess of 15-17 mm Hg.
As used herein "primary pulmonary hypertension" refers to pulmonary
25 hypertension not caused by another underlying disease.
As used herein "secondary pulmonary hypertension" refers to
pulmonary hypertension resulting from another underlying disease. Typically,
the
underlying disease causing secondary pulmonary hypertension is a pulmonary or
cardiac disease.
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As used herein "palliative therapy" refers to therapy that alleviates the
symptoms of a disease without curing that disease.
As used herein "cardiac index" refers to the ratio of cardiac output to
body weight.
As used herein "a pharmaceutically acceptable vehicle" is intended to
include solvents, carriers, diluents and the like, which are used as additives
to
preparations of the recombinant DNA molecules containing the NOS gene of the
invention so as to provide a carrier or adjuvant for the administration of
such
compounds.
As used herein "treatment" or "treating" is intended to include the
administration of therapeutic compositions of the invention to a subject for
purposes which may include prophylaxis, amelioration, prevention, or cure of a
medical disorder, such as pulmonary hypertension.
As used herein "nitric oxide synthase" refers to an enzyme capable of
catalyzing the formation of nitric oxide. For example, NOS can catalyze the
formation of nitric oxide from the terminal guanidine nitrogen of arginine,
with
the stoichiometric production of citrulline. A nitric oxide synthase of this
invention can be a constitutively expressed or inducible form of nitric oxide
synthase.
As used herein "constitutive endothelial nitric oxide synthase" or
"endothelial nitric oxide synthase" refers to a nitric oxide synthase having
the
enzymatic properties of the endothelial nitric oxide synthase encoded by a
sequence depicted in Figure 6, or a sequence having significant sequence
homology with the sequence of Figure 6. Typically, an endothelial nitric oxide
synthase of this invention is encoded by a nucleic acid exhibiting greater
than
90% sequence homology with the sequence of Figure 6. In particular
embodiments of this invention, an endothelial nitric oxide synthase of this
invention is encoded by a nucleic acid exhibiting greater than 95% sequence
homology with the sequence of Figure 6.
As used herein a "vector" refers to a plasmid, phage, or other DNA
molecule, which provides an appropriate nucleic acid environment for a
transfer
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of a gene of interest into a host cell. A vector will ordinarily be capable of
replicating autonomously in eukaryotic hosts, and may be further characterized
in terms of endonuclease restriction sites where the vector may be cut in a
determinable fashion. The vector may also comprise a marker suitable for use
in
identifying cells transformed with the cloning vector. For example, markers
can
be antibiotic resistance genes.
As used herein "operable linkage" refers to the position, orientation, and
linkage between a structural gene and expression control elements) such that
the
structural gene can be expressed in any host cell. The term "expression
control
element" includes promoters, enhancers, ribosome binding sites, etc.
II. Detailed Description
A. The Nitric Oxide Synthase Gene
This invention relates to gene therapy methods using the nitric oxide
synthase gene to induce pulmonary vasodilation. In particular embodiments,
this
invention relates to methods of treating pulmonary hypertension.
The human endothelial isoform of the ceNOS gene has been cloned. See
Janssens et al., J. Biol. Chem. 267:14519-14522 (1992). The ceNOS gene
contains a 3609 by open reading frame encoding a 1203 amino acid protein. The
predicted molecular weight of this protein is about 133 kDa. At the amino acid
level, this protein shares about 52 percent sequence homology with the
neuronal
isoform of NOS. This homology is most evident within regions corresponding
to the flavin mononucleotide, flavin adenine dinucleotide, and NADPH binding
sites, and less evident in the amino and carboxy terminal regions.
Plasmid hNOS3C containing the endothelial isoform of the ceNOS gene
was deposited at the American Type Culture Collection, 12301 Parklawn Drive,
Rockville, Maryland 20832, USA on July 17, 1996, as Accession Number 98106.
In addition to using this cloned ceNOS gene having the sequence of
Figure 6 to induce pulmonary vasodilation and treat pulmonary hypertension,
this
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invention also relates to the use of ceNOS genes having similar but not
identical
sequences. Thus, this invention relates to the use of any ceNOS gene capable
of
synthesizing nitric oxide having a similar structure to the ceNOS gene
depicted
in Figure 6. For example, due to the degeneracy of the genetic code, a ceNOS
gene of this invention can encompass any nucleotide sequence encoding the
amino acid sequence of ceNOS, as described in Janssens et al., supra. A ceNOS
gene of this invention can also encompass genes encoding amino acid additions,
substitutions, or deletions, so long as these changes do not significantly
affect the
structural or functional properties of the protein.
This invention also relates to the use of different classes or isoforms of
NOS to induce pulmonary vasodilation and treat pulmonary hypertension. For
example, inducible nitric oxide synthase (iNOS) can be used. Alternatively,
the
neuronal isoform (nNOS) can also be used. As with the ceNOS gene, this
invention also relates to the use of iNOS and nNOS genes having similar but
not
identical sequences to those described supra at 3. For example, iNOS and nNOS
genes useful to practice this invention can encode amino acid additions,
substitutions, or deletions. The invention also encompasses the use of any
iNOS
or nNOS nucleotide sequence encoding the amino acid sequence of iNOS or
nNOS, respectively.
Amino acid sequence deletions generally range from about 1 to 30
residues, more preferably 1 to 10 residues, and typically are contiguous. The
deletions would typically be outside of the flavin mononucleotide, flavin
adenine
dinucleotide, and NADPH binding sites. For example, deletions may be in the
amino or carboxy terminal regions of the protein.
Amino acid sequence insertions include amino and/or carboxy-terminal
fusions of from one residue to polypeptides of essentially unrestricted
length, as
well as intrasequence insertions of single or multiple amino acid residues.
Intrasequence insertions, i.e., insertions within the complete NOS molecule
sequence, generally range from about 1 to 10 residues, more preferably 1 to 5.
An
example of a terminal insertion includes a fusion of a signal sequence,
whether
heterologous or homologous to the host cell, to the N-terminus or C-terminus
of
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the molecule. A fusion sequence often facilitates the secretion of the NOS
functional derivative from recombinant hosts.
A NOS of this invention also relates to a sequence in which at least one
amino acid residue in the NOS molecule has been removed and a different
residue
inserted in its place. Such substitutions preferably are made in accordance
with
the following Table.
Table 1
Original Residue Exemplary Substitutions
Ala Gly; Ser
Arg Lys
Asn Gln; His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Ala; Pro
His Asn; Gln
Ile Leu; Val
Leu Ile; Val
Lys Arg; Gln; Glu
Met Leu; Tyr; Ile
Phe Met; Leu; Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp; Phe
Val IIe: Leu
Substantial changes in functional or immunologicaI identity are made by
selecting substitutions that are less conservative than those in Table 1,
e.g.,
selecting residues that differ more significantly in their effect on
maintaining
(a) the structure of the polypeptide backbone in the area of substitution, for
example, as a sheet or helical conformation, (b) the charge or hydrophobicity
of
the molecule at the target site, or {c) the bulk of the side chain. The
substitutions
that in general are expected to those in which {a} glycine and/or proline is
substituted by another amino acid or is deleted or inserted; {b) a hydrophilic
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residue, e.g., Beryl or threonyl, is substituted for (or by) a hydrophobic
residue,
e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine
residue is
substituted for (or by) any other residue; (d) a residue having an
electropositive
side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) a
residue
having an electronegative charge, e.g., glutamyl or aspartyl; or (e) a residue
having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one
not
having such a side chain, e.g., glycine.
B. Gene Transfer Vectors for the NOS Genes
Once an appropriate NOS gene has been selected, it must be inserted into
an appropriate gene transfer vector for use in gene therapy. Appropriate gene
transfer vectors include retroviral vectors, adenovirus vectors, and non-viral
vectors that can be targeted to specific cell surface receptors for
internalization.
For example, Sendai virus (HVJ) can be used as a carrier for a cDNA-liposome
complex.
Retroviral gene transfer vectors are retroviruses that have been rendered
non-pathogenic by removal or alteration of viral genes so that little or no
viral
proteins are made in cells infected with the vector. Viral replication
functions are
provided through the use of packaging cells that produce viral protein but not
infectious virus. Following infection of packaging cells with a retroviral
vector,
virions are produced that can infect target cells, but no further viral spread
occurs.
The major advantages of retroviral vectors for gene therapy include a high
efficiency of gene transfer into replicating cells, the precise integration of
the
transferred genes into cellular DNA, and the lack of further spread of the
sequences following transduction. However, the retroviral vectors are
typically
not made synthetically but should be produced by cultured cells, and these
vectors
are complex mixtures that are not purified to homogeneity after production.
For
a more detailed discussion of retroviral gene transfer vectors, see Miller,
Nature
357:455-60 (1992).
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Liposome mediated gene transfer can also be used with commercially
available liposornes. However, the efficacy of gene transfer can be increased
by
combining the liposome with the HVJ virus.
Adenovirus gene transfer vectors are normally replication defective.
These gene transfer vectors have the capacity to carry large segments of DNA,
up
to 8-10 kb. The adenovirus genome is about 36 kb in size. Other advantages
include a very high titre (10" ml-'), the ability to infect nonreplicating
cells, and
the ability to infect tissues in situ. Moreover, adenovirus gene transfer
vectors do
not integrate into the target chromosomal DNA.
An adenovirus gene transfer vector typically contains expression
regulatory sequences such as promoters and enhancers. For example, the
constitutive cytomegalovirus (CMV) early gene promoter/enhancer and/or the
SV40 polyadenylation signal sequence may be used. Other alternative expression
regulatory sequences are known and could be selected by one skilled in the
art.
The NOS gene is then inserted into a plasmid containing appropriate regulatory
elements using standard recombinant DNA techniques such that the regulatory
elements are operably linked to the NOS gene. This expression cassette can
then
be inserted into a vector containing adenovirus sequences that permit
homologous
recombination with the adenovirus genome. By way of example, a suitable vector
is pACCMVpLpA. This plasmid can then be cotransfected with a vector
comprising the full-length adenovirus genome into a suitable host cell, which
include transformed human embryonic kidney cells, containing an integrated
copy
of the left most 12% of the adenovirus 5 genome. The vector comprising the
full-
length adenovirus genome preferably contains an insert within the genome in
order to exceed the packaging limit for adenovirus, rendering the full-length
adenovirus containing vector replication defective.
Construction of recombinant adenovirus vectors is not only possible
through homologous recombination in a suitable cell line, but also through
direct
in vitro ligation of fragments containing virion DNA and the recombinant viral
vector. Suitable host cells for the cotransformation include human embryonic
kidney cells, 911 cells (Introgene, b.v., Rij swijk, Netherlands) and PER 6
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cells (Introgene). Alternatively, adenovirus vectors showing decreased
immunogenicity can also be used.
Homologous recombination between the NOS containing plasmid and the
plasmid containing the adenovirus genome results in an adenovirus genome of
packageable size where the NOS gene has replaced a portion of the adenovirus
genome necessary for viral replication. In specific embodiments of this
invention, the adenovirus early region 1 is replaced by the cloned chimeric
gene,
rendering the virus replication defective. The resulting virus can be used as
a
gene transfer vector for the NOS gene. In addition, adenoviruses with
insertions
in the early region 3 (E3) and second generation adenoviral vectors with
insertions into the early gene 2 or early gene 4 regions can be used for gene
transfer purposes.
C. Introduction of tJie NOS Transfer Vector into Lung Tissue
A suitable NOS gene transfer vector of the invention can be delivered to
the lungs using various delivery systems. For example, the gene transfer can
occur either ex vivo or in vivo. For ex vivo gene transfer, macrophages can be
transduced in vitro, and reintroduced into the patient. When gene transfer
occurs
in vivo, the vector can be introduced by intratracheal, intravenous,
intraperitoneal,
intramuscular, or intraarterial injections. In order to target the vector to
lung
tissue, adenovirus vectors that are selectively taken up by pulmonary
endothelial
cells may be used. Alternatively, a pulmonary tissue specific promoter can be
used. However, aerosol delivery is preferred since it is non-invasive and
results
in deeper penetration of the material into the lungs. Aerosolized material can
be
deposited throughout the airways and alveoli of subjects to be treated. See
Stribling et al., Proc. Natl. Acad. Sci (USA) 89:11277-11281 {1992).
Typically,
the vector is diluted to the required concentration in an isotonic physiologic
buffer solution. To optimize distal intro-alveolar delivery a surfactant can
added
to the solution.
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The vector may also be combined with drugs that would lengthen the
clearance time of the vector in the patient. For example, effective
concentrations
of immunosuppressive agents, sufficient to lengthen the clearance time of the
vector, such as cyclosporin or steroids could be used. The vector can also be
combined with phosphodiesterase inhibitors such as Zaprinast. See Cohen et al.
,
J. Clin. Invest. 97:172-179 (1996).
The recombinant adenovirus vector comprising the NOS gene can be
administered to patients in need of treatment as an aerosol. The concentration
ranges would typically be Sx 10' and Sx 109 plague forming units (pfu) per ml.
In
specific embodiments of this invention, the concentration range would be from
Sx l Og - 1 x 109 pfu/ml.
When the recombinant adenovirus vector comprising the NOS gene is
administered to patients in need of treatment by intratracheal installation,
concentration ranges administered are similar to the concentration ranges for
aerosol administration.
The gene transfer vector can be formulated according to known methods
to prepare pharmaceutically useful compositions, whereby the transfer vector
is
combined with a pharmaceutically acceptable carrier vehicle. These
formulations
may vary depending on the nature of the transfer vector, the mode of
administration, and the indication. Suitable vehicles and their formulation
are
described, for example, in Remington's Pharmaceutical Science ( 18th ed. Mack
Publ. Co. ( 1990)), incorporated herein by reference.
Having now generally described the invention, the same will now be more
readily understood by reference to the following examples, which are provided
by way of illustration and are not intended to be limiting.
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Examples
Example 1
Transduction of Rat Fetal Lung Fibroblasts with AdCMVceNOS
To study whether high levels ceNOS protein expression could be
achieved, rat fetal lung fibroblasts were grown to 60-70% confluence, infected
with AdCMVceNOS or AdCMVHirudin, fixed, and stained with a monoclonal
anti-ceNOS antibody.
In order to construct AdCMVceNOS, a 3.7 kb EcoRIlBamHI fragment
of the human endothelial nitric oxide synthase cDNA, Janssens, S.P. et al., J.
i 0 Biol. Chem. 267: I45 I 9- I 4522 ( 1992), was constructed by ligating a
3.4 kb
EcoRIINcoI fragment to a 0.3 kb PCR fragment containing an
additional 3'-BamHI restriction site. The latter was amplified using
a 5'-CGGCGATGTTACCATGGCAACCAACGT-3' primer corresponding
to the Nco1 site at position 3398 and a 5'-
CGGATCCCGGCTCTCAGGGGCTGTTGGTG-3' primer and including an
additional BamHI site at the 3' end of the cDNA.
For the recombinant virus construction, the 3.7 kb fragment comprising
the entire protein coding region of ceNOS was cloned between the immediate
early CMV promoter/enhancer and the SV40 polyadenylation signal of the
bacterial plasmid pACCMVpLpA. Gomez-Foix, A. et al., J. Biol. Chem.
267:25129-25134 (1992). pACCMVpLpA was obtained from Dr. R. D. Gerard,
Center for Transgene Technology and Gene Therapy, Leuven, Belgium. This
plasmid contains the E 1 A-deleted sequences of type 5 adenovirus, including
the
origin of replication, the packaging signal, the pUC 19 polylinker, and the
strong
enhancer/promoter of the immediate early genes of cytomegalovirus (CMV). A
recombinant adenovirus was generated by homologous recombination with
pJM 17, obtainable from Microbix Biosystems, Inc., Toronto, Ontario, Canada,
a bacterial plasmid containing the full-length adenoviral genome, following
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cotransfection in E 1 A-transformed human embryonic kidney cells. These cells
are available from the American Type Culture Collection, Rockville, Maryland
and the Microbix Biosystems, Inc., Toronto, Ontario, Canada.
The presence of the human ceNOS insert in virion DNA isolated from
infected 293 cells was confirmed by PCR analysis using the 5'
CGGCGATGTTACCATGGCAACCAACGT-3' primer complementary to the
ceNOS 3'-NADPH-adenine site and the 5'CTCTGTAGGTAGTTTGTCCA-3'
primer corresponding to the SV40 splice/polyA site. Southern blot analysis of
recombinant viral DNA isolates probed with the primer complementary to the
ceNOS 3'-NADPH-adenine site was also performed. ceNOS-containing viral
isolates were amplified on confluent 293 cells and, after appearance of
cytopathic
effects, isolated, precipitated, and concentrated by discontinuous CsCI
gradient.
Gerard, C. and Meidell, R., Adenovirus vectors in DNA cloning -- A practical
approach, in Mammalian Systems, Hames, B.D. & Glover, D., eds., Oxford
University Press, Oxford, England ( 1995). These viral isolates were named
AdCMVceNOS. Viral titers were determined by infection of monolayers of 293
cells with serial diiutions of the recombinant adenovirus.
Recombinant adenovirus carrying the LacZ gene encoding a nuciear
localizing variant of the E. coli (3-galactosidase gene were prepared,
amplified
and titered as for AdCMVceNOS. See Herz, J. & Gerard, R.D., Proc. Natl. Acad.
Sci. USA 90:2812-2816 (1993). Similarly, a viral construct containing the cDNA
of the thrombin inhibitor hirudin was prepared for use as a control virus. For
all
in vivo studies, viral titers were adjusted to 5 x 109 pfu/mI. For
transduction of
rat fetal lung fibroblasts, multiplicities of infection (MOI) of 10 and 100
were
selected, since a higher MOI was associated with cytopathic effects.
To detect ceNOS protein in the transduced rat fetal lung fibroblasts, the
rat fetal lung fibroblasts (RFL-6) were cultured in DMEM supplemented with
10% fetal bovine serum (GIBCO), 50 units/ml penicillin, and 50 mg/ml
streptomycin. The cells were grown in chamber slides (Nune, Naperville, IL) to
about 60% confluence and infected with AdCMVceNOS and AdCMVHirudin
diluted in DMEM with 2% fetal bovine serum at 10 and 100 pfu/cell. After 12
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hours, the viral suspension was removed and the cells were maintained in
culture
for 3 days. The presence of the ceNOS gene product was detected by
immunostaining. Three days following in vitro infection with the adenoviral
vectors, the cells were washed with phosphate-buffered saline, fixed for 20
minutes in 4% paraformaldehyde and washed twice in 1 mM Tris, 0.9% NaCI,
0.1% Triton X-100, pH 7.6 (Tris-buffered saline, TBS). Cells were pre-
incubated
with swine serum at a 1:5 dilution in TBS for 45 minutes and exposed overnight
to anti-ceNOS pAB, a rat polyclonal antibody that recognizes human ceNOS
(Transduction Laboratories, Exeter, UK) at a concentration of 2 mg/ml. After a
one-hour incubation with horseradish peroxidase-labeled swine anti-rabbit
second
antibody, Prosan, diluted 1:50, and pre-absorbed overnight with 10% rat serum
and 3% bovine serum albumin, antibody binding was visualized with
diaminobenzedine tetrahydrochloride in 0.1 M Tris buffer, pH 7.2, containing
0.01 % HZO 2 Harris' hematoxylin was used as counterstain and slides were
dehydrated and mounted with dePex mountant medium {Prosan, Gent, Belgium).
After 3 days, abundant ceNOS was observed in the AdCMVceNOS but
not in the AdCMV hirudin-infected cells. See Figure 1. ceNOS expression was
detectable after 24 hours, and the number of cells staining positive peaked at
3
days following infection.
Example 2
Measurement of cGMP Levels in Transduced Rat Feta! Lung Fibroblasts
RFL-6 cells from the American Type Culture Collection, Rockville,
Maryland, which contain abundant soluble guanylate cyclase, were grown to 90%
confluence in 12-well tissue-culture plates ( 105 cells/well) and infected for
4
hours with either AdCMVceNOS or AdCMVHirudin at 100 pfu/cell or medium
only {DMEM with 2% fetal bovine serum) [MOI=100]. Following infection,
cells were cultured for 3 days in DMEM with 10% fetal bovine serum. Cells
were pretreated for 10 min. with 0.3 mM 3-isobutyl-1-methylxanthine (IBMX)
to inhibit phophodiesterase activity in the presence of the calcium ionophore
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A23I87 (2 mM) to stimulate ceNOS activity. 1 mM sodium nitroprusside for 5
minutes was used as a positive control. Intracellular eGMP was extracted in
ice-
cold 15% trichloroacetic acid (TCA), pH 4Ø TCA was extracted in H20-
saturated ether and, following lyophilization, cGMP was quantitated by a
commercial enzyme-immuno assay (Amersham Lifescience, Gent, Belgium).
The specificity of cGMP formation following soluble guanylate cyclase
stimulation by transduced NOS was confirmed by inhibition with 0.5 mM
N°-
nitro-L-arginine methyl ester (L-NAME).
Intracellular cGMP was measured in cells exposed to the calcium
ionophore A23187 (2mM) and IBMX. cGMP levels did not differ between
uninfected RFL-6 cells and RFL-6 cells infected with AdCMVHirudin. cGMP
levels were markedly increased in RFL-6 cells infected with AdCMVceNOS, and
in cells exposed to sodium nitroprusside, a NO donor compound. See Figure 2.
Preincubation of RFL-6 cells with 0.5 mM L-NAME for 30 minutes markedly
reduced cGMP levels in AdCMVceNOS-infected cells. The L-NAME-
inhibitable increase in cGMP levels in AdCMVceNOS-infected RFL-6 cells
suggested that the transgene encoded a biologically active NOS.
Example 3
Transduction of Lung Tissue After Aerosolization of
Recombinant AdCMV~l3gal and AdCMVceNOS
To achieve high levels of transgene expression in peripheral pulmonary
tissues, recombinant adenoviruses were aerosolized in vivo in rat lungs during
mechanical ventilation. Recombinant adenovirus carrying the LacZ gene was
used to study the distribution of transgene expression.
Wistar rats (300-350 grams body weight) were anesthetized by
intraperitoneal injection of pentobarbital (50 mg/kg), intubated with a
polyethylene tube (number PE-240 tubing; 1.67 mm ID), and mechanically
ventilated with room air (Model 683; Harvard Apparatus, South Natick, MA).
During mechanical ventilation, 600 p l solution of recombinant adenovinls
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(AdCMV~3gal and AdCMVceNOS, 5 x 109 pfu/ml) were aerosolized into the
lungs via a silastic catheter introduced via a midline neck incision into the
trachea
distal from the endotracheal tube. Using a tuberculin syringe, a total volume
of
600 ~l viral solution was administered drop by drop during the inspiratory
phase
S of the ventilatory cycle {50 ~.l/10 minutes). Tidal volume was set at 2.5
ml, and
frequency at 60/minute. After viral delivery, the catheter was removed from
the
trachea. Control rats were given an equal volume of sterile saline solution.
No
side effects were observed during aerosol delivery or following extubation.
Four days after administration of AdCMV~igal into the lungs, animals
were sacrificed by an overdose of pentobarbital, and the lungs were perfused
and
fixed through the airways in 4% (wt/vol) formaldehyde. 2 mm-thick segments
from the central and peripheral areas of all lobes were incubated in 20%
sucrose
overnight, overlaid with O.C.T. compound and frozen in liquid nitrogen. Seven
micrometer cryostat sections were mounted on poly-L-lysine-coated slides, and
the presence and distribution ofthe LacZ gene was detected using (3-
galactosidase
staining (5 mmol/L K4Fe(CN) 6 5 mmol/L K ~e(CN) ~ 1 mmol/L MgCI 2 and
1 mg/ml 5-Bromo-4-chloro-3-indolyl-[3-D-galactopyranoside (Boehringer
Mannheim GmbH, Germany) in PBS) for 4-6 hours, and counter-stained with
eosin. To estimate gene transfer efficiency in the histological sections,
positive
cells were identified by their nuclear blue coloration. AdCMV(3gal-infected
lungs
showed diffuse transduction of airway epithelial cells, alveolar lining cells,
and
adventitial cells in medium and small-sized pulmonary vessels after 5 days
(see
Figure 3A).
To measure the localization of ceNOS immunoreactivity in
AdCMVceNOS-transduced lung tissue, lungs from AdCMVceNOS-infected
animals were perfused through the pulmonary artery with PBS, and 4%
formaldehyde was instilled into the airways. Lungs were divided in small
central
and peripheral segments corresponding to the different lobes, and the segments
were overlaid with O.C.T. compound and frozen in liquid nitrogen. Seven pm
cryostat sections were mounted on slides, washed twice with TBS and blocked
with normal rat serum, diluted 1:5 in TB S for 45 minutes. The sections were
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incubated overnight with the anti-ceNOS antibody (2 mg/ml) followed by
incubation for 1 hour with a rabbit anti-mouse IgG peroxidase conjugate
{dilution
1:50; preabsorbed overnight at 4 °C with 10% preimmune rat serum and 3%
bovine serum albumin). Antibody binding was visualized with 3,3'-
diaminobenzadine tetrahydrochloride (DAB, Sigma Chemicals) in 0.1 M Tris
buffer, pH 7.2, containing 0.01 % H20z. Sections were counter-stained with
Harris' hematoxylin, dehydrated, and mounted with dePex-mountant medium.
The efficacy and distribution of ceNOS expression in the lungs following
aerosolization of AdCMVceNOS was studied at various times (3, 4, 5, 8 and 12
days) after gene transfer by immunostaining with monoclonal antibodies
directed
against human ceNOS. Diffuse ceNOS immunostaining was observed in large
airways, lung parenchyma, and endothelial cells of the medium-sized and small
pulmonary vessels. See Figures 3B and 3C. The intensity of staining was
maximal after 5 days, but was still detected after 2 weeks. In control and
AdCMV~igal-treated rats, no ceNOS immunoreactivity was detected in large
airways, alveolar epithelial cells, or in small pulmonary vessels. See Figure
3D.
Endogenous ceNOS immunoreactivity was predominantly detected in endothelial
cells of large, fully muscular vessels. There was little variation in the
staining
pattern between animals infected with AdCMVceNOS. Infection with a titer of
5 x 1 O9 pfu/ml AdCMVceNOS was not associated with any significant pulmonary
infiltrates and did not affect body weight.
Example 4
Activation of tl:e Inducible Form of NOS (iNOS)
by Transduction of AdCMVceNOS
Cytokines released during local inflammatory reactions or in response to
adenoviral infection could activate the inducible isoform of NOS (iNOS).
Therefore the effect of gene transfer and the associated immune response
against
adenovirus on the stimulation of iNOS gene expression in rat lungs was
investigated. No iNOS immunoreactivity was observed with a specific anti-iNOS
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antiserum (Transduction Laboratories) on sections from AdCMVceNOS or
AdCMV(3gal-treated rats. These results show that recombinant adenovirus
infection itself does not appreciably stimulate NOS production via
inflammation
and induction of the inducible isoform of NOS.
Example S
ceNOS Protein Levels in Adenovirus-Infected and Control Luhgs
ceNOS protein levels in adenovirus-infected and control lungs were
measured by immunoblot analysis of extracts from control, AdCMV~3gal, and
AdCMVceNOS-treated rat lungs.
Expression of ceNOS in rat lungs was assessed on day 4 after gene
transfer. Animals were sacrificed and the lungs were excised and processed
immediately or quick-frozen in liquid nitrogen. To extract total protein,
lungs
were homogenized in ice-cold buffer (5 mM Hepes, pH 7.9; 26% glycerol (v/v);
1.5 mM MgCl2; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM phenylmethanesulfonyl
fluoride (PMSF); and 300 mM NaCI) and incubated on ice for 30 minutes. After
centrifugation at 100,000 g at 4 ° C for 20 minutes, the supernatant
containing
crude enzyme preparations was mixed with an equal volume of 2% SDS/1% ~i-
mercaptoethanol and fractionated using 8% SDSIPAGE {70 p.l/lane). Proteins
were then transferred to a nitrocellulose membrane (Hybond-ECL, Amersham
Lifesciences, Gent, Belgium) by semi-dry electroblotting for one hour. The
membranes were blocked by incubating for one hour at room temperature with
Motto-Tween (5% nonfat dry milk, 0.1 % Tween-20) and incubated with a primary
monoclonal mouse anti-ceNOS IgGl antibody (mAb, 0.25 mg/ml, dilution
1:1000, Transduction Laboratories, Exeter, UK). Bound antibody was detected
with horseradish peroxidase-labeled rat anti-mouse IgG second antibody
(Prosan,
dilution 1:2000 in Blotto/Tween) and visualized using enhanced
chemiluminescence (ECL, Amersham, Gent, Belgium).
The antibody detected abundant levels of the 135 kDa protein in the lung
extracts of rats 4 days after treatment with AdCMVceNOS (Figure 4). Only very
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low levels were detected after aerosolization of AdCMV (gal, or in lung
extracts
from untreated control rats.
Example G
ceNOS Enzymatic Activity in Adenovirus-Infected ar:d Control Lungs
ceNOS enzymatic activity as defined by [3H]L-arginine to [3H]L-citrulline
conversion was measured in extracts from AdCMV~3ga1 and AdCMVceNOS
transduced lungs.
To measure ceNOS enzymatic activity, L-arginine to L-citrulline
conversion was assayed in pulmonary extracts using a modification of the
method
described by Xue et al., Amer. J. Phys. 267:L667-L678 ( 1994). Lung protein
extracts prepared as described above were purified by affinity chromatography
on
2',3'-ADP Sepharose and incubated for 30 minutes at 37°C in a solution
of
10 mM L-[2,3 3H] argenine (59 Ci/mmol: l Ci=37 Gbq), 1 mM NADPH, 100 nM
calmodulin, 2 mM CaCl2, 200 pM tetrahydrobiopterin in a final volume of 1 ml.
To inhibit NOS activity, duplicate samples were incubated in the presence of
0.5
mM L-NAME. The reaction was stopped by adding I ml of stop buffer (2 mM
EGTA, 2 mM EDTA, 20 mM Hepes buffer, pH 5.5) to 200 ~l aliquots of the
reaction mixture. The total volume was then applied to a I ml Dowex AG
SOWX-8 column (Na+ form, Bio-Rad Laboratories, Nazareth Eke, Belgium)
preequilibrated with the stop buffer. L-[2,3-3H]citrulline was eluted with 2
ml of
distilled water and the radioactivity was determined by liquid scintillation
counting. Enzyme activity was expressed as citrulline production in
pmolminutes''~mg protein'.
[3H]L-citrulline formation was 86% greater in lung extracts from
AdCMVceNOS treated rats compared to AdCMV~igal treated animals, and this
increased conversion was blocked in the presence of L-NAME. Simultaneously,
biological activity of the expressed ceNOS was evaluated by measuring
intrapulmonary cGMP.
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Example 7
cGMP Levels in Adenovirars-Infected a~td Control Lungs
Intrapulmonary cGMP levels were measured in extracts from
AdCMVceNOS, AdCMV(3gal, and control lungs. For cGMP determinations
lungs were frozen in liquid nitrogen and 400 to 700 mg tissue samples
subsequently homogenized in 1 ml icecold 6% trichloroacetic acid (TCA), pH
4Ø The sample was then centrifuged at 10,000 x g for 15 minutes at 4
° C. The
supernatant was transferred to a 30 ml glass centrifuge tube and TCA was ether
extracted 4 times. A 500 ul aliquot of the sample was then lyophilized,
resuspended in 500 pl of O.OSM sodium acetate buffer (pI-i 5.8) and assayed
for
cellular cGMP utilizing a nonradioactive enzyme-immunoassay kit (Amersham
Life Science). Pulmonary cGMP levels were described as picomoles cGMP per
mg of TCA precipitatiable protein.
cGMP levels were about 10-fold greater in AdCMVceNOS transduced
lungs compared to control lungs (59 + 9 pmol/mg protein vs. 7 + I pmol/mg
protein and 3 ~ 1 pmol/mg protein, respectively, P< 0.05).2
Example 8
Aerosolization of Recombinant AdCMVceNOS Attenuates
Hypoxia-Induced Pulmonary Vasoconstriction
The effect of increased production of NO following AdCMVceNOS gene
transfer on hypoxic pulmonary vasoconstriction was investigated.
After recovery from the aerosol gene delivery procedure, rats were
maintained in room air for three days and reanesthesitized on the fourth day
after
gene transfer. To measure pulmonary artery pressure (PAP), a silastic catheter
Analysis of variance (ANOVA) in the Examples was determined
by the Student-Neumann-Keuls post test to determine significant differences in
multiple comparison testing between groups. All values are expressed as means
~ SEM. For all experiments, statistical significance was assumed at P < 0.05.
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(0.30 mm ID, 0.64 mm OD) was introduced into the right jugular vein and
advanced through the right atrium and ventricle into the pulmonary artery. To
measure systemic arterial pressure, a 2 F catheter was positioned in the right
carotid artery. Both carotid arterial and pulmonary artery catheters were
connected to a pressure transducer (Model AA 016 Baxter, Uden, Holland), a
display oscilloscope (Press Ampl. 863, Siemens, Germany) and a thermal
recorder {Mingograf 82, Siemens, Soina, Sweden). The position of the
pulmonary artery catheter was verified by the characteristic pressure tracing
on
the oscilloscope and was confirmed by autopsy. Electronically meaned PAP and
right ventricular systolic pressures were recorded.
Cardiac output was measured by the thermodilution technique which was
validated in rodents in previous studies. See Janssens et al., J. Applied
Physiol.
77:1101-1108 (1994). Briefly, a 1.5 F thermodilution probe was inserted in the
thoracic aorta via the right carotid artery and connected to a thermal
dilution
computer (Model REF-1, Edwards, USA), and a strip-chart recorder. Through
the silastic pulmonary artery catheter, 0.15 ml saline was injected and
cardiac
output was read directly from the computer display. All values were measured
in triplicate and varied <15%. Cardiac index (CI) was defined as the ratio of
cardiac output over body weight in kilograms (ml~miri'~kg '). Total pulmonary
vascular resistance index (TPRI) was computed by dividing mean pulmonary
artery pressure by cardiac index (mm Hg~miri'~ml-'~kg-')
Rats were initially mechanically ventilated with room air and baseline
PAP, systemic blood pressure, and cardiac output were recorded. To study the
hypoxic pulmonary vasoconstrictor response, rats were ventilated with 10% O2,
90% Nz, and PAP was monitored continuously for 25 minutes. After 5, 15, and
25 minutes of hypoxia, cardiac output was measured. Rats were then ventilated
with room air and hemodynarnic measurements were repeated after 20 minutes.
During acute hypoxia, PAP increased from 18+1 mm Hg to 27+1 and
28~1 mm Hg in saline control and AdCMV(3gal transduced rats, respectively. In
contrast, the rise in PAP was markedly attenuated in AdCMVceNOS transduced
rats (23+2 mm Hg, P<0.05) and was significantly decreased as early as 5
minutes
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of hypoxia. See Figure 5, left panel. Cardiac index did not differ between
AdCMVceNOS treated rats and AdCMV(3ga1 infected rats 185+1 S ml/min/kg vs.
176~18 ml/min/kg, respectively). The reduction in total pulmonary resistance
index (TPRI) during acute hypoxia in AdCMVceNOS treated rats was to 0.157
mm Hg~ml-' ~miri '~kg ' and to 0.122 mm Hg~ml-' ~miri ' ~kg-' for AdCMV (3 gal
and
saline control rats, respectively. This reduction was therefore due to a
direct
pulmonary vasodilatory effect rather than an indirect effect on cardiac
output. See
Figure 5, right panel. Systemic blood pressure was similar in AdCMVceNOS
treated and in AdCMV(3ga1 treated,rats (153+6 mm Hg vs. 150+6 mm Hg,
respectively, n=4). Taken together, aerosolization of AdCMVceNOS and the
resulting overexpression of the ceNOS gene product significantly reduced acute
hypoxic vasoconstriction without affecting systemic blood pressure or cardiac
index.
ceNOS gene transfer thus locally produces NO which selectively
vasodilates pulmonary vessels in a fashion similar to that observed with NO
gas
therapy in hypoxic newborn rats and in human volunteers breathing 12 % OZ in
N~. Frostell et al. , Anesthesiol o~ 78:427-43 5 ( 1993 ). Inhaled NO did not
affect
pulmonary hemodynamics during room air breathing and caused no systemic
hemodynamic effects, possibly because any NO which diffuses into the blood
stream is rapidly inactivated by hemoglobin. Rimar et al., Circulation 88:2884-
2887 {1993). Similarly, AdCMVceNOS aerosol did not affect systemic blood
pressure. However, NO gas inhalation only has an immediate and shortlasting
effect on pulmonary hemodynamics, and requires continuous administration.
Pepke-Zaba et al., Lancet 338:1173-1174 (1991 ). By contrast, these results
demonstrate that a single aerosol does of AdCMVceNOS was able to attenuate
hypoxic pulmonary vasoconstriction even when the hypoxic challenge was
applied 4 to 7 days after gene delivery. Thus, these results are indicative
that
ceNOS expression in lungs is safe and has clinical applications as adjunctive
treatment in some pulmonary hypertensive disease states responsive to inhaled
NO, including persistent pulmonary hypertension of the newborn, perioperative
pulmonary hypertension, and adult respiratory distress syndrome.
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SEQUENCE LISTING
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(i) APPLICANT: The General Hospit al Corporation
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(ii) TITLE OF INVENTION: Method of :Lnducing Vasodilation and
Treating Pulmonary Hypertension Using Adenoviral-Mediated
Transfer of the Nitric Oxidc=_ Synthase Gene
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
CTCTGTAGGT AGTTTGTCCA 20
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4099 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N(~:5:
GAATTCCCAC TCTGCTGCCT GCTCCAGCAG ACGGAC(sCAC AGTAACATGG GCAACTTGAA 60
GAGCGTGGCC CAGGAGCCTG GGCCACCCTG CGGCCT(~GGG CTGGGGCTGG GCCTTGGGCT 120
GTGCGGCAAG CAGGGCCCAG CCACCCCGGC CCCTGA(JCCC AGCCGGGCCC CAGCATCCCT 180
ACTCCCACCA GCGCCAGAAC ACAGCCCCCC GAGCTC(~CCG CTAACCCAGC CCCCAGAGGG 240
GCCCAAGTTC CCTCGTGTGA AGAACTGGGA GGTGGG(~AGC ATCACCTATG ACACCCTCAG 300
CGCCCAGGCG CAGCAGGATG GGCCCTGCAC CCCAAGACGC TGCCTGGGCT CCCTGGTATT 360
TCCACGGAAA CTACAGGGCC GGCCCTCCCC CGGCCC(:CCG GCCCCTGAGC AGCTGCTGAG 420
TCAGGCCCGG GACTTCATCA ACCAGTACTA CAGCTC(:ATT AAGAGGAGCG GCTCCCAGGC 480
CCACGAACAG CGGCTTCAAG AGGTGGAAGC CGAGGTGGCA GCCACAGGCA CCTACCAGCT 540
TAGGGAGAGC GAGCTGGTGT TCGGGGCTAA GCAGGC(~TGG CGCAACGCTC CCCGCTGCGT 600
GGGCCGGATC CAGTGGGGGA AGCTGCAGGT GTTCGATGCC CGGGACTGCA GGTCTGCACA 660
GGAAATGTTC ACCTACATCT GCAACCACAT CAAGTATGCC ACCAACCGGG GCAACCTTCG 720
CA 02260771 1999-07-12
28.4
CTCGGCCATCACAGTGTTCCCGCAGCGCTGCCCTGGCCGA GGAGACTTCCGAATCTGGAA 780
CAGCCAGCTGGTGCGCTACGCGGGCTACCGGCAGCAGGAC GGCTCTGTGCGGGGGGACCC 840
AGCCAACGTGGAGATCACCGAGCTCTGCATTCAGCACGGC TGGACCCCAGGAAACGGTCG 900
CTTCGACGTGCTGCCCCTGCTGCTGCAGGCCCCAGA'rGAGCCCCCAGAACTCTTCCTTCT 960
GCCCCCCGAGCTGGTCCTTGAGGTGCCCCTGGAGCACCCC ACGCTGGAGTGGTTTGCAGC 1020
CCTGGGCCTGCGCTGGTACGCCCTCCCGGCAGTGTCCAAC ATGCTGCTGGAAATTGGGGG 1080
CCTGGAGTTCCCCGCAGCCCCCTTCAGTGGCTGGTACATG AGCACTGAGATCGGCACGAG 1140
GAACCTGTGTGACCCTCACCGCTACAACATCCTGGA(~GATGTGGCTGTCTGCATGGACCT 1200
GGATACCCGGACCACCTCGTCCCTGTGGAAAGACAAGGCA GCAGTGGAAATCAACGTGGC 1260
CGTGCTGCACAGTTACCAGCTAGCCAAAGTCACCATCGTG GACCACCACGCCGCCACGGC 1320
CTCTTTCATGAAGCACCTGGAGAATGAGCAGAAGGCc~AGGGGGGGCTGCCCTGCAGACTG 1380
GGCCTGGATCGTGCCCCCCATCTCGGGCAGCCTCAC~CCCTGTTTTCCATCAGGAGATGGT 1440
CAACTATTTCCTGTCCCCGGCCTTCCGCTACCAGCCAGAC CCCTGGAAGGGGAGTGCCGC 1500
CAAGGGCACCGGCATCACCAGGAAGAAGACCTTTAAAGAA GTGGCCAACGCCGTGAAGAT 1560
CTCCGCCTCGCTCATGGGCACGGTGATGGCGAAGCGAGTG AAGGCGACAATCCTGTATGG 1620
CTCCGAGACCGGCCGGGCCCAGAGCTACGCACAGCA(iCTGGGGAGACTCTTCCGGAAGGC 1680
TTTTGATCCCCGGGTCCTGTGTATGGATGAGTATGA(:GTGGTGTCCCTCGAACACGAGAC 1740
GCTGGTGCTGGTGGTAACCAGCACATTTGGGAATGG(~GATCCCCCGGAGAATGGAGAGAG 1800
CTTTGCAGCTGCCCTGATGGAGATGTCCGGCCCCTA(:AACAGCTCCCCTCGGCCGGAACA 1860
GCACAAGAGTTATAAGATCCGCTTCAACAGCATCTC(:TGCTCAGACCCACTGGTGTCCTC 1920
TTGGCGGCGGAAGAGGAAGGAGTCCAGTAACACAGA(;AGTGCAGGGGCCCTGGGCACCCT 1980
CAGGTTCTGTGTGTTCGGGCTCGGCTCCCGGGCATA(:CCCCACTTCTGCGCCTTTGCTCG 2040
TGCCGTGGACACACGGCTGGAGGAACTGGGCGGGGAGCGG CTGCTGCAGCTGGGCCAGGG 2100
CGACGAGCTGTGCGGCCAGGAGGAGGCCTTCCGAGG(;TGGGCCCAGGCTGCCTTCCAGGC 2160
CGCCTGTGAGACCTTCTGTGTGGGAGAGGATGCCAAGGCC GCCGCCCGAGACATCTTCAG 2220
CCCCAAACGGAGCTGGAAGCGCCAGAGGTACCGGCT(~AGCGCCCAGGCCGAGGGCCTGCA 2280
CA 02260771 1999-07-12
28.5
GTTGCTGCCAGGTCTGATCCACGTGCACAGGCGGAAGATG TTCCAGGCTACAATCCGCTC 2340
AGTGGAAAACCTGCAAAGCAGCAAGTCCACGAGGGCCACC ATCCTGGTGCGCCTGGACAC 2400
CGGAGGCCAGGAGGGGCTGCAGTACCAGCCGGGGGACCAC ATAGGTGTCTGCCCGCCCAA 2460
CCGGCCCGGCCTTGTGGAGGCGCTGCTGAGCCGCGTGGAG GACCCGCCGGCGCCCACTGA 2520
GCCCGTGGCAGTAGAGCAGCTGGAGAAGGGCAGCCC'rGGTGGCCCTCCCCCCGGCTGGGT 2580
GCGGGACCCCCGGCTGCCCCCGTGCACGCTGCGCCAGGCT CTCACCTTCTTCCTGGACAT 2640
CACCTCCCCACCCAGCCCTCAGCTCTTGCGGCTGCTCAGC ACCTTGGCAGAAGAGCCCAG 2700
GGAACAGCAGGAGCTGGAGGCCCTCAGCCAGGATCCCCGA CGCTACGAGGAGTGGAAGTG 2760
GTTCCGCTGCCCCACGCTGCTGGAGGTGCTGGAGCA(:,TTCCCGTCGGTGGCGCTGCCTGC 2820
CCCACTGCTCCTCACCCAGCTGCCTCTGCTCCAGCC(~CGGTACTACTCAGTCAGCTCGGC 2880
ACCCAGCACCCACCCAGGAGAGATCCACCTCACTGTAGCT GTGCTGGCATACAGGACTCA 2940
GGATGGGCTGGGCCCCCTGCACTATGGAGTCTGCTCCACG TGGCTAAGCCAGCTCAAGCC 3000
CGGAGACCCTGTGCCCTGCTTCATCCGGGGGGCTCC(:TCCTTCCGGCTGCCACCCGATCC 3060
CAGCTTGCCCTGCATCCTGGTGGGTCCAGGCACTGG(~ATTGCCCCCTTCCGGGGATTCTG 3120
GCAGGAGCGG CTGCATGACA TTGAGAGCAA AGGGCTGCAG CCCACTCCCA TGACTTTGGT 3180
GTTCGGCTGC CGATGCTCCC AACTTGACCA TCTCTA('CGC GACGAGGTGC AGAACGCCCA 3240
GCAGCGCGGGGTGTTTGGCCGAGTCCTCACCGCCTT(:TCCCGGGAACCTGACAACCCCAA 3300
GACCTACGTGCAGGACATCCTGAGGACGGAGCTGGC~CGCGGAGGTGCACCGCGTGCTGTG 3360
CCTCGAGCGGGGCCACATGTTTGTCTGCGGCGATGTTACC ATGGCAACCAACGTCCTGCA 3420
GACCGTGCAGCGCATCCTGGCGACGGAGGGCGACATGGAG CTGGACGAGGCCGGCGACGT 3480
CATCGGCGTGCTGCGGGATCAGCAACGCTACCACGAAGAC ATTTTCGGGCTCACGCTGCG 3540
CACCCAGGAGGTGACAAGCCGCATACGCACCCAGAG(:TTTTCCTTGCAGGAGCGTCAGTT 3600
GCGGGGCGCAGTGCCCTGGGCGTTCGACCCTCCCGG(:TCAGACACCAACAGCCCCTGAGA 3660
GCCGCCTGGCTTTCCCTTCCAGTTCCGGGAGAGCGG(:TGCCCGACTCAGGTCCGCCCGAC 3720
CAGGATCAGCCCCGCTCCTCCCCTCTTGAGGTGGTG(:CTTCTCACATCTGTCCAGAGGCT 3780
GCAAGGATTCAGCATTATTCCTCCAGGAAGGAGCAAAACG CCTCTTTTCCCTCTCTAGGC 3840
CA 02260771 1999-07-12
-28.6-
CTGTTGCCTC GGGCCTGGGT CCGCCTTAAT CTGGAAGGCC CCTCCCAGCA GCGGTACCCC 3900
AGGGCCTACT GCCACCCGCT TCCTGTTTCT TAGTCCGAAT GTTAGATTCC TCTTGCCTCT 3960
CTCAGGAGTA TCTTACCTGT AAAGTCTAAT CTCTAA:~TCA AGTATTTATT ATTGAAGATT 4020
TACCATAAGG GACTGTGCCA GATGTTAGGA GAACTACTAA AGTGCCTACC CCAGCTCAAA 4080
P,~~~.AAAAAA 4 0 9 9
