Nucleic acids encoding nitric oxide synthase variants

Details Nucleic acids encoding nitric oxide synthase variants Nucleic acids encoding nitric oxide synthase variants

2000-09-13

2003-09-16

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S252300, C435S252330, C435S320100, C435S189000, C536S023200

Reexamination Certificate

active

06620616

ABSTRACT:

BACKGROUND
The free radical nitric oxide (NO) is a chemical messenger that is involved in regulating blood pressure, homeostasis, platelet aggregation, immuno-integrity and neurotransmission. NO is produced in many cell types, including endothelial cells, neurons, airway epithelial cells and macrophages. In blood vessels, NO mediates endothelium-dependent vasodilation. NO is also involved in maintaining basal vascular tone and regulating regional blood flow. In the nervous system NO plays a role in neurotransmission, synaptic plasticity, peristalsis, penile erection, neuro-degenerative disease, and excitotoxicity. In the immune system, NO is produced by activated macrophages and neutrophils as a cytotoxic agent against tumor cells and pathogens.
The formation of NO is catalyzed by a family of enzymes termed nitric oxide synthases. The nitric oxide synthases (NOSs) are encoded by three different genes. Two of the genes encode isoforms that bind calmodulin in a reversible Ca
2+
dependent manner. These two isoforms are known as neuronal NOS (nNOS) or NOS I and endothelial NOS (eNOS) or NOS III. nNOS and eNOS are constitutively expressed. nNOS and eNOS have approximately 55% amino acid sequence identity. The third gene encodes an inducible isoform known as inducible NOS (iNOS) or NOS II. iNOS is constitutively expressed only in select tissues such as lung epithelium, and is more typically synthesized in response to inflammatory or pro-inflammatory mediators. Each NOS is comprised of an N-terminal oxygenase domain and a C-terminal reductase domain, with a Ca
2+
— calmodulin (CaM) binding region of approximately 30 amino acids located between the two domains. (See
FIG. 1.
)
Recent studies have shown that in vivo gene transfer of polynucleotides encoding different NOS isoforms may represent a therapeutic strategy for diseases characterized by decreased bioavailability of NO, such as vascular diseases. Specifically, Lloyd-Jones and Bloch have shown that gene therapy employing an nNOS-expressing adenoviral vector increased the sensitivity of a normal rabbit's carotid arteries to acetylcholine and also reversed the deficit in endothelium-dependent vascular relaxation in cholesterol-fed rabbits. (Lloyd-Jones and Bloch (1996)
Annual Rev. Med
. 47:365-75.) Quian et. al. showed that in vivo transfer of the nNOS gene into cholesterol-fed rabbits reduced vascular adhesion molecule expression, lipid deposition, and inflammatory cell infiltration in the carotid arteries of such animals. (Qian, et. al. (1999)
Circulation
98, 2979-2982.) Von der Leyen et al have shown that in vivo transfer of an eNOS-expressing plasmid to balloon denuded rat carotids significantly limited the subsequent development of neointimal hyperplasia. The eNOS-expressing plasmids were delivered via a liposome-Sendai virus hemagglutinin protein complex. (Von der Leyen, et. al. (1995)
PNAS
92:1137-1141.) Kullo et al showed that adenovirus-mediated transfer of the gene for eNOS to the adventitia of rabbit carotid arteries had a favorable effect on vascular reactivity (Kullo, et. al. (1996)
Circulation
96:7, 2254-2261.) Shears et al have shown that in vivo transfer of an adenovirus expressing iNOS reduced vasculopathy in rat aortic allografts. (Shears, et. al. (1997)
J. Clin. Invest
. 100:8, 2035-2042.) Thus, all three major NOS isoforms appear to be reasonable candidates for in vivo applications to achieve or augment NO production.
Accordingly, it is desirable to have polynucleotides which encode NOS. It is especially desirable to have polynucleotides which encode NOS proteins or polypeptides that are fully active at all possible intracellular concentrations of O
2
.
SUMMARY OF THE INVENTION
The present invention provides polynucleotides which encode variants of NOS. The “NOS variant” comprises a substitution mutant, wherein the tryptophan that is normally located on the alpha 3 helix, six residues upstream from the cysteine which binds heme in the corresponding non-variant NOS, is replaced with one of the other 19 naturally-occurring amino acid residues. Preferably, the targeted tryptophan is replaced with an amino acid residue that forms no or weaker hydrogen bonds with the NOS heme thiolate.
The present invention also relates to vectors and recombinant cells comprising a nucleic acid which encodes a NOS variant. The present invention also relates to NOS variant proteins and polypeptides.


REFERENCES:
patent: 5132407 (1992-07-01), Stuehr et al.
patent: 5268465 (1993-12-01), Bredt et al.
patent: 5468630 (1995-11-01), Billiar et al.
patent: 5594032 (1997-01-01), Gonzalez-Cadavid et al.
patent: 5766909 (1998-06-01), Xie et al.
patent: 5830848 (1998-11-01), Harrison et al.
patent: 5834306 (1998-11-01), Webster et al.
patent: 5882908 (1999-03-01), Billiar et al.
patent: 5919682 (1999-07-01), Masters et al.
Adak S, Crooks C, Wang Q, Crane BR, Tainer JA, Getzoff ED, Stuehr DJ. Tryptophan 409 controls the activity of neuronal nitric-oxide synthase by regulating nitric oxide feedback inhibition. J Biol Chem. Sep. 17, 1999;274(38):26907-11.*
Shimanuki T, Sato H, Daff S, Sagami I, Shimizu T. Crucial role of Lys(423) in the electron transfer of neuronal nitric-oxide synthase. J Biol Chem. Sep. 17, 1999;274(38):26956-61.*
Ausbel, F.M. et al, Current Protocols in Molecular Biology (Jan. 28, 1999) Chapter 8; Unit 8.1 http://www.mrw2.interscience.wiley.com/cponline/tserver.dll?command=doGetDoc&sUI=&database=CP&useScheme=WIS_Framed.Scheme&getDoc=cp_toc_fs.html.*
Yumoto T, Sagami I, Daff S, Shimizu T. Roles of the heme proximal side residues tryptophan409 and tryptophan421 of neuronal nitric oxide synthase in the electron transfer reaction. J Inorg Biochem. Nov. 2000;82.*
Sullivan ME, Thompson CS, Dashwood MR, Khan MA, Jeremy JY, Morgan RJ, Mikhailidis DP. Nitric oxide and penile erection: is erectile dysfunction another manifestation of vascular disease? Cardiovasc Res. Aug. 15, 1999;43(3):658-65. Review.*
von der Leyen, GH Gibbons, R Morishita, NP Lewis, L Zhang et al (1995) Gene Therapy Inhibiting Neointimal Vascular Lesion: In vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene. Proc Natl Acad Sci 92: 1137-41.*
Qian H, Neplioueva V, Shetty GA, Channon KM, George SE. Nitric oxide synthase gene therapy rapidly reduces adhesion molecule expression and inflammatory cell infiltration in carotid arteries of cholesterol-fed rabbits. Circulation. Jun. 15, 1999;99(23):2979.*
“Tryptophan 409 Controls the Activity of Neuronal Nitric-oxide Synthase by Regulating Nitric Oxide Feedback Inhibition” by Adak, et al.The Journal of Biological Chemistry, vol. 274, No. 38, Sep. 17, 1999, pp. 26907-26911.
“Molecular Basis for Hyperactivity in Tryptophan 409 Mutants of Neuronal NO Synthase” by Adak, et al.,The Journal of Biological Chemistry, vol. 275, No. 23, Jun. 9, 2000, pp. 17434-17439.
“A Kinetic Simulation Model that Describes Catalysis and Regulation in Nitric-Oxide Synthase” by Santolini, et al.The Journal of Biological Chemistry, vol. 276, 2001, pp. 1-11.
“Nitric Oxide Synthase Gene Therapy Rapidly Reduces Adhesion Molecule Expression and Inflammatory Cell Infiltration in Carotid Arteries of Cholesterol-Fed Rabbits” by Qian, et al.,Circulation, vol. 98(18), Nov. 3, 1998, pp. 1905-1911.
“Gene therapy inhibiting neointimal vascular lesion: In vivo transfer of endothelial cell nitric oxide synthase gene” by Von Der Leyen, et al.,Proc. Natl. Acad. Sci. USA, vol. 92, Feb. 1995, pp. 1137-1141.
“Adventitial Gene Transfer of Recombinant Endothelial Nitric Oxide Synthase to Rabbit Carotid Arteries Alters Vascular Reactivity” by Kullo, et al.,Circulation, vol. 96, No. 7, Oct. 7, 1997, pp. 2254-2261.
“Inducible Nitric Oxide Synthase Suppresses the Development of Allograft Arteriosclerosis” by Shears, et al,J. Clin. Invest., vol. 100, No. 8, Oct. 997, pp. 2035.-2042.
“Nitric Oxide Synthesis in the Lung” by Dweik, et al.,J. Clin. Invest., vol. 101, No. 3, Feb., 1998, pp. 660-666.
“Inducible Nitric Oxide Synthase Requires both the Canonical Calmodulin-binding Domain and Additional Se

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