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WO1998002170A1 - Procede servant a provoquer la vasodilatation et a traiter l'hypertension pulmonaire au moyen du transfert provoque par adenovirus du gene de synthase d'oxyde nitrique - Google Patents

Procede servant a provoquer la vasodilatation et a traiter l'hypertension pulmonaire au moyen du transfert provoque par adenovirus du gene de synthase d'oxyde nitrique Download PDF

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Publication number
WO1998002170A1
WO1998002170A1 PCT/US1997/012510 US9712510W WO9802170A1 WO 1998002170 A1 WO1998002170 A1 WO 1998002170A1 US 9712510 W US9712510 W US 9712510W WO 9802170 A1 WO9802170 A1 WO 9802170A1
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pulmonary
nitric oxide
oxide synthase
pulmonary hypertension
lungs
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PCT/US1997/012510
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English (en)
Inventor
Stefan Janssens
Kenneth D. Bloch
Désiré COLLEN
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The General Hospital Corporation
Leuven Research And Development, V.Z.W.
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Priority to EP97933529A priority Critical patent/EP0979088A4/fr
Publication of WO1998002170A1 publication Critical patent/WO1998002170A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C12N9/0075—Nitric-oxide synthase (1.14.13.39)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • 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.
  • 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.
  • 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. 3:151 -3064 ( 1992).
  • the constitutiveiy 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.
  • the neuronal NOS isoform 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 547:768-770 (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.
  • iNOS inducible nitric oxide synthase
  • the inducible form of ⁇ OS is not calcium regulated, but its expression is induced by cytokines. This form of ⁇ OS functions as a cytotoxic agent, and NO produced by inducible NOS targets tumor cells and pathogens.
  • hypoxia induces a significant reduction in contractile responses to acetylcholine and to inhibitors of NOS.
  • hypoxia induces a significant reduction in contractile responses to acetylcholine and to inhibitors of NOS.
  • hypoxia suppresses basal and agonist- stimulated release of NO. Johns et al, Circ. Res. (55:1 08-1515 (1989); Shaul et al, J. Cardiovasc. Pharmacol 22:819-827 (1993).
  • hypoxia inhibits NO production by reducing ceNOS mRNA levels and ceNOS mRNA stability. McQuillan et al, Am. J. Physiol 267: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. 555:214-221 (1995). Therefore, hypoxia-induced hypertension may correlate with reduced NO generation from pulmonary endothelium affecting the balance between pulmonary vasoconstrictive and vasodilatory stimuli.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 Ca 3 (PO 4 ) 2 precipitation and DEAE dextran. Retroviral vector transfer systems also have the capacity to integrate transferred genes stably into a wide variety of cell types.
  • retro viruses 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).
  • these non- viral vectors have a relatively low in vivo transduction efficiency.
  • 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 lungs of a patient in need of pulmonary vasodilation.
  • the nitric oxide synthase can be a constitutiveiy expressed or an inducible nitric oxide synthase gene.
  • the pulmonary vasodilation is selective
  • the vector is an adenovirus vector
  • the nitric oxide synthase gene is the endothelial nitric oxide synthase gene
  • this vector is transduced into lung tissue as an aerosol.
  • 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 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.
  • 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
  • a pharmaceutical composition comprising the nitric oxide synthase gene operably linked to an expression control element and a means for transducing said gene into pulmonary tissue.
  • the pharmaceutical composition comprises AdCMVceNOS in admixture with a pharmaceutically acceptable carrier.
  • 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 shows the expression of ceNOS in rat lungs. Protein extracts were obtained from the lungs of rats aerosolized with AdCMVceNOS (lane 2) and AdCMV ⁇ gal (lane 3). 70 ⁇ g 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. Figure 3.
  • FIG 3 depicts ceNOS and ⁇ -galactosidase gene expression in the lungs of rats transduced with AdCMVceNOS and AdCMV ⁇ gal.
  • AdCMV ⁇ gal transduced rat lungs show nuclear localized ⁇ -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 magnification; C, 400-fold magnification).
  • FIG. 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.
  • SNP Sodium nitroprusside
  • AdCMVHirudin expressing the thrombin inhibitor, hirudin
  • the data depicted are means ⁇ SEM of five determinations, except for AdCMVHirudin, which are means ⁇ SEM of three determinations.
  • Figure 6 depicts the nucleotide sequence of the endothelial isoform of the ceNOS gene. Detailed Description of the Preferred Embodiments
  • 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.
  • 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.
  • transduction 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.
  • 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 30 mm Hg systolic and greater than 12 mm Hg diastolic, or a mean pulmonary artery pressure in excess of 15-17 mm Hg.
  • primary pulmonary hypertension refers to pulmonary hypertension not caused by another underlying disease.
  • second pulmonary hypertension refers to pulmonary hypertension resulting from another underlying disease.
  • the underlying disease causing secondary pulmonary hypertension is a pulmonary or cardiac disease.
  • proliferative therapy refers to therapy that alleviates the symptoms of a disease without curing that disease.
  • cardiac index refers to the ratio of cardiac output to body weight.
  • 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.
  • 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.
  • nitric oxide synthase refers to an enzyme capable of catalyzing the formation of nitric oxide.
  • 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 constitutiveiy expressed or inducible form of nitric oxide synthase.
  • 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.
  • an endothelial nitric oxide synthase of this invention is encoded by a nucleic acid exhibiting greater than
  • 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.
  • a "vector” refers to a plasmid, phage, or other DNA molecule, which provides an appropriate nucleic acid environment for a transfer 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.
  • operable linkage refers to the position, orientation, and linkage between a structural gene and expression control element(s) such that the structural gene can be expressed in any host cell.
  • expression control element includes promoters, enhancers, ribosome binding sites, etc.
  • This invention relates to gene therapy methods using the nitric oxide synthase gene to induce pulmonary vasodilation.
  • 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 bp open reading frame encoding a 1203 amino acid protein.
  • the predicted molecular weight of this protein is about 133 kDa.
  • 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,
  • this invention also relates to the use of ceNOS genes having similar but not identical sequences.
  • 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.
  • 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.
  • inducible nitric oxide synthase iNOS
  • nNOS neuronal isoform
  • this invention also relates to the use of iNOS and nNOS genes having similar but not identical sequences to those described supra at 3.
  • 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 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.
  • Substantial changes in functional or immunological 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.
  • 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 residue, e.g., seryl 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
  • an appropriate NOS gene 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.
  • Sendai virus HVJ
  • 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.
  • 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.
  • 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.
  • Liposome mediated gene transfer can also be used with commercially available liposomes. 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 "1 ), 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.
  • CMV constitutive cytomegalovirus
  • 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.
  • 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.
  • adenovirus vectors are 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., Rijswijk, Netherlands) and PER 6 cells (Introgene).
  • adenovirus vectors showing decreased immunogenicity can also be used.
  • 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.
  • 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.
  • a suitable NOS gene transfer vector of the invention can be delivered to the lungs using various delivery systems.
  • the gene transfer can occur either ex vivo or in vivo.
  • macrophages can be transduced in vitro, and reintroduced into the patient.
  • the vector can be introduced by intratracheal, intravenous, intraperitoneal, intramuscular, or intraarterial injections.
  • adenovirus vectors that are selectively taken up by pulmonary endothelial cells may be used.
  • a pulmonary tissue specific promoter can be used.
  • 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- 1 1281 (1992).
  • the vector is diluted to the required concentration in an isotonic physiologic buffer solution.
  • a surfactant can added to the solution.
  • 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. ,
  • the recombinant adenovirus vector comprising the NOS gene can be administered to patients in need of treatment as an aerosol.
  • concentration ranges would typically be 5xl0 7 and 5x10 9 plague forming units (pfu) per ml. In specific embodiments of this invention, the concentration range would be from
  • 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.
  • rat fetal lung fibroblasts were grown to 60-70% confluence, infected with AdCMVceNOS or AdCMVHirudin, fixed, and stained with a monoclonal anti-ceNOS antibody.
  • AdCMVceNOS a 3.7 kb EcoRl/BamHl fragment of the human endothelial nitric oxide synthase cDNA, Janssens, S.P. et al, J. Biol. Chem. 267:14519-14522 (1992), was constructed by ligating a 3.4 kb
  • 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.
  • pACCMVpLpA was obtained from Dr. R. D. Gerard, Center for Transgene Technology and Gene Therapy, Leuven, Belgium.
  • This plasmid contains the El A-deleted sequences of type 5 adenovirus, including the origin of replication, the packaging signal, the pUC19 polylinker, and the strong enhancer/promoter of the immediate early genes of cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • a recombinant adenovirus was generated by homologous recombination with pJM17, obtainable from Microbix Biosystems, Inc., Toronto, Ontario, Canada, a bacterial plasmid containing the full-length adenoviral genome, following cotransfection in El 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.
  • AdCMVceNOS AdCMVceNOS. Viral titers were determined by infection of monolayers of 293 cells with serial dilutions of the recombinant adenovirus.
  • Recombinant adenovirus carrying the LacZ gene encoding a nuclear- localizing variant of the E. coli ⁇ -galactosidase gene were prepared, amplified and titered as for AdCMVceNOS. See Herz, J. & Gerard, R.D., Proc. Natl Acad.
  • a viral construct containing the cDNA of the thrombin inhibitor hirudin was prepared for use as a control virus.
  • viral titers were adjusted to 5 x 10 9 pfu/ml.
  • multiplicities of infection (MOI) 10 and 100 were selected, since a higher MOI was associated with cytopathic effects.
  • 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 (Nunc, 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 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.
  • the cells were washed with phosphate-buffered saline, fixed for 20 minutes in 4% paraformaldehyde and washed twice in 1 mM Tris, 0.9% NaCl,
  • 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, Singer, UK) at a concentration of 2 mg/ml.
  • 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 (10 5 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.
  • IBMX 3-isobutyl-l -methylxanthine
  • IBMX 3-isobutyl-l -methylxanthine
  • 1 mM sodium nitroprusside for 5 minutes was used as a positive control.
  • Intracellular cGMP was extracted in ice- cold 15%) trichloroacetic acid (TCA), pH 4.0.
  • TCA was extracted in H 2 O- saturated ether and, following lyophilization, cGMP was quantitated by a commercial enzyme-immuno assay (Amersham Lifescience, Gent, Belgium).
  • 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.
  • 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
  • pentobarbital 50 mg/kg
  • intubated with a polyethylene tube number PE-240 tubing; 1.67 mm ID
  • room air Model 683; Harvard Apparatus, South Natick, MA
  • 600 ⁇ l solution of recombinant adenovirus (AdCMV ⁇ gal and AdCMVceNOS, 5 x 10 9 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.
  • a tuberculin syringe a total volume of 600 ⁇ l viral solution was administered drop by drop during the inspiratory phase 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.
  • 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 ⁇ m cryostat sections were mounted on slides, washed twice with TBS and blocked with normal rat serum, diluted 1:5 in TBS for 45 minutes.
  • the sections were 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%> H 2 O 2 .
  • Sections were counter-stained with Harris' hematoxylin, dehydrated, and mounted with dcPex-mountant medium.
  • 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 ⁇ gal-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 10 9 pfu/ml AdCMVceNOS was not associated with any significant pulmonary infiltrates and did not affect body weight.
  • 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 antiserum (Transduction Laboratories) on sections from AdCMVceNOS or AdCMV ⁇ gal-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.
  • ceNOS protein levels in adenovirus-infected and control lungs were measured by immunoblot analysis of extracts from control, AdCMV ⁇ gal, 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.
  • lungs were homogenized in ice-cold buffer (5 mM Hepes, pH 7.9; 26% glycerol (v/v); 1.5 mM MgCl 2 ; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM phenylmethanesulfonyl fluoride (PMSF); and 300 mM NaCl) 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% ⁇ - mercaptoethanol and fractionated using 8% SDS/PAGE (70 ⁇ l/lane).
  • ice-cold buffer 5 mM Hepes, pH 7.9; 26% glycerol (v/v); 1.5 mM MgCl 2 ; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM phenylmethanesulfon
  • 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 blotto-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, Wales, 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 low levels were detected after aerosolization of AdCMV ⁇ gal, or in lung extracts from untreated control rats.
  • ceNOS enzymatic activity as defined by [ 3 H]L-arginine to [ 3 H]L-citrulline conversion was measured in extracts from AdCMV ⁇ gal and AdCMVceNOS transduced lungs.
  • the reaction was stopped by adding 1 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 1 ml Dowex AG 50WX-8 column (Na + form, Bio-Rad Laboratories, Nazareth Eke, Belgium) preequilibrated with the stop buffer. L-[2,3- 3 H]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 pmol minutes "1 mg protein "1 .
  • Intrapulmonary cGMP levels were measured in extracts from AdCMVceNOS, AdCMV ⁇ gal, 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% trichioroacetic acid (TCA), pH
  • Pulmonary cGMP levels were described as pico oles 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 ⁇ 1 pmol/mg protein and 3 ⁇ 1 pmol/mg protein, respectively, P ⁇ 0.05). 2
  • thermodilution technique Cardiac output was measured by the thermodilution technique which was validated in rodents in previous studies. See Janssens et a , J. Applied Physiol. 77:1 101 -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 was defined as the ratio of cardiac output over body weight in kilograms (ml min '1 kg “1 ).
  • Total pulmonary vascular resistance index was computed by dividing mean pulmonary artery pressure by cardiac index (mm Hgmin "1 ml "1 kg “1 ).
  • Rats were initially mechanically ventilated with room air and baseline PAP, systemic blood pressure, and cardiac output were recorded.
  • rats were ventilated with 1 % O 2 , 90%) N 2 , and PAP was monitored continuously for 25 minutes. After 5, 15, and
  • TPRI total pulmonary resistance index
  • AdCMVceNOS aerosol did not affect systemic blood pressure.
  • NO gas inhalation only has an immediate and shortlasting effect on pulmonary hemodynamics, and requires continuous administration.
  • Pepke-Zaba et al Lancet 555:1173-1174 (1991).
  • 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.
  • 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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Abstract

L'invention concerne un procédé de thérapie génique servant à provoquer la vasodilatation pulmonaire. Elle consiste, plus spécifiquement, à introduire le gène de synthase d'oxyde nitrique dans les poumons, ce qui provoque la vasodilatation pulmonaire. Ceci exerce un effet d'hypotension sur la circulation pulmonaire et n'affecte pratiquement pas la pression sanguine systémique ni l'indice cardiaque. Ce procédé est utile pour traiter l'hypertension pulmonaire primaire ou consécutive à différentes maladies.
PCT/US1997/012510 1996-07-17 1997-07-17 Procede servant a provoquer la vasodilatation et a traiter l'hypertension pulmonaire au moyen du transfert provoque par adenovirus du gene de synthase d'oxyde nitrique WO1998002170A1 (fr)

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EP1016727A1 (fr) * 1998-12-30 2000-07-05 Introgene B.V. Thérapie génique ayant pour but la promotion de l' angiogenèse
US6146887A (en) * 1994-03-31 2000-11-14 Jurgen Schrader DNA expression vectors for use in the gene therapeutic treatment of vascular disorders
WO2002017897A3 (fr) * 2000-08-30 2003-01-30 Primecyte Inc Procedes de therapie antitumorale
EP1348434A1 (fr) * 2002-03-27 2003-10-01 Fujisawa Deutschland GmbH Utilisation de pyridylamides comme inhibiteurs de l'angiogenèse
WO2004058293A1 (fr) * 2002-12-24 2004-07-15 Northern Therapeutics Inc. Procedes de diagnostic, de prevention et de traitement de l'apparition precoce de l'hypertension pulmonaire
WO2000040740A3 (fr) * 1998-12-30 2007-05-18 Crucell Holland Bv Therapie genique favorisant l'angiogenese
EP1839667A3 (fr) * 1999-09-23 2010-04-14 Northern Therapeutics Inc. Thérapie de gène à base de cellules pour le système pulmonaire
DE102012220693A1 (de) 2012-11-13 2014-05-15 Trumpf Medizin Systeme Gmbh + Co. Kg Schutzeinrichtung für ein Bediengerät

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DATABASE MEDLINE ON DIALOG, US NATIONAL LIBRARY OF MEDICINE, (Bethesda, MD, USA), No. 96295016, TZENG et al., "Vascular Gene Transfer of the Human Inducible Nitric Oxide Synthase: Characterization of the Activity and Effects on Myointimal Hyperplasia"; & MOLECULAR MEDICINE, March 1996, Vol. 2, No. 2, pages 211-225. *
DATABASE MEDLINE ON DIALOG, US NATIONAL LIBRARY OF MEDICINE, (Bethesda, MD, USA), No. 96435198, SETOGUCHI et al., "Transfer of Endothelial Nitric Oxide Synthase Gene in Purpose of Gene Therapy for Pulmonary Arterial Hypertension"; & JAPANESE JOURNAL OF CLINICAL MEDICINE, February 1996, Vol. 54, No. 2, pages 369-376. *
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6146887A (en) * 1994-03-31 2000-11-14 Jurgen Schrader DNA expression vectors for use in the gene therapeutic treatment of vascular disorders
US6149936A (en) * 1994-03-31 2000-11-21 Joseph Schrader DNA expression vectors for the use in the gene therapeutic treatment of vascular disorders
WO2000040740A3 (fr) * 1998-12-30 2007-05-18 Crucell Holland Bv Therapie genique favorisant l'angiogenese
EP1016727A1 (fr) * 1998-12-30 2000-07-05 Introgene B.V. Thérapie génique ayant pour but la promotion de l' angiogenèse
EP1839667A3 (fr) * 1999-09-23 2010-04-14 Northern Therapeutics Inc. Thérapie de gène à base de cellules pour le système pulmonaire
WO2002017897A3 (fr) * 2000-08-30 2003-01-30 Primecyte Inc Procedes de therapie antitumorale
US6716879B2 (en) 2000-08-30 2004-04-06 Compass Pharmaceuticals, Llc Methods for anti-tumor therapy
WO2003080054A1 (fr) * 2002-03-27 2003-10-02 Fujisawa Deutschland Gmbh Utilisation d'amides pyridyliques en tant qu'inhibiteurs de l'angiogenese
EP1348434A1 (fr) * 2002-03-27 2003-10-01 Fujisawa Deutschland GmbH Utilisation de pyridylamides comme inhibiteurs de l'angiogenèse
WO2004058293A1 (fr) * 2002-12-24 2004-07-15 Northern Therapeutics Inc. Procedes de diagnostic, de prevention et de traitement de l'apparition precoce de l'hypertension pulmonaire
DE102012220693A1 (de) 2012-11-13 2014-05-15 Trumpf Medizin Systeme Gmbh + Co. Kg Schutzeinrichtung für ein Bediengerät
EP3223111A1 (fr) 2012-11-13 2017-09-27 TRUMPF Medizin Systeme GmbH + Co. KG Dispositif de protection pour un appareil de commande
US10194545B2 (en) 2012-11-13 2019-01-29 Trumpf Medizin Systeme Gmbh + Co. Kg Protecting medical operator devices

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