WO2013154771A1 - Redox-resistant nitric oxide synthase - Google Patents

Abstract

A redox-resistant endothelial nitric oxide synthase and methods of its use are provided. The disclosed redox-resistant endothelial nitric oxide synthase has reduced dimer disruption in response to oxidation compared to wild-type enzymes. The redox-resistant endothelial nitric oxide synthase can be used to maintain nitric oxide product under oxidative conditions, for example in the treatment of cardiovascular disease.

Description

REDOX-RESISTANT NITRIC OXIDE SYNTHASE

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under

Agreements HL60190, HL67841, and HL084739 awarded by the National Heart, Lung and Blood Institute division of the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention is generally directed to recombinant proteins and there use in treating cardiovascular disease.

BACKGROUND OF THE INVENTION

The impact of cardiovascular disease in the United States is enormous. It is the leading cause of death in the United States (2009

Mortality Multiple Cause Micro-data Files, Centers for Disease Control and Prevention). According to the American Heart Institution, the cost of medical care for heart disease (in 2008 dollar values) will rise from $273 billion to $818 billion. New medicines for treating and preventing cardiovascular disease are in high demand.

Cardiovascular disease is associated with a number of different disorders including hypercholesterolaemia, hypertension and diabetes. The underlying pathology for most cardiovascular diseases is atherosclerosis, which is in turn associated with endothelial dysfunctional. Nitric oxide (NO) plays an important role in the protection against the onset and progression of cardiovascular disease by regulating blood pressure and vascular tone, inhibiting platelet aggregation and leukocyte adhesion, and preventing smooth muscle cell proliferation. Reduced bioavailability of NO is thought to be one of the central factors common to cardiovascular disease, although it is unclear whether this is a cause of, or result of, endothelial dysfunction. Disturbances in NO bioavailability leads to a loss of the cardioprotective actions and in some cases may even increase disease progression. Nassem, K., Mol Aspects Med., 26(l-2):33-65 (2005). For example, nitric oxide present in the blood vessels is involved in vasodilation and increasing blood flow. A reduction in nitric oxide production is attributed to many diseases such as atherosclerosis, diabetes or hypertension. Therefore, proper nitric oxide production is important for normal physiology of the body. Nitric oxide is synthesized from a family of enzymes known as nitric oxide synthases (NOS). One NOS is endothelial nitric oxide synthase (eNOS). eNOS is found primarily in endothelial cells in the vasculature and is associated with several vascular diseases, such as atherosclerosis, hypertension and erectile dysfunction.

Current treatments for eNOS dysfunction include the use of stem cells to deliver active eNOS so that nitric oxide can be produced. However, a problem with this approach is that the same stimulus, such as oxidants, that caused the original eNOS dysfunction would still be present and would also inhibit eNOS activity of the stem cell delivered eNOS.

Therefore, it is an object of the invention to provide methods and compositions for treating diseases associated with eNOS dysfunction.

It is another object of the invention to provide methods and compositions for increasing nitric oxide production in a cell under oxidative stress.

SUMMARY OF THE INVENTION

Redox-resistant nitric oxide synthases are provided. The redox- resistant nitric oxide synthases have been engineered to replace the oxidant sensitive ZnS₄ cluster with a redox stable tetra-arginine cluster. The resulting enzyme is fully functional, is resistant to dimer disruption by oxidative stress, and retains the ability to produce NO under conditions in which the wildtype enzyme is severely inhibited. An exemplary redox- resistant nitric oxide synthase contains a C94R and C99R substitution relative to SEQ ID NO: 1 also represented as SEQ ID NO:2. In one embodiment, the redox-resistant nitric oxide synthase forms a dimer, for example a homodimer. The dimer forms a complex with P0₄ ^"3 _. Cells that contain the redox resistant nitric oxide synthase are also described.

Exemplary cells are mammalian cells that have been genetically engineered to express one or more redox-resistant nitric oxide synthases. The mammalian cell is preferably a human endothelial progenitor cell.

Methods of treating a disease associated with the NO signaling transduction pathway are also provided. Representative diseases that can be treated include, but are not limited to diseases related to eNOS dysfunction. Preferred diseases to be treated include cardiovascular disease.

Cardiovascular disease includes atherosclerosis and hypertension. Preferred methods of treatment include administering to a subject cells that have been genetically engineered to express a redox-resistant eNOS. The cells are preferably progenitor cells including autologous endothelial progenitor cells.

Other methods of treatment include delivering redox-resistant eNOS protein or nucleic acids encoding the redox-resistant eNOS to the subject.

Methods of increasing nitric oxide (NO) production in a cell are also provided. The increase in NO production is relative to cells that have not been treated with the disclosed compositions. An increase in NO production in a cell can be accomplished by administering an effective amount of a composition comprising a redox-resistant eNOS or nucleic acids encoding the redox-resistant eNOS.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the structure of Zn-tetrathiolate cluster. The cluster is composed of two cysteine residues (C94 and C99, human nomenclature) from each subunit of eNOS. Oxidation of cysteine residues within the Zn4 cluster requires less energy due to formation of three electron S-S bond in the tetrahedral coordination.

Figure 2 shows an ion cluster inversion. The upper panel

demonstrates that wildtype eNOS contains a Zn cluster with four negatively charged sulfur atoms surrounding the Zn²⁺ cation in a tetrahedral configuration. In the C94R/C99R redox-resistant eNOS the introduction of four arginine residue are proposed to stabilize the dimeric interface of eNOS via electrostatic interactions with a P0₄ ^3" anion. The lower panel shows both the crystal structure of the Zn cluster (PDB ID 3NOS) in wildtype eNOS and computer simulated structure. Figures 3A and 3B are line graphs of absorbance (at λ260ηηι) versus filtration fraction. Figure 3A shows a gel filtration profile (absorbance at λ260ηηι) of WT (solid)-, C94R/C99R (dash)- and C94A/C99A(dot)-eNOS. WT- and C94R/C99R-eNOS form dimeric proteins in contrast to the C94A/C99A redox-resistant eNOS. Figure 3B shows a gel filtration analysis of WT (solid)- and C94R/C99R (dash)-eNOS treated with 0.5mM H202. H2O2 treatment disrupts the dimeric structure of WT eNOS, but the

C94R/C99R redox-resistant eNOS is resistant to monomerization. Figure 3C shows bar graphs of NO production (pmol/h^g of protein) versus untreated or H2O2 treated eNOS (either WT or redox-resistant eNOS). Hydrogen peroxide alters NO generation in the WT eNOS, but not in C94R/C99R eNOS redox-resistant protein. NO production in C94R/C99R eNOS was increased under basal conditions but the generation of NO by WT eNOS is reduced by H2O2 treatment while it is maintained in the C94R/C99R redox- resistant eNOS. (Mean ± SEM, N=3, * p<0.05 vs WT).

Figures 4A and 4B show western blots of WT and redox-resistant eNOS exposed to increasing levels of H2O2 as well as bar graphs showing dimer/monomer ration (fold untreated) versus increasing H2O2

concentrations. The western blots and bar graphs show eNOS dimer stability under oxidative stress in COS-7 cells. The upper panels of Figure 4A and 4B show western blot analysis using low temperature SDS-PAGE of cells treated with different doses of H2O2 (30 min). Figure 4A shows a dose dependant decrease in dimer content for WT eNOS while Figure 4B shows that the C94R/C99R redox-resistant eNOS was resistant to dimer disruption even at 400μΜ H₂02. Figure 4C is a bar graph of NO production

(pmol/30min^g of protein) versus cells treated in the presence or absence of 300 μΜ H₂0₂. The cells either contain WT eNOS or redox-resistant eNOS. The graph shows that H2O2 treatment reduced NO levels in COS-7 cells expressing WT eNOS but not in those expressing the C94R/C99R resistant (Mean ± SEM, N=3, * p<0.05 vs untreated,†vs. WT eNOS +H₂0₂). DETAILED DESCRIPTION OF THE INVENTION

I. Definitions and Abbreviations

As used herein, the term "effective amount" or "therapeutically effective amount" means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic and/or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected. Typically, an effective amount of the disclosed compositions is an amount that increases NO production in a subject in amount effective to reduce or alleviate one or more symptoms of cardiovascular disease.

As used herein, the term "allogeneic" is meant to refer to any materia] derived from a different mamma] of the same species.

The term "eNOS" refers to endothelial nitric oxide synthase, preferably human endothelial nitric oxide synthase.

The term "NO" refers to nitric oxide.

As used herein, the term "nucleic acid molecule encoding," refers to the order or sequence of nucleotides along a strand of nucleotides. The order of these nucleotides determines the order of amino acids along the polypeptide (protein) chain. The nucleotide sequence thus codes for the amino acid sequence.

As used herein, the term "protein" includes proteins, protein variants, peptides and peptide variants.

A redox-resistant eNOS refers to an eNOS enzyme that has been modifed to be more resistant to oxidative stress relative to unmodified eNOS or wildtype eNOS. For example, redox-resistant eNOS is shown in Figures 4A-C and demonstrates increased dimer formation relative to wildtype eNOS in the presence of H2O2. The term includes truncated proteins relative to SEQ ID NO: l that retain eNOS enzymatic activity and redox-resistance.

The phrase "stem cell" refers both to the earliest renewable cell population responsible for generating cell mass in a tissue or body and the very early progenitor cells, which are somewhat more differentiated, yet are not committed and can readily revert to become a part of the earliest renewable cell population. Methods for the ex- vivo culturing of stem cells are well known in the art of cell culturing ("Culture of Animal Cells— A Manual of Basic Technique" by Freshney, Wiley -Liss, N.Y. (1994), Third Edition).

The term "subject", "individual" or "patient" refers to any individual who is the target of administration. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The term does not denote a particular age or sex.

By "treatment" and "treating" is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

"Protein Transduction Domain" or PTD refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compounds that facilitate traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule traversing membranes, for example going from extracellular space to intracellular space, or cytosol to within an organelle. Exemplary PTDs include, but are not limited to, HIV TAT,

YGRKKRRQRRR (SEQ ID NO: 3), or RKKRRQRRR (SEQ ID NO: 4); 1 1 Arginine residues, or positively charged polypeptides or polynucleotides having 8-15 residues, preferably 9-11 residues.

II. Redox Resistant Nitric Oxide Synthase

It has been discovered that fully functional redox-resistant nitric oxide synthases can be produced by replacing the oxidant sensitive ZnS₄ cluster with a redox stable cluster. The redox stable cluster is preferably a tetra-arginine cluster. The redox-resistant nitric oxide synthases can be used to treat one or more symptoms of diseases related to the NO signal transduction pathway. The disclosed redox-resistant nitric oxide synthases produce NO under conditions that typically result in the down-regulation of NO production, i.e., oxidative stress. Thus, the redox-resistant nitric oxide synthases can function in the disease state to treat one or more symptoms of cardiovascular disease.

A. Endothelial nitric oxide synthase (eNOS)

Nitric oxide synthases (NOSs) are a family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule involved in many physiological and pathological processes affecting nearly every organ system in the body. Endothelial nitric oxide synthase (eNOS) is a key producer of NO found in the vascular system. eNOS is expressed in vascular endothelium, airway epithelium, and certain other cell types where it generates the key signaling molecule NO. Diminished NO availability contributes to systemic and pulmonary hypertension, atherosclerosis, and airway dysfunction.

eNOS dependant vasodilation is an important mechanism regulating vascular tone, the oxidative stress induced disruption of normal eNOS function can induce pathological changes in blood vessels.

Wild type eNOS, the sequence of which is shown below, contains cysteine residues at positions 94 and 99. These cysteines play a role in the formation of eNOS dimers and zinc tetrathiolate. MGNLKSVAQE PGPPCGLGLG LGLGLCGKQG PATPAPEPSR APASLLPPAP EHSPPSSPLT 60

QPPEGPKFPR VKN EVGSIT YDTLSAQAQQ DGPCTPRRCL GSLVFPRKLQ GRPSPGPPAP

120

EQLLSQARDF INQYYSSIKR SGSQAHEQRL QEVEAEVAAT GTYQLRESEL VFGAKQA RN

180

APRCVGRIQ GKLQVFDARD CRSAQEMFTY ICNHIKYATN RGNLRSAITV FPQRCPGRGD

240

FRI NSQLVR YAGYRQQDGS VRGDPANVEI TELCIQHG T PGNGRFDVLP LLLQAPDDPP

300

ELFLLPPELV LEVPLEHPTL E FAALGLR YALPAVSNML LEIGGLEFPA APFSG YMST

360

EIGTRNLCDP HRYNILEDVA VCMDLDTRTT SSL KDKAAV EINVAVLHSY QLAKVTIVDH

420

HAATASFMKH LENEQKARGG CPAD A IVP PISGSLTPVF HQEMVNYFLS PAFRYQPDP

480

KGSAAKGTGI TRKKTFKEVA NAVKISASLM GTVMAKRVKA TILYGSETGR AQSYAQQLGR

540

LFRKAFDPRV LCMDEYDWS LEHETL LW TSTFGNGDPP ENGESFAAAL MEMSGPYNSS

600

PRPEQHKSYK IRFNSISCSD PLVSS RRKR KESSNTDSAG ALGTLRFCVF GLGSRAYPHF

660

CAFARAVDTR LEELGGERLL QLGQGDELCG QEEAFRG AQ AAFQAACETF CVGEDAKAAA

720

RDIFSPKRS KRQRYRLSAQ AEGLQLLPGL IHVHRRKMFQ ATIRSVENLQ SSKSTRATIL

780

VRLDTGGQEG LQYQPGDHIG VCPPNRPGLV EALLSRVEDP PAPTEPVAVE QLEKGSPGGP

840

PPG VRDPRL PPCTLRQALT FFLDITSPPS PQLLRLLSTL AEEPREQQEL EALSQDPRRY

900

EE K FRCPT LLEVLEQFPS VALPAPLLLT QLPLLQPRYY SVSSAPSTHP GEIHLTVAVL

960

AYRTQDGLGP LHYGVCST L SQLKPGDPVP CFIRGAPSFR LPPDPSLPCI LVGPGTGIAP

1020

FRGF QERLH DIESKGLQPT PMTLVFGCRC SQLDHLYRDE VQNAQQRGVF GRVLTAFSRE

1080

PDNPKTYVQD ILRTELAAEV HRVLCLERGH MFVCGDVTMA TNVLQTVQRI LATEGDMELD

1140

EAGDVIGVLR DQQRYHEDIF GLTLRTQEVT SRIRTQSFSL QERQLRGAVP AFEPPGSDT

1200

NSP

(SEQ ID NO: l)

Other embodiments provide redox-resistant eNOS from other mammalian species. These redox-resistant eNOS proteins can be produced by replacing the oxidant sensitive ZnS₄ cluster with a redox stable tetra- arginine cluster.

1. Sensitivity to Oxidation

Protein-protein interactions and different signal transduction events play a role in modifying eNOS activity. For example, the active form of eNOS enzyme exists as two identical subunits that form a head to tail homodimer. It has been previously shown that cysteines 94 and 99 of eNOS form a zinc tetra-coordinated (ZnS₄) cluster between each subunit. Zinc bound to the tetrathiolate cluster has also been shown to stabilize the dimer interface on the N-terminal region of eNOS (Raman et al. Cell 95:939- 950, 1998; Hemmens et al. Journal of Biological Chemistry 275 35786- 35791, 2000). It has been shown that the ZnS₄ cluster is highly sensitive to oxidants such as ONOO , NO, and ¾(¾ (Zou et al. J. of Clinical

Investigation 109:817-826, 2002; Ravi et al. PNAS 101 :2619-2624, 2004; Tummala et al. DNA and Cell Biol. 27:25-33, 2008), and the oxidation of the ZnS cluster results in monomerization of eNOS and inhibition of catalytic activity.

2. The ZnS₄ cluster

Metallo-enzymes can coordinate zinc ions (Zn) through cysteine or histidine residues. The Zn ion can coordinate four ligands in a tetrahedral structure. The ZnS₄ cluster in endothelial nitric oxide synthase (eNOS) can be important for eNOS activity. Endothelial NOS, like all NOS isoforms, is a homodimeric enzyme with the ZnS₄ cluster at the dimer interface. The cluster is formed by four sulfur atoms from two cysteine residues C94 and C99 from each monomer (Fig. 1). It is well established that the dimeric configuration is required for NO formation (Raman, et al. Cell 1998, 95, 939-50; Li, et al. J Biol Chem 1999, 274, 21276-84). Thus, the ZnS₄ cluster is an important contributor to the proper function of eNOS. However, the four sulfur atoms in the tetrahedral configuration are very sensitive to oxidation, leading to eNOS dimer disruption and attenuated NO production (Fonseca, et al. DNA Cell Biol 2010, 29, 149-60; Zou, et al. J Clin Invest 2002, 109, 817-26).

The distance between sulfur atoms in the ZnS cluster is equal to the distance between sulfur atoms in (S-S) disulfide bond. Therefore, the formation of an intermediate with a two-center three-electron bond between two sulfur atoms is very favorable (Fig.1) (Lu, et al. Biochim BiophysActa 2001, 1525, 89-96). Further oxidation of the three-electron (S-S) intermediate requires significantly less energy, therefore, oxidation of cysteine residues in ZnS4 cluster can occur even under mild oxidative stress.

B. Redox-resistant eNOS Proteins and Peptides

As noted above, it has been discovered that fully functional redox- resistant nitric oxide synthases can be produced by replacing the oxidant sensitive ZnS₄ cluster with a redox stable cluster. The redox stable cluster is preferably a tetra-arginine cluster. One embodiment provides a redox- resistant eNOS containing the double mutant C94R/C99R relative to wildtype (SEQ ID NO: 1). The wildtype ZnS₄ cluster can be modified by replacing one or both cysteine residues with arginine or other amino acid residues that reduces the sensitivity of the protein to oxidative stress. SEQ ID NO:2 shows the amino acid sequence of eNOS containing the

C94R/C99R substitutions.

MGNLKSVAQE PGPPCGLGLG LGLGLCGKQG PATPAPEPSR APASLLPPAP EHSPPSSPLT

60

QPPEGPKFPR VKN EVGSIT YDTLSAQAQQ DGPRTPRRRL GSLVFPRKLQ GRPSPGPPAP

120

EQLLSQARDF INQYYSSIKR SGSQAHEQRL QEVEAEVAAT GTYQLRESEL VFGAKQA RN

180

APRCVGRIQ GKLQVFDARD CRSAQEMFTY ICNHIKYATN RGNLRSAITV FPQRCPGRGD

240

FRI NSQLVR YAGYRQQDGS VRGDPANVEI TELCIQHG T PGNGRFDVLP LLLQAPDDPP

300

ELFLLPPELV LEVPLEHPTL E FAALGLR YALPAVSNML LEIGGLEFPA APFSG YMST

360

EIGTRNLCDP HRYNILEDVA VCMDLDTRTT SSL KDKAAV EINVAVLHSY QLAKVTIVDH

420

HAATASFMKH LENEQKARGG CPAD A IVP PISGSLTPVF HQEMVNYFLS PAFRYQPDP

480

KGSAAKGTGI TRKKTFKEVA NAVKISASLM GTVMAKRVKA TILYGSETGR AQSYAQQLGR

540

LFRKAFDPRV LCMDEYDWS LEHETL LW TSTFGNGDPP ENGESFAAAL MEMSGPYNSS

600

PRPEQHKSYK IRFNSISCSD PLVSS RRKR KESSNTDSAG ALGTLRFCVF GLGSRAYPHF

660

CAFARAVDTR LEELGGERLL QLGQGDELCG QEEAFRG AQ AAFQAACETF CVGEDAKAAA

720

RDIFSPKRS KRQRYRLSAQ AEGLQLLPGL IHVHRRKMFQ ATIRSVENLQ SSKSTRATIL

780

VRLDTGGQEG LQYQPGDHIG VCPPNRPGLV EALLSRVEDP PAPTEPVAVE QLEKGSPGGP

840

PPG VRDPRL PPCTLRQALT FFLDITSPPS PQLLRLLSTL AEEPREQQEL EALSQDPRRY

900

EE K FRCPT LLEVLEQFPS VALPAPLLLT QLPLLQPRYY SVSSAPSTHP GEIHLTVAVL

960

AYRTQDGLGP LHYGVCST L SQLKPGDPVP CFIRGAPSFR LPPDPSLPCI LVGPGTGIAP

1020

FRGF QERLH DIESKGLQPT PMTLVFGCRC SQLDHLYRDE VQNAQQRGVF GRVLTAFSRE

1080

PDNPKTYVQD ILRTELAAEV HRVLCLERGH MFVCGDVTMA TNVLQTVQRI LATEGDMELD

1140

EAGDVIGVLR DQQRYHEDIF GLTLRTQEVT SRIRTQSFSL QERQLRGAVP AFEPPGSDT

1200

NSP

(SEQ ID NO:2) The redox-resistant eNOS can include the full length eNOS protein or eNOS peptides that are able to form homodimers and produce NO. In other words, the redox-resistant eNOS can contain fewer amino acids than full length eNOS. In some embodiments the redox-resistant eNOS may be longer than full length eNOS. Active forms of the eNOS protein or redox- resistant eNOS can be used. Active eNOS is any variant of eNOS that is capable of producing NO. Redox-resistant eNOS peptides retain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of enzyme activity. In some embodiments the redox-resistant eNOS can have greater than 100% of wild type enzyme activity. Redox-resistant eNOS can form dimers with full length eNOS, an eNOS peptide, another redox-resistant eNOS or a redox-resistant eNOS peptide.

The disclosed peptides can be in isolated form. As used herein in reference to the disclosed peptides, the term "isolated" means a peptide that is in a form that is relatively free from material such as contaminating polypeptides, lipids, nucleic acids and other cellular material that normally is associated with the peptide in a cell or that is associated with the peptide in a library or in a crude preparation. The disclosed peptides can have any suitable length sufficient to produce nitric oxide.

Redox-resistant eNOS from other species can also be used. One of skill in the art would understand what mutations in an eNOS from another species would correspond to the mutations described for human eNOS.

It has been previously reported that arginine rich structures can be stabilized by formation of strong electrostatic interactions between arginine residues and negative ions such as phosphate or chloride (Mrabet, et al. Biochemistry 1992, 31, 2239-53). Therefore, substituting the cysteine generated Zn cluster with arginines can be used to still form an active eNOS homodimer, but one that is redox insensitive.

1. Monomers and Dimers

Redox-resistant eNOS proteins can be monomeric or dimeric, and only dimers are enzymatically active. In some embodiments, the dimer is composed of two redox-resistant eNOS protein monomers containing the same mutations. Dimers containing the same monomers are homodimers.

In other embodiments the dimer is composed of two redox-resistant eNOS proteins wherein one protein monomer contains the C94R/C99R mutations and the other protein monomer contains one or more different mutations. Dimers containing different monomers are heterodimers. The redox-resistant eNOS dimers retain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of wild type activity enzymatic activity.

Heterodimers between the redox-resistant eNOS monomer and wildtype eNOS monomer can also form.

The dimer can form a complex with a Zn²⁺ ion. In some

embodiments, the dimer can form a complex with a phosphate ion (P0₄ ^~3).

2. Variants and Derivatives

The disclosed proteins, redox-resistant proteins, and redox-resistance peptides can be further modified as variants of the original (i.e., wild type or redox-resistant eNOS) protein. As an example, a "methylated derivative" of a protein refers to a form of the protein that is methylated. Unless the context indicates otherwise, reference to a methylated derivative of a protein does not include any modification to the base protein other than methylation. Methylated derivatives can also have other modifications, but such modifications generally will be noted. For example, conservative variants of an amino acid sequence would include conservative amino acid substitutions of the based amino acid sequence. Thus, reference to, for example, a "methylated derivative" of a specific amino acid sequence "and conservative variants thereof would include methylated forms of the specific amino acid sequence and methylated forms of the conservative variants of the specific amino acid sequence, but not any other modifications of derivations. As another example, reference to a methylated derivative of an amino acid segment that includes amino acid substitutions would include methylated forms of the amino acid sequence of the amino acid segment and methylated forms of the amino acid sequence of the amino acid segment include amino acid substitutions.

Protein variants and derivatives are well understood by those of skill in the art and can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross- linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific

mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.

As used herein in reference to a specified amino acid sequence, a "conservative variant" is a sequence in which a first amino acid is replaced by another amino acid or amino acid analog having at least one biochemical property similar to that of the first amino acid; similar properties include, for example, similar size, charge, hydrophobicity or hydrogen-bonding capacity. Conservative variants are also referred to herein as "conservative amino acid substitutions," "conservative amino acid variants," "conservative substitutions," and similar phrase. A "conservative derivative" of a reference sequence refers to an amino acid sequence that differs from the reference sequences only in conservative substitutions. As an example, a conservative variant can be a sequence in which a first uncharged polar amino acid is conservatively substituted with a second (non-identical) uncharged polar amino acid such as cysteine, serine, threonine, tyrosine, glycine, glutamine or asparagine or an analog thereof. A conservative variant also can be a sequence in which a first basic amino acid is conservatively substituted with a second basic amino acid such as arginine, lysine, histidine, 5 -hydroxy lysine, N-methyllysine or an analog thereof. Similarly, a conservative variant can be a sequence in which a first hydrophobic amino acid is conservatively substituted with a second hydrophobic amino acid such as alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine or tryptophan or an analog thereof. In the same way, a conservative variant can be a sequence in which a first acidic amino acid is conservatively substituted with a second acidic amino acid such as aspartic acid or glutamic acid or an analog thereof; a sequence in which an aromatic amino acid such as phenylalanine is conservatively substituted with a second aromatic amino acid or amino acid analog, for example, tyrosine; or a sequence in which a first relatively small amino acid such as alanine is substituted with a second relatively small amino acid or amino acid analog such as glycine or valine or an analog thereof. For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein. It is understood that conservative variants of the disclosed amino acid sequences can encompass sequences containing, for example, one, two, three, four or more amino acid substitutions relative to the reference sequence, and that such variants can include naturally and non-naturally occurring amino acid analogs.

Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Examples of such substitutions, referred to as conservative substitutions, can generally in accordance with the following Table 1.

TABLE 1: Amino Acid Substitutions Original Residue Exemplary Conservative

Substitutions, others are known in the art.

Ala Ser

Arg Lys; Gin

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn, Lys

Glu Asp

Gly Pro

His Asn;Gln

He Leu; Val

Leu He; Val

Lys Arg; Gin

Met Leu; He

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val He; Leu

Substantial changes in function or immunological identity can be made by selecting substitutions that are less conservative, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the 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 which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an

electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation. These can be referred to as less conservative variants.

Peptides can have a variety of modifications. Modifications can be used to change or improve the properties of the peptides. For example, the disclosed peptides can be N-methylated, O-methylated, S-methylated, C- methylated, or a combination at one or more amino acids.

The amino and/or carboxy termini of the disclosed peptides can be modified. Amino terminus modifications include methylation (e.g.,— HCH₃ or— (CH₃)₂), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as a -chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO- or sulfonyl functionality defined by R-SO₂-, where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N-terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the peptide compound. In preferred embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride.

Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also cyclize the disclosed peptides, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. C- terminal functional groups of the disclosed peptides include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.

One can replace the naturally occurring side chains of the genetically encoded amino acids (or the stereoisomeric D amino acids) with other side chains, for instance with groups such as alkyl, lower (Ci_₆) alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic. In particular, proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.

morpholino), oxazolyl, piperazinyl (e.g., 1 -piperazinyl), piperidyl (e.g., 1- piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

The proteins can contain modified chemical linkages. For example, linkages for amino acids or amino acid analogs can include CH₂NH~, -CH₂S-, -CH2--CH2 --, ~CH=CH~ (cis and trans), -COCH₂ -,

— CH(OH)CH₂— , and— CHH₂SO-(These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and

Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);

Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463- 468; Hudson, D. et al, Int J Pept Prot Res 14: 177-185 (1979) (~CH₂NH~, CH₂CH₂); Spatola et al. Life Sci 38: 1243-1249 (1986) (-CH H₂-S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (-CH-CH-, cis and trans);

Almquist et al. J. Med. Chem. 23: 1392-1398 (1980) (-COCH₂-); Jennings- White et al. Tetrahedron Lett 23 :2533 (1982) (~COCH₂~); Szelke et al.

European Appln, EP 45665 CA (1982): 97:39405 (1982) (~CH(OH)CH₂~); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (~C(OH)CH₂~); and Hruby Life Sci 31 : 189-199 (1982) (-CH₂-S-); each of which is

incorporated herein by reference. A particularly preferred non-peptide linkage is— CH₂NH— . It is understood that peptide analogs can have more than one atom between the bond atoms, such as β-alanine, γ-aminobutyric acid, and the like.

It is understood that one way to define the variants and derivatives of the disclosed amino acids sequences, amino acid segments, peptides, proteins, etc. herein is through defining the variants and derivatives in terms of homology/identity to specific known sequences. For example, specifically disclosed are variants of these and other amino acids sequences, amino acid segments, peptides, proteins, etc. herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software

Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183 :281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment.

It is understood that the description of conservative variants and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative variants. C. Nucleic Acids Encoding Redox-Resistant eNOS Proteins

Nucleic acids encoding the disclosed redox-resistant eNOS proteins and peptides are readily obtained based on the amino acid sequence of SEQ ID NO: l and other eNOS proteins sequences known in the art using conventional techniques.

The nucleic acids typically contain expression control systems. For example, the inserted sequences encoding the redox-resistant eNOS in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the protein. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. Thus, also disclosed are nucleic acids encoding the disclosed peptides operably linked to an expression control sequence.

Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably

cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al, Nature, 273: 1 13 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment (Greenway, P. J. et al, Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species can also be used.

Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al, Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M. L., et al, Mol. Cell. Bio. 3 : 1 108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J. L. et al, Cell 33 : 729 (1983)) as well as within the coding sequence itself (Osborne, T. F., et al, Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 by in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

The promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

In certain embodiments the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and contains of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.

D. Pharmaceutical Compositions

In some embodiments, it may be beneficial to administer the redox resistant NO synthases as a pharmaceutical composition. The

pharmaceutical compositions containing the recombinant proteins can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The administration can be done according to standard procedures used by those skilled in the art.

E. Antibodies that Recognize Redox-Resistant eNOS

Also disclosed are antibodies that can recognize a redox-resistant eNOS and do not recognize wild type eNOS. The antibody can be polyclonal, monoclonal, bifunctional, humanized, or single chain antibodies.

These antibodies can be used to determine the presence of a redox- resistant eNOS in a solution, such as a cell lysate. The antibody can be tagged with a label such as a fluorescent label. Labeled antibodies can be used for in vitro studies.

F. Cells Containing Redox-Resistant eNOS

Cells that contain a redox-resistant eNOS are also provided. The cell can be autologous or allogeneic. The cells can be primary cells or cell lines. In some embodiments, the cells are stem cells or progenitor cells. In particular, the progenitor cell can be an endothelial progenitor cell.

The redox-resistant eNOS can be introduced into the cell in a variety of ways which are known to those of skill in the art. For example, either the redox-resistant eNOS protein or a nucleic acid encoding the redox-resistant eNOS protein can be used.

A cell expressing a nucleic acid encoding redox-resistant eNOS, for example SEQ ID NO:2 can be used to provide the redox-resistant eNOS to another cell, tissue, or whole mammal where a higher level of redox-resistant eNOS can be useful to treat or alleviate a disease, disorder or condition associated with eNOS dysfunction. Therefore, disclosed are compositions used to make genetically modified cells expressing redox-resistant eNOS that can b useful to treat or alleviate a disease, disorder or condition involving eNOS dysfunction.

i. Delivery of Nucleic Acids and Proteins

Nucleic acids and proteins can be delivered alone or in a

pharmaceutical composition with or without an adjuvant.

Delivery systems can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate

precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, these delivery methods can be used to target certain cell populations by using targeting signals.

Electroporation can be used to get proteins into cells. In some embodiments, a protein or nucleic acid can be targeted to a specific cell type using a targeting signal. In some embodiments, a carrier such as a protein transduction domain can be used to get a protein into a cell. a. Protein Transduction Domains

In some embodiments, the eNOs C94R/C99R proteins can include one or more domains for enhancing delivery of the polypeptide across the plasma membrane in into the interior of cells. The eNOs C94R/C99R proteins can be fusion proteins that include a protein transduction domain (PTD), also known as a cell penetrating peptide (CPP). PTDs are known in the art, and include, but are not limited to, small regions of proteins that are able to cross a cell membrane in a receptor-independent mechanism

(Kabouridis, P., Trends in Biotechnology (1 1):498-503 (2003)). Although several of PTDs have been documented, the two most commonly employed PTDs are derived from TAT (Frankel and Pabo, Cell, 55(6): 1 189-93(1988)) protein of HIV and Antennapedia transcription factor from Drosophila, whose PTD is known as Penetratin (Derossi et al, J Biol Chem.,

269(14): 10444-50 (1994)).

The Antennapedia homeodomain is 68 amino acid residues long and contains four alpha helices. Penetratin is an active domain of this protein which consists of a 16 amino acid sequence derived from the third helix of Antennapedia. Several modifications to TAT, including substitutions of Glutatmine to Alanine, i.e., Q-^ A, have demonstrated an increase in cellular uptake anywhere from 90% (Wender et al, Proc Natl Acad Sci US A., 97(24): 13003-8 (2000)) to up to 33 fold in mammalian cells. (Ho et al, Cancer Res., 61(2):474-7 (2001)) The most efficient uptake of modified proteins was revealed by mutagenesis experiments of TAT-PTD, showing that an 1 1 arginine stretch was several orders of magnitude more efficient as an intercellular delivery vehicle. Thus, some embodiments include PTDs that are cationic or amphipathic. Additionally exemplary PTDs include, but are not limited to, poly-Arg - RRRRRRR (SEQ ID NO:5); PTD-5 - RRQRRT SKLMKR (SEQ ID NO:6); Transportan

GWTLNSAGYLLGKTNLKALAALAKKIL (SEQ ID NO:7); KALA - WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:8); and RQIKIWFQNRRMKWKK (SEQ ID NO:9).

In some embodiments, the fusion protein includes an endosomal escape sequence that improves delivery of the protein to the interior of the cell. Endosomal escape sequences are known in the art, see for example, Barka, et al, Histochem. Cytochem., 48(11): 1453-60 (2000) and Wadia and Stan, Nat. Med., 10(3):310-5 (2004).

III. Methods for Treating Diseases Associated with eNOS

Dysfunction

Preferred methods for treating diseases using the redox resistant NO synthase involve transfecting cells to express the redox resistant NO synthase and administering the transfected cells into a subject in need thereof. In other embodiments, the redox resistant NO synthase is administered to a subject in need thereof.

A. Cell Therapy

In some embodiments, the compositions disclosed herein are used to modify cells in culture and then these modified cells are administered to a patient. The cells can be isolated from the subject to be treated (autologous) or from an allogeneic host. The cells can be stem cells, progenitor cells, somatic cells, or embryonic cells. Stem cells are cells capable of producing cell types of more than one lineage. Progenitor cells are cells that can produce different cell types of a single lineage. Cell lineages refer to endoderm, mesoderm, or ectoderm. Exemplary cells that can be used in the disclosed methods include adult stem cells, embryonic stem cells, progenitor cells, adipose-derived stem cells, and somatic cells.

Stem cells or progenitor cells can be obtained or isolated from any of a variety of sources, including, but not limited to bone marrow, umbilical cord blood, and/or donor cells from peripheral, circulating blood of any mammal, preferably a human. The stem cells or progenitor cells can be endothelial progenitor cells. An endothelial progenitor cell is a cell that is capable of developing into an endothelial cell or a functional equivalent of an endothelial cell.

In one embodiment, the stem and/or progenitor cells are derived from a source selected from the group consisting of hematopoietic cells, umbilical cord blood cells, and mobilized peripheral blood cells. Cells can be selected by positive and negative selection techniques. Methods of preparation of stem cells or progenitor cells are well known in the art, commonly selecting cells expressing one or more stem cell markers such as CD34, CD133, etc, or lacking markers of differentiated cells. Endothelial progenitor cells can have specific markers such as CD34, CD31, CD146, Tie2, VE-Cadherin, VEGFR2 or VEGFR3/FLT-4. One or several markers can be used during the selection process. Selection is usually by FACS, or immunomagnetic separation. Embryonic stem cells and methods of their retrieval are well known in the art and are described, for example, in Trounson A O (Reprod Fertil Dev (2001) 13: 523), Roach M L (Methods Mel Biol (2002) 185: 1), and Smith A G (Annu Rev Cell Dev Biol (2001) 17:435). Adult stem cells are stem cells, which are derived from tissues of adults and are also well known in the art. Methods of isolating or enriching for adult stem cells are described in, for example, Miraglia, S. et al. (1997) Blood 90: 5013, Uchida, N. et al. (2000) Proc. Natl. Acad. Sci. USA 97: 14720, Simmons, P. J. at al. (1991) Blood 78: 55, Prockop D J (Cytotherapy (2001) 3 : 393), Bohmer R M (Fetal Diagn Ther (2002) 17: 83) and Rowley S D et al. (Bone Marrow Transplant (1998) 21 : 1253), Stem Cell Biology Daniel R. Marshak (Editor) Richard L. Gardner (Editor), Publisher: Cold Spring Harbor Laboratory Press, (2001) and Hematopoietic Stem Cell Transplantation. Anthony D. Ho (Editor) Richard Champlin (Editor), Publisher: Marcel Dekker (2000).

Techniques for culturing cells in culture are well known in the art.

A nucleic acid encoding a redox-resistant eNOS can be introduced into the cell. In some embodiments a redox-resistant eNOS protein is introduced in the cell.

Once the modified cell expressing a redox-resistant eNOS is administered to a subject, the redox-resistant eNOS can aid in nitric oxide production. The redox-resistant eNOS produces nitric oxide under conditions in which wild type eNOS is oxidized and has reduced or no enzymatic activity.

C. Diseases Associated with eNOS Dysfunction

Several diseases are associated with eNOS dysfunction. The methods herein preferably treat those diseases in which the eNOS dysfunction is due to oxidative stress. Diseases associated with eNOS dysfunction include, but are not limited to, cardiovascular disease, atherosclerosis, diabetes, erectile dysfunction and vascular disease.

1. Cardiovascular Disease

Nitric oxide (NO) plays an important role in the protection against the onset and progression of cardiovascular disease by regulating blood pressure and vascular tone, inhibiting platelet aggregation and leukocyte adhesion, and preventing smooth muscle cell proliferation. Reduced bioavailability of NO is thought to be one of the central factors common to cardiovascular disease, although it is unclear whether this is a cause of, or result of, endothelial dysfunction. Disturbances in NO bioavailability leads to a loss of the cardioprotective actions and in some case may even increase disease progression. Nassem, K., Mol Aspects Med., 26(l-2):33-65 (2005). Therefore, increasing nitric oxide production with the disclosed

compositions can provide an essential therapeutic benefit.

2. Atherosclerosis

Atherosclerosis occurs when there is an accumulation of fatty materials leading to a thickening of the artery walls. Disease complications include the slowing or stopping of blood flow in the arteries. Because nitric oxide in blood vessels is involved in vasodilation and increasing blood flow, the ability to increase nitric oxide production provides a therapeutic effect for atherosclerosis.

3. Diabetes

Individuals with insulin-dependent diabetes are at a high risk of having a vascular disorder, such as hypertension. Because nitric oxide is an endothelium-derived relaxing factor, it can have therapeutic effects on hypertension which is a common complication of diabetes. Therefore, redox-resistant eNOS can be used to treat diabetic complications.

4. Erectile Dysfunction

Erectile dysfunction is a multifactorial disorder that involves the blood flow into sponge-like bodies within the penis. Nitric oxide acts as one of the main vasoactive neurotransmitters. Therefore, reduced nitric oxide plays a role in erectile dysfunction. Redox-resistant eNOS can provide therapeutic effects on penile erection by increasing nitric oxide production.

5. Vascular Disease

Vascular disease includes those diseases affecting the circulatory pathway, such as the blood vessels. Vascular disease can be characterized as reduced endothelium-mediated vasodilation. Nitric oxide is a key element in endothelial cells that aids in vasodilation. Therefore, in vascular diseases that have reduced vasodilation, administering a redox-resistant eNOS could have beneficial effects.

D. Methods of Increasing NO Production

One embodiment provides a method for increasing NO production in a subject by administering the redox-resistant NO synthase, nucleic acids encoding the redox-resistant synthase, cells expressing the redox-resistant NO synthase or a combination thereof. The increase of NO production is relative to the amount of NO production in the subject in the absence of treatment. Methods of increasing NO production in a cell under oxidative stress include administering an effective amount of an oxidant insensitive eNOS.

A preferred oxidant insensitve NO synthase is the wild type eNOS containing the C94R and C99R mutations. In some embodiments, redox- resistant eNOS dimers can be administered.

E. Methods of Reducing or Inhibiting eNOS Dimer

Disruption

Methods of inhibiting eNOS dimer disruption in a cell under oxidative stress include administering an effective amount of an oxidant insensitive eNOS. eNOS dimers are necessary for NO production and therefore reducing or inhibiting the disruption of the dimer can be beneficial. Because oxidation can lead to eNOS dimer disruption, the presence of an oxidant insensitive eNOS maintains NO production under oxidative conditions.

F. Combination Therapy

In certain embodiments, the disclosed methods can include administering additional therapeutic agents. The disclosed compositions can be administered in combination with any known agents for treating diseases such as, but not limited to, vascular disease, atherosclerosis, diabetes and erectile dysfunction. They can also be administered with known compositions that increase nitric oxide production. For example, the additional therapeutic agents can be, but are not limited to, vasodialating β-blockers that produce NO release. Examples include nebivolol, labertolol, carvedilol, and bucindolol. Sulfhydryl ACE inhibition, for example zofenopril, can also be used. Statins, high blood pressure medications, anti-platelet medications, blood thinners, and pain medications can all be administered in combination with the disclosed compositions.

For combination therapies, the therapeutic treatment can be coadministered with another therapeutic agent. Co-administration involves the administration of two or more agents to a subject so that both agents and/or their metabolites are present in the subject at the same time. Co- administration includes simultaneous administration in separate

compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

Examples

Example 1: Protein engineering to develop a redox resistant endothelial NO synthase

Materials and Methods

To begin to analyze the properties of C94R/C99R redox-resistant protein, a histidine tagged protein was expressed and purified using a bacterial expression system and Ni-NTA affinity chromatography. The protein was further purified by sequential gel-filtration through Sephadex™ G-200 and G-25 in the presence of phosphate buffer to enhance the formation of the tetra-arginine cluster.

In order to evaluate the effect of the C94R/C99R mutation on dimeric assembly and susceptibility to oxidative mediated disruption in cells, plasmids encoding WT- and the C94R/C99R redox-resistant eNOS were transiently transfected into COS-7 cells using the Effectene transfection reagent (Qiagen). Twenty-four hours after transfection cells were harvested and lysed at 4°C and subjected to low temperature SDS-PAGE and Western blot analysis as previously described (Fonseca, et al. DNA Cell Biol 2010, 29, 149-60).

Results

The oxidative stress induced disruption of normal eNOS function can induce pathological changes in blood vessels that can lead to a number of generation in WT, but not the C94R/C99R redox-resistant eNOS (Fig. 3 C). Interestingly, the level of NO production was higher in the C94R/C99R redox-resistant compared with WT eNOS (Fig. 3) although the levels of superoxide production measured by EPR (Magnettech) were not different (data not shown). This increase in eNOS activity could be due to the fact that C94R/C99R redox-resistant eNOS is not susceptible to the NO-mediated dimer disruption previously shown in diseases including atherosclerosis, diabetes mellitus and hypertension (Knowles, Jet al. J Clin Invest 2000, 105, 451-8; Cai, et al. Diabetologia 2005, 48, 1933-40; Escobales, et al. Curr Vase Pharmacol 2005, 3, 231-46). Thus, maintaining NO production is a primary goal in the treatment of cardiovascular disorders.

Molecular dynamic (MD) experiments were initially undertaken using the available crystal stucture of human eNOS (PDB ID 3NOS) to determine if mutation of the two tetrathiolate cluster generating cysteine residues to arginines (C94R/C99R) would grossly alter the eNOS structure. Also a phosphate ion (P0₄ ^3") was introduced as a replacement for the Zn²⁺ ion. The resulting redox-resistant protein was simulated in a water filled cube for 100ns using the MD simulation module in Yasara. The Amber94 force field was also applied. After minimization of free energy, the structure of eNOS was still predicted to be dimeric and that three arginine residues were found to interact with the phosphate ion (Fig. 2). Four arginines did not form compact tetrahedral structure, however, the structure of catalytic center and substrate channel did not appear to be disturbed in the C94R/C99R redox- resistant eNOS.

Gel filtration analysis demonstrated that the level of dimer in the purified C94R/C99R eNOS redox-resistant protein is equivalent to that found in WT eNOS (Fig 3 A). This is in contrast to an eNOS modified in which the ZnS4 cluster is disrupted by insertion of alanine residues (Rafikov, et al. J. Endocrinol 201 1, 210, 271-84) (C94A/C99A, Fig. 3 A). Oxidation of the ZnS₄ cluster by hydrogen peroxide (H2O2) treatment resulted in the disruption of the WT eNOS dimeric structure as previously published (Fonseca, et al. DNA Cell Biol 2010, 29, 149-60) while the C94R/C99R redox-resistant eNOS was resistant to H₂02-mediated dimer disruption (Fig. 3 B). Using a chemilumenescent method of NO detection (GE, NOA 280i) it was determined that hydrogen peroxide treatment (300μΜ) decreased NO to occur in wildtype eNOS (Ravi, et al. Proc Natl Acad Sci US A 2004, 101, 2619-24). Together these data indicate that the inversion of the ion cluster from cation centered ZnS₄ cluster to an anion centered tetra-arginine cluster does not appear to affect the dimeric interface and catalytic properties of the recombinant protein. Moreover, the presence of the tetraarginine cluster enhances the redox stability of the dimeric eNOS.

Figures 4A and 4B demonstrated that COS-7 cells expressing the C94/C99R redox-resistant eNOS had a higher dimenmonomer ratio than those expressing wildtype eNOS. Treatment with H2O2, which disrupts the ZnS4 cluster inducing eNOS monomerization (Fonseca, et al. DNA Cell Biol 2010, 29, 149-60), leads to a dose dependant decrease in eNOS dimer levels in wildtype but not C94R/C99ReNOS expressing cells (Fig. 4 A & B). Thus, the replacement of redox sensitive tetrathiolate cluster with redox stable four arginines results in a stablization of the enzyme to oxidative stress. NO generation was also measured for both WT- and the C94R/C99R redox- resistant eNOS when exposed to H2O2. WT- and C99R-eNOS produced similar amounts of NO_x under basal conditions (Fig. 4 C). H2O2 exposure significantly decreased the rate of NOx generation in WT eNOS (Fig. 4 C). However, the same rate of NO_x generation in the C94R/C99R eNOS expressing cells was unaffected by H2O2 (Fig. 4 C).

Together these data demonstrate that replacement of the oxidant sensitive ZnS₄ cluster with a redox stable tetra-arginine cluster produces a fully functional enzyme that is resistant to dimer disruption by oxidative stress and retains the ability to produce NO under conditions in which the wildtype enzyme is severely inhibited. There is controversy in the literature with regards to the main contributor in stabilizing the dimeric interface in NOS, the Zn-tetrathiolate cluster or BH₄ (Tummala, et al. DNA Cell Biol 2008, 27, 25-33; Chen, et al. Biochemistry 2010, 49, 3129-37; Cai, et al. Cardiovasc Res 2005, 65, 823-31; Ravi, et al. Proc Natl Acad Sci USA 2004, 101, 2619-24; Erwin, et al. JBiol Chem 2005, 280, 19888-94). Both of these are redox sensitive, and the binding site of BH₄ is located in close proximity to the ZnS₄ cluster. Thus, it has been difficult to differentiate the effects mediated by ZnS₄ cluster from those of BH4. However, the data presented here provide evidence that the ZnS₄ cluster is more important for dimer sensitivity to oxidative stress than BH₄.

Claims

We claim:

1. A recombinant endothelial nitric oxide synthase having an amino acid sequence according to SEQ ID NO:2.

2. The recombinant endothelial nitric oxide synthase according to claim 2 further comprising a protein transduction domain.

3. The recombinant endothelial nitric oxide synthase according to claim 2, wherein the protein transduction domain is selected from the group consisting of

4. A dimer comprising the recombinant endothelial nitric oxide synthase of claim 1.

5. The dimer of claim 2 , wherein the dimer is a homodimer.

6. The dimer of claim 2, wherein the dimer forms a complex with P0₄ ³

7. A cell comprising the protein of claim 1.

8. A recombinant endothelial progenitor cell comprising a nucleic acid encoding the protein of claim 1.

9. A method of treating a cardiovascular disease comprising administering an effective amount of a composition comprising a redox resistant eNOS, wherein the cardiovascular disease is associated with endothelial nitric oxide synthase (eNOS) dysfunction due to oxidative stress.

10. The method of claim 9, wherein the oxidant insensitive eNOS is the C94R/C99R redox-resistant eNOS.

1 1. The method of claim 9, wherein the oxidant insensitive eNOS is a protein or a nucleic acid that encodes the oxidant insensitive eNOS.

12. The method of claim 9, wherein the composition is administered using a cell therapy approach.

13. The method of claim 9, wherein the disease associated with eNOS dysfunction is vascular disease.

14. A method of increasing nitric oxide production in a cell comprising administering an effective amount of a composition comprising an oxidant insensitive eNOS.

15. The method of claim 14, wherein the cell is under oxidative stress.

16. The method of claim 15, wherein the cell is an endothelial cell.

17. The method of claim 15, wherein the cell is a stem cell.

18. A method of inhibiting eNOS dimer disruption in a cell comprising administering an effective amount of a composition comprising an oxidant insensitive eNOS.

19. The method of claim 18, wherein the cell is under oxidative stress.