Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/55—Protease inhibitors
  • A61K38/57—Protease inhibitors from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/55—Protease inhibitors
  • A61K38/553—Renin inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/55—Protease inhibitors
  • A61K38/556—Angiotensin converting enzyme inhibitors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
• • 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

Definitions

• This invention relates to gene therapy for treatment of diseases not caused by genetic deficiencies.
• genes e.g.. globin
• Some genes will need to be expressed in the appropriate cells at precise levels in order to have a therapeutic effect and to avoid killing the cell.
• the most suitable diseases for gene therapy are life threatening diseases where the levels and lineage specificity of expression need not be stringently regulated.
• One such candidate is adenosine deaminase deficiency disease which leads to a severe combined immunodeficiency (SCID). id.- at 169-170.
• SCID severe combined immunodeficiency
• Anderson, 216 Science 401 , 1984 describes gene therapy as insertion of a normal gene into an organism to correct a genetic defect. It describes various techniques available for such therapy in humans and other organisms.
• Costantani, et al.. 233 Science 1192, 1986 described correction of murine ⁇ -thalassemia by gene transfer to a germ line as an example of gene therapy, and as a preliminary step toward gene therapy in humans.
• BioVenture View (Feb. 1992, not admitted to be prior art to the present invention) describes a method for insertion of foreign genes into fibroblasts without use of viral vectors. They suggest delivery of insulin, LDL receptors and calcitonin.
• This invention concerns in vivo expression of polypeptides active as agonists or as inhibitors of protein activity. The activity inhibited includes that of proteinases, anti-proteinases, and other enzymes or hormones as well as the activity of receptors for hormones or other endogeneous molecules, into a living animal by introduction of nucleic acid encoding such an agonist or inhibitor. Applicant has recognized that there are a variety of protein activities involved in various pathologic states.
• pathologic states occur in individuals without genetic defects. That is, the state is not caused by a severe lack of a protein activity within an individual caused by lack of naturally occurring nucleic acid encoding that activity (i.e.. nucleic acid present in normal individuals within a population). Rather, the pathological state reflects an aberrant level of protein activity caused by some other mechanism or by more subtle regulations of protein activity or that the pathological state can be manipulated in spite of normal protein activity. Applicant's invention is based upon the therapeutic benefit that is achieved by delivering a gene or other nucleic acid that encodes an agonist or inhibitor of such a protein activity.
• the invention features a method for treatment of a patient having a normal level of activity of a naturally occurring protein, or having a condition characterized by the presence of an elevated or reduced level of that natural protein.
• the method includes administering to the patient nucleic acid which encodes an agonist or inhibitory polypeptide which mimics or inhibits the biological activity of the natural protein or the naturally occurring effector of that natural protein.
• the agonist or inhibitor is a polypeptide (i.e.. an amino acid chain including at least 2 amino acids, and generally up to about 500 amino acids) expressed from the nucleic acid when that nucleic acid is present in the patient.
• This agonist or inhibitory polypeptide acts in vivo to reduce the specific biological activity of the natural protein or the naturally occurring effector in the patient.
• the agonists may also act as enhancers of protein activity.
• naturally occurring protein is meant to include proteins produced by the majority of the group of animals to which the patient belongs, and includes those proteins produced at abnormal levels in an individual to be treated, but not those proteins which are produced at abnormal levels because of a genetic defect in the structural gene encoding such a protein in the individual to be treated.
• proteins include various cell receptors, cytokines, hormones, proteases and other enzymes. They do not include those proteins introduced by extraneous organisms such as viruses, bacteria or other infectious organisms.
• polypeptide is defined as a polypeptide which acts as an agonist, i.e..
• polypeptides may inhibit enzymatic formation of a deleterious effector compound (e.g.. inhibitors of the formation of renin, angiotensin I, or angiotensin II), may inhibit deleterious protein degradation (e.g.. degradation by stromelysin, or collagenase), or may inhibit enzymatic degradation of beneficial effector compounds (e.g.. neutral endopeptidase 24.11 degradation of atrial natriuretic factor, ANF, or angiotensin converting enzyme (ACE) degradation of bradykinin).
• a deleterious effector compound e.g. inhibitors of the formation of renin, angiotensin I, or angiotensin II
• deleterious protein degradation e.g.. degradation by stromelysin, or collagenase
• beneficial effector compounds e.g. neutral endopeptidase 24.11 degradation of atrial natriuretic factor, ANF, or angiotensin converting enzyme
• the invention features inhibitory polypeptides active against various proteases, including: (a) inhibition of proteases that lead to disease, inhibition of proteases involved in the biosynthesis of peptide hormones (including inhibition of proteases involved in hypertension, e.g., the production of renin, angiotensin I, angiotensin II, and endothelin, such as inhibition of proteases including prorenin protease, renin, ACE, and endothelin converting enzyme (ECE)); (b) inhibition of proteases directly involved in the disease process, including inhibition of proteases involved in inflammation processes, inhibition of proteases such as stromelysin, collagenases and other metalloproteases, inhibition of serine proteases involved in emphysema or arthritis (e.g..).
• proteases that lead to disease inhibition of proteases involved in the biosynthesis of peptide hormones (including inhibition of proteases involved in hypertension, e.g., the production of
• the invention also features polypeptides that antagonize peptide binding receptors and thereby produce a beneficial effect, including antagonizing the C-receptor of ANF to produce higher levels of ANF, antagonizing the angiotensin II receptor, and antagonizing the vasoactive intestinal peptide (VIP) receptor.
• VIP vasoactive intestinal peptide
• the presence of the normal, elevated, or reduced level of the natural protein is a causative element in diseases such as hypertension (high blood pressure), cancer, inflammation, congestive heart failure, Alzheimer's disease and arthritis.
• a protease involved in control of blood pressure is inhibited, e.g.. angiotensin - converting enzyme, renin, prorenin protease, neutral endopeptidase
• the inhibitory polypeptide may reduce the specific biological activity by direct interaction with the natural protein (e.g.. by 5 binding to the protein and thus inhibiting it in a competitive or non- competitive fashion).
• the agonist or inhibitory polypeptide may increase or decrease the specific biological activity of the natural protein by interacting with a naturally occurring effector compound and thus inhibiting the effect that 0 such an effector has upon the activity of the natural protein or to the active site of the protein itself.
• the natural protein may be a protease, and the effector an anti-protease. Inhibition of the anti-protease effector will increase the specific activity of the protease.
• An example includes the increased 5 activity of atrial natriuretic factor upon i hibition of neutral endopeptidase 24.11 (ANFase or enklephalinase).
• the inhibitory polypeptide may also reduce the specific biological activity (or increase such activity) by other competitive mechanisms, for example, by binding to a receptor for the natural 0 protein.
• the natural protein may be a clearance receptor, e.g.. for atrial natriuretic factor, and the inhibitory polypeptide binds to that receptor and thereby raises the level of atrial natriuretic factor in the body.
• the inhibitory polypeptide may reduce the specific biological activity by reducing 5 the rate of formation of the natural protein by inhibiting activity of a protease involved in production of that natural protein.
• the natural protein may be angiotensin II and the inhibitory polypeptide may act upon the converting enzyme which affects production of angiotensin II.
• the invention also features supplementation (by an agonist polypeptide) of a naturally occurring inhibitory polypeptide in those patients where increased levels of the inhibitory polypeptide will have benefit in patients without genetic deficiency.
• a naturally occurring inhibitory polypeptide for example the level of naturally occurring ⁇ ** -antitrypsin (trypsin as well as elastase inhibitor) can be increased by the method of the invention (by introducing a gene encoding this inhibitor) and thus is useful in ameliorating disease in patients with conditions such as emphysema, chronic obstructive pulmonary disease, and vascular wall weakenings (such as arterial aneurysms and dissections), ⁇ *-- antitrypsin can also be used to inhibit other related proteinases for the prevention and/or treatment of inflammatory diseases and arthritis.
• inhibitory polypeptides of proteinases are useful in treatment and prevention of arthritis and other inflammatory conditions, including rheumatoid arthritis and osteoarthritis.
• arthritis and other inflammatory conditions including rheumatoid arthritis and osteoarthritis.
• joint destruction in rheumatoid arthritis and other inflammatory joint diseases is reported to be mediated by various proteases (C.E. Brinckerhoff, Arthritis Rheum, 34:1073-5 (1991)).
• One group of degradative enzymes that is particularly important is the metalloproteinase family (including stromelysin and collagenase) (J.F. Woessner, Jr., FASEB J, 5:2145-2154 (1991)).
• TIMP tissue inhibitors of metalloproteinases
• the ratio of metalloproteinase and TIMP gene expression is thought to be a critical determinant of the net tissue destruction in arthritis.
• T1MP-1 gene is expressed by the rheumatoid arthritis synovium, the amount of protein produced appears to be insufficient to block extracellular matrix destruction.
• the present invention features delivery of a TIMP-1 gene to joint tissues in order to increase local TIMP production.
• genes encoding smaller inhibitory peptides could be delivered to the joint to accomplish the same goal.
• Another way to deliver genes to the joint to increase proteinase inhibitor production is to remove small amounts of synovial fluid or tissue and transfect or infect fibroblasts isolated from these samples with the gene. The cells could be expanded in vitro and then injected into inflamed joints where they would produce the inhibitory polypeptide.
• a similar method has been used to deliver a member of the IL-1 family into rabbit joints (G.
• proteases important in arthritis could also be targeted by gene therapy.
• Protease nexin-1 (PN-1) and protease nexin-2 (PN-2) as well as hirudin are examples of proteins that reportedly inhibit these enzymes (B. Bergman, et al., Proc Natl Acad Sci, 83:996-1000 (1986); M. Schapira and P. Patston, Trends Cardiovasc Med, 1 :146 - 151
• Alzheimer's disease which is characterized by the presence of beta amyloid deposits, may be treated by inhibition of proteases responsible for generating such beta amyloid.
• Other inflammatory and non-inflammatory diseases marked by tissue destruction like inflammatory bowel disease or emphysema can also be treated by inhibiting metalloproteinases or serine proteases.
• Alternative strategies for using gene therapy to prevent or treat inflammation include the use of a gene encoding peptide inhibitors of cellular adhesion, such as soluble integrins, solubilized forms of integrin ligands, or peptides that directly interfere with integrin-mediated adhesion.
• Genes that encode anti- inflammatory cytokines, such as IL-10, or that encode polypeptides that inhibit pro-inflammatory cytokines like IL-1 or TNF alpha will also be beneficial in the treatment of inflammatory diseases.
• Proteinases are involved in proliferation or spread of some cancer cells. Such proteinases aid survival of a tumor cell since they promote tissue and vascular invasion. Inhibition of one or more of these tumor proteinases by introduction of a tumor proteinase inhibitory polypeptide (such as TIMP-1 or TIMP-2) using the method of this invention will reduce or prevent tumor growth, spread and metastasis.
• a tumor proteinase inhibitory polypeptide such as TIMP-1 or TIMP-2
• proteinase inhibitory polypeptides are used to constantly inhibit proteinases which elevate blood pressure, e.g.. angiotensin converting enzyme, renin or endothelin converting enzyme. Inhibition of the latter proteinase reduces or prevents vasospasm associated with migraine headaches, or rheumatic diseases such as Raynaud's phenomena, scleroderma, and systemic lupus erythematosus. Such inhibition of vasospasm is also useful in treating ischemic diseases such as angina pectoris, heart attack, stroke, transient ischemic attack, peripheral vascular disease and bowel, skin and renal ischemia. Inhibition of angiotensin converting enzyme is also useful for treatment of angina pectoris, congestive heart failure and the prevention of heart attack and stroke.
• angiotensin converting enzyme is also useful for treatment of angina pectoris, congestive heart failure and the prevention of heart attack and stroke.
• Inhibition of this and other proteases by the method of this invention is useful in prevention of vascular remodeling associated with atherosclerosis and coronary reocclusion following percutaneous transluminal angioplasty and coronary artery bypass grafting administered either at the desired site or tissue where needed, or systemically.
• inhibition of this and other proteases by the method of this invention is useful in prevention of cardiac remodeling associated with pressure overload, post myocardial infarction hypertrophy, and other forms of heart failure.
• the invention includes delivery and expression of one or more genes encoding natural agonist or inhibitory polypeptides of naturally occurring proteins derived from animals, e.g.. humans.
• the invention includes delivery of one or more genes encoding novel agonist or inhibitory polypeptides, which can be created de novo or as derivatives of known agonists or inhibitors to be used in gene delivery, as described below.
• Genes encoding such agonists or inhibitors can be delivered in any form, including direct injection of nucleic acid, for example, as DNA or RNA, ex vivo treatment of cells with re-implantation of treated cells, or vector mediated transfer, including such vectors as retroviral, vaccinia, adenovirus, polio, adeno-associated virus, and herpes virus.
• This invention can also be used to enhance the antithrombic activity of endothelial cells which can be transfected or infected in vivo.
• the present invention inserts a sequence encoding an inhibitor or antagonist of prothrombotic mediators.
• inhibitors may bind at either the proteolytic (active) site or the fibrinogen binding site of the thrombin molecule.
• Additional inhibitors include PPACK (D-Phe-Pro-ArgCH 2 CI; Kettner and Sahw, Thrombosis Res., 14, 969-973 (1979)). or serine protease inhibitors (Schapira and Patston, Trends Cardiovasc.
• Peptide antagonists containing RGD or KGD sequences have been described (see for example, Nichols, et al., Trends in Pharmacological Sciences, 13, 413-417 (1992)) and claims are made for derivatives thereof or other inhibiting sequences.
• genes encoding such polypeptides or the natural RGD-containing polypeptides derived from the venom of various vipers such as trigramin (Huang, et aL, Biochemistry, 28, 661-666 (1989)), echistatin (Gan, e al., J. Biol. Chem., 263, 19827-832 (1988)), bitstatin (Shebuski, et al., J.
• Another use of this invention is the transfection or infection of endothelial cells in vitro or ex vivo (for example, those obtained from human umbilical vessels or from autologous veins) with genes encoding antithrombotic peptides or proteins. These endothelial cells can then be grafted on cardiac valves, vascular stents, or other appropriate prostheses to from an antithrombotic surface when implanted in vivo, utilizing the antithrombotic approaches outlined above.
• the method of this invention and delivery of polypeptide agonist and inhibitors is useful in treating not only humans and animals, but also for treatment of plants.
• the invention provides a broad means by which a number of conditions or diseases can be treated.
• a suitable purified nucleic acid is introduced into a cell (e.g.. an endothelial cell, a kidney, blood element, fibroblast, heart, or liver cell) and expression of an agonist or inhibitory polypeptide caused by use of an appropriate gene expression system.
• the level of activity of the agonist or inhibitory polypeptide can be regulated using standard methodology.
• a promoter and enhancer region can be selected and provided to be suitable for expression in such a cell, and under certain circumstances to recognize the level of one or more factors relevant to a desired level or expression of the inserted gene.
• nucleic acid sequences can be introduced not only into patients suffering from those diseases or conditions discussed above, but also into normal patients as a preventative or prophylactic treatment.
• any particular person is known to be susceptible to a specific disease later in life (e.g.. susceptibility to cancer growth because of a family predilection, or because of occurrence of a cancer earlier in life)
• that person may have a gene encoding an appropriate polypeptide introduced into their body so that the polypeptide is active to prevent occurrence or manifestation of the disease.
• those patients thought to be susceptible to Alzheimer's disease may be treated by provision of an inhibitory polypeptide which inhibits a protease which may become elevated in such patients. Occurrence of Alzheimer's disease has been linked to the presence of proteases responsible for generating beta amyloid, thus inhibition of such proteases will prevent early manifestation of
• Alzheimer's disease these patients are prevented from suffering from this disease by introducing the inhibitory polypeptide-encoding nucleic acid before the patient is at a stage of life when the disease may be manifest, for example, at age 50 or 60.
• purified nucleic acid sequences is meant to include any nucleic acid produced by recombinant DNA or RNA technology that is synthesized or isolated from nucleic acid with which it naturally occurs.
• a vector system such as a phage, plasmid or virus system, so that it might be introduced into any desired cell.
• the vector be formed from a viral system which can be introduced into endothelial cells or fibroblasts to cause expression of a desired polypeptide within such cells or secreted from such cells.
• endothelial cells may be infected with a desired gene in a virus or other carrier, and then placed into a patient.
• This invention is advantageous over prior methods for inhibiting protein activities in vivo which involved direct administration of an inhibitory polypeptide.
• the present invention allows controlled and continuous regulation of expression of the inhibitory polypeptide so that sufficient polypeptide is produced as needed by a patient. Since such polypeptides can be expressed constitutively it can be an effective in vivo inhibitor even if the in vivo half-life of the polypeptide is short.
• Hypertension and high blood pressure are regulated by a complex system including an interrelationship between hormones and various proteinases.
• a complex system including an interrelationship between hormones and various proteinases.
• the method of this invention can be used to interfere with the biological activity of at least one of these protein activities and thus affect occurrence of hypertension.
• Renin and Angiotensin An outline of one system known to be involved in regulation of blood pressure is termed "the renin and angiotensin system” and is described by Cushman and Ondetti, Progress in Medicinal Chemistry, eds. Ellis and West, Elsevier/North-Holland Biomedical Press 42, 1980, hereby incorporated by reference herein.
• Renin and angiotensin converting enzyme are involved in production of angiotensin II, which acts at angiotensin receptors to regulate blood pressure.
• Renin is an endopeptidase released into the blood from the kidney and causes cleavage of the glycoprotein, angiotensinogen, to produce angiotensin I. This is converted to an octapeptide, angiotensin II, by angiotensin converting enzyme.
• Angiotensin II is a potent hypertensive and salt-retaining peptide. Angiotensins I and II are also hydrolyzed by peptidases referred to as angiotensinases.
• angiotensinogen to angiotensin II (or the later produced product angiotensin III)
• angiotensin II or the later produced product angiotensin III
• antibodies to the various components have been used in treatment to inhibit the various activities, but are depleted from the body and do not necessarily penetrate to the site of action of the target factor.
• Other inhibitors include various receptor antagonists which act in a competitive fashion, and inhibitors of the various proteases such as both angiotensin converting enzyme and renin.
• Effective inhibitors include snake venom peptides and orally active inhibitors which bind specifically at the active site of the enzyme.
• Other such inhibitors are small polypeptides having between 5 and 9 amino acids, and are known to be efficient inhibitors of angiotensin converting enzyme.
• Angiotensin converting enzyme is also inhibited to varying degrees by polypeptides that bind to the enzyme in the same manner as substrates or the polypeptide or dipeptide products of the enzyme's action.
• peptide inhibitors of this enzyme activity are presently known.
• Angiotensin converting enzyme is also able to cause breakdown of bradykinin.
• inhibition of the activity of angiotensin converting enzyme is beneficial to a hypertensive patient in two ways since it (a) reduces the level of angiotensin II, and thus reduces blood pressure, and (b) increases the level of bradykinin which is thought to be responsible for vasodilation, and thus also alleviates high blood pressure.
• Renin Inhibitors chapter 11 , page 483 (which describes use of substrate analogs as inhibitors of renin, as well as inhibitory peptides based on the structure of prorenin), Cushman et al., Enzyme Inhibitors as Drugs , Ed. Sandier, p. 231 , Cushman et al., 17 Progress in Medicinal Chemistry 42, 1980, and by Douglas, et al.. 116 Endocrinology. 1598, 1985 (which alsp describes the efficacy of octa- and heptapeptide receptor antagonists of angiotensin II).
• Such inhibitors are said to be rapidly cleaved by peptidases, and thus have a short duration of action upon intravenous administration.
• Two particularly useful examples of polypeptides inhibitory to angiotensin converting enzyme include Pro-Lys-Trp-Ala-Pro and Glu-Trp-Pro-Arg-Pro-Gln-lle-Pro-Pro.
• Another useful target is a protease involved in production of renin from preprorenin or prorenin.
• Inhibitors useful in this invention will include polypeptides having an amino acid sequence similar to the proteolytic target of preprorenin or prorenin. Such an inhibitor is best produced (e.g.. by viral transfection) within the juxtaglomerular cell in the kidney so that the peptide inhibitor is within the cells where the target protease is located.
• Boger 20 Annual Reports in Medicinal Chemistry. 257, describes inhibition of renin using antibodies to purified renin, and competitive inhibitors based upon the 13 or 14 amino acid substrate sequence of renin.
• This structure can be used as an aid to designing useful polypeptide inhibitors.
• Angiotensin converting enzyme is a particularly useful target for inhibition since blockage of the enzyme is possible with minimal ill effect (a cough may occur in some patients). Renin may also be totally blocked, but even reduction of its activity by about 50% within a patient may be sufficient to regulate blood pressure.
• the level of renin and angiotensin within most patients having high blood pressure is not significantly different from normal, however, the above-noted peptide inhibitors provide a significant lowering of blood pressure.
• Inhibition of renin in normotensive individuals does not lower blood pressure.
• regulating the levels of renin and angiotensin also will have utility in the treatment of heart failure. Endothelin
• Another useful target system for treatment of hypertension and heart failure is the hormone endothelin described generally by Yanagisawa and Masaki, 38 Biochemical Pharmacology. 1877, 1989.
• This hormone causes stimulation of atrial natriuretic peptide secretion and inhibits renin release.
• the hormone itself acts as a vasoconstrictor and has implications in the treatment of hypertension. It is formed from a precursor which is proteolytically cleaved by endothelin converting enzyme.
• a useful target for treatment of hypertension is endothelin converting enzyme to prevent production of endothelin, and thus reduce hypertension. Since endothelin regulates the level of renin, inhibition of production of endothelin will be particularly useful for treatment of hypertension.
• Inhibitors of any target enzyme or hormone or receptor can be identified by methodology equivalent to that used for identifying inhibitors of renin and angiotensin converting enzyme discussed above, and in the art cited above.
• Immunopharmaceutics, Inc. states that it has synthesized a potential human therapeutic and endothelin receptor antagonist by use of computer aided antibody directed drug design technology. This antagonist is stated to be useful in development of a treatment for hypertension.
• polypeptides which act as antagonists to the production or actions of endothelin will be useful in the present invention.
• Inhibitors at the angiotensin II receptor are described by Douglas, et al. 116 Endocrinology. 1598 (1985) where angiotensin II analogs containing modifications of the N-terminus and C-terminus are shown to act as angiotensin II receptor antagonists.
• angiotensin II analogs containing modifications of the N-terminus and C-terminus are shown to act as angiotensin II receptor antagonists.
• One example is Arg-Val-Tyr-lle-His-Pro-lle.
• Atrial Natriuretic Factor C- receptor Maack, et al.. 238 Science. 675 (1987) describe atrial natriuretic factor C-receptor inhibitors.
• polypeptide analogs of atrial natriuretic factor are described that lower blood pressure by binding to the atrial natriuretic factor clearance receptor and thus raise endogenous levels of atrial natriuretic factor.
• Other Receptors are described by Douglas,
• polypeptides active at a receptor for neuropeptide Y include polypeptides active at a receptor for neuropeptide Y, and other such polypeptides e.g.. vasoactive intestinal polypeptide, which are co-released from sympathetic nerves and amplify nerve activity, and thus contribute to high blood pressure.
• Peptides of the present invention can be administered to block the activity of these target polypeptides on their receptors. Since such receptors are known, such inhibitory polypeptides can be modeled on the target polypeptides themselves to provide inhibition in a competitive manner.
• any enzyme, hormone, or polypeptide which acts to elevate blood pressure can be inhibited by the method of the present invention by blocking the activity of that enzyme, hormone or polypeptide by a competitive or non-competitive mechanism using a polypeptide inhibitor produced intracellularly or inhibitor secreted into the bloodstream or interstitial fluid.
• an enzyme, hormone or receptor which is useful for reducing blood pressure may be introduced by a method of this invention to act as an agonist to a natural protein.
• a polypeptide is produced in the endothelial cells, heart cells, or other cells which are in contact with the blood, e.g.. the liver, kidney (particularly, for renin) and vascular smooth muscle cells. It is preferable that such cells be replicating cells.
• the inhibitors may be as short as dipeptides, for example, Phe-Ala described by Llorens, et al.. 96 BBRC. 1710 (1980). Kow and Pfaff, 28 Annual Review Pharmacological Toxicology. 163 (1988) describe the neuromodulatory actions of various peptides and notes that vasopressin may be useful in regulation of blood pressure.
• polypeptide inhibitors and agonists are already known which are suitable for reducing hypertension.
• Other useful polypeptides can be identified using existing technology to determine those most useful in regulation of hypertension and blood pressure. As described above, such polypeptides may act by inhibiting protease activity, or by binding competitively at a receptor involved in regulation of hypertension (for example, by preventing hormonal binding to that receptor), or may inhibit activity of enzymes which reduce the level of peptides or hormones which reduce hypertension.
• Peptides produced by the present invention are active in vivo. Their production can be maintained at a constant or varied level. Thus, such activity can be maintained for an extended period of time by such continuous production, while exogenous peptide administration would be impractical because of the short half-life of many such peptides.
• the structure of the inhibitory polypeptide and target enzyme is studied and modifications of the structure of the inhibitory polypeptide made using rational design criteria to design a optimum polypeptide.
• modified polypeptides i.e.. one having a slightly different amino acid sequence from the lead compound
• Such libraries can be screened by assay of protease inhibitory activity using protocols designed for specific detection of a desired polypeptide inhibitor. Such technology is described generally by Brown, Genetic Engineering News. 1 (January 1992) hereby incorporated by reference herein. The three major methods described in the literature are: a) screening of a peptide library generated from bacteriophage vectors containing random oligonucleotide sequences: (Parmley and Smith, 73 Gene. 305, 1988, Scott and Smith, 249 Science. 386, 1991 , Cwirla, et al.. 87 Proc. Nat. Acad.
• nucleic acid sequence which encodes the desired inhibitory polypeptide, and provide that nucleic acid sequence adjacent other sequences useful for in vivo expression of the polypeptide.
• the nucleic acid encoding an inhibitor may be introduced by direct gene transfer or other gene therapy techniques referenced above (and incorporated herein by reference).
• the inhibitor is produced as a secreted peptide and thus is provided with pro and prepro sequences and any other modifications necessary for its useful production in vivo.
• pro and prepro sequences and any other modifications necessary for its useful production in vivo There follows examples of such procedures known in the art. See e.g.. Dorner & Kaufman, 185 Meths. Enz. 577, 1990. Of course, the actual methodology used will depend upon the organism being treated, e.g.. bacterium or human. Those skilled in the art will recognize the description below as a preferable guide for in vivo human production. Equivalent techniques can be used in other organisms.
• Protein or peptide secretion typically requires the protein to have 16-30 residues (signal sequence) attached to the N-terminus, which enables the protein to be translocated into the endoplasmic reticulum. During this process the signal sequence is cleaved by specific proteases.
• signal sequence an example of a signal sequence as described in the literature is Met-Arg-(Leu) n -Pro-Xaa-Ala-Leu-
• preproinsulin Long Term Evolution
• preproalbumin a protein that undergoes processing in either the cell or in whole blood to generate the active polypeptide.
• the polypeptide or protein is initially translated into a prepropeptide (i.e.. an N-terminal signal sequence, the active peptide and an N-terminal or C-terminal peptide).
• Secretion of the active polypeptide involves: (1) translocation into the endoplasmic reticulum and cleavage of the signal peptide and (2) cleavage of the propeptide to the active polypeptide (see In Brain Peptides, (Eds. Krieger, et al.) "Biosynthesis and processing of neuropeptides by Loh and Gainer). The latter process can occur in a variety of ways. Typically, endocrine cells sort propeptides to a secretory granule which contains the processing enzyme. These enzymes can have high specificities and are localized only in certain cell types (e.g..).
• prorenin is cleaved by a thioprotease in secretory granules found in juxtaglomerular cells).
• a second secretion pathway (primarily used by non-endocrine cells) involves transfer of the propeptide to membrane-bound vesicles, which mediate constitutive secretion of the propeptide.
• cleavage to the active peptide requires extracellular enzymes (either in the blood or on the surfaces of cells that make sufficient contact with propeptides that are secreted into the blood).
• procollagen is an example.
• Inhibitory peptides of this invention can be expressed in a system modelled upon any of these types of systems.
• Sequences of some cleavage sites are known with the classical site being the dibasic amino acid sequence Lys-Arg (see e.g.. Hosaka, et al.. 266 J. Biol. Chem.. 12,127, 1991). Cleavage may also appear to depend on other parts of the sequence as well the dibasic group. Sequence specificities for some extracellular proteases are known (e.g.. for neutral endopeptidase 24.11 , and Factor X).
• the polypeptide inhibitor can be biosynthesized as a part of a propeptide where the cleaved product would be the peptide inhibitor and a peptide (e.g.. N-terminal fragment of insulin, ANF, or a serum protein such as albumin).
• a peptide e.g.. N-terminal fragment of insulin, ANF, or a serum protein such as albumin.
• Another strategy is to create a peptide containing numerous copies of the peptide inhibitor separated by a peptide sequence containing a cleavable site.
• the polypeptide may be produced as a protein with the inhibitory polypeptides positioned within the protein such that inhibition of the desired target is still attainable. Possible sites are at the C-terminus, N-terminus or in a solvent-exposed region of the protein, e.g.. within a loop of the protein.
• An example of such a construction is the binding of hexapeptides expressed on the phage coat protein to receptors or enzymes as described in Parmley and Smith, 73 Gene 305, 1988.
• Suitable systems for expression and delivery of nucleic acid encoding the above inhibitory polypeptides are described by Nabel et al.. 249 Science. 1285, 1990, Dick et al.. Trends in Genetics. June 1986, Breakfield, EPA 453242, and by Buttrick, et al.. 70 Circulation Research. 193, 1992, all hereby incorporated by reference herein.
• the level of expression of a desired inhibitory polypeptide is chosen to provide a desired level of in vivo inhibition. In one extreme a gene may be expressed constitutively. Alternatively, a promoter or other regulatory region can be chosen to cause gene expression only when a specific level of a chosen signal is present in the expressing cell.
• control and level of expression can be determined by choice of a particular promoter (e.g.. with low or high expression level, regulated or not regulated) and by selection of a vector with a copy number of suitable magnitude or by the quantity of vector or transduced cells used.
• TIMP-1 cDNA (Nature 315:768 (1985)) is cloned into the polylinker site of pAFVXM. This plasmid can be used to generate infectious vector particles (see below).
• a dominant selectable marker comprised of an SV40 early promoter driving expression of neomycin phosphotransferase is inserted into the vector to facilitate isolation of infected or transfected cell lines.
• a promoter selected from the RSV promoter, SV40 early or late promoter, the CMV immediate early promoter, human beta-bactin promoter, or Moloney murine MLV SL3-3 promoter, is inserted upstream to the TIMP-1 gene in the construct or alternately to insert the TIMP-1 gene into an amphotrophic retroviral vector using the LTR as a promoter and containing the packaging signal.
• plasmids when placed in a suitable packaging cell, express a retroviral vector construct which contains a packaging signal.
• the packaging signal directs packaging of the vector construct into a capsid and envelope along with all further proteins required for viable retroviral particles.
• the capsid, envelope, and other proteins are preferably produced from one or more plasmids containing suitable genomes placed in the packaging cell.
• Such genomes may be a proviral construct, which in a simple case may merely have the packaging signal deleted. As a result, only the vector will be packaged. Suitable packaging cell lines and the genome necessary for accomplishing such packaging, are previously described (Mol Cell Bio 6:2895 (1986)).
• nucleic acid thus, produced can be suitably administered to the animal in fashions known to those of skill in the art, e.g., by injection into the requisite site.
• Isolated viral constructs containing either the rodent TIMP-1 cDNA, an inactive TIMP-1 mutant cDNA, or an irrelevant rodent protein cDNA are injected into the rear ankle joints of anesthetized Lewis rats or a susceptible mouse strain.
• cultured rodent synoviocytes are infected in vitro and subsequently injected into the joints.
• the rats are immunized with complete Freund's adjuvant (0.75 mg of mycobacterium butyricum in 100 uL of mineral oil) in the base of the tail.
• the invention includes implantation of cells in a region that can be removed, or use of a second gene which will destroy the cell in which it is resident when it is no longer desired, by, for example, the use of a benign compound activated to a toxic form by the conditional lethal gene.
• administration can be topically in the nose, eyes, urethra, ureter or renal papilla, e.g.. for anti-inflammatory treatment or anti-allergy treatment.
• Nucleic acid which does not integrate into DNA can be used in place of the conditional lethal genes discussed above.
• a lethal gene may be activated by shining a light on the patient to turn on a light-activated promoter regulating the lethal gene, e.g.. thymidine kinase.
• Other diseases or conditions which can be treated by the methods of this invention include those affected by (1) anticoagulants, e.g.. a tPA inhibitor or other coagulation factor inhibitors such as protein C or S, (2) complement inhibitors, (3) inhibitors of adhesion molecules such as E-selectin, ICAM or platelet receptor llb/llla, (4) inhibitors of cytokine binding, such as IL-1 , (e.g.. to treat rheumatoid arthritis) or TNF, for lowering homocysteine levels, or protective apolipoprote ⁇ ns (e.g.. for treatment of atherosclerosis or hyperlipidemia), and (6) various antibodies.
• anticoagulants e.g.. a tPA inhibitor or other coagulation factor inhibitors such as protein C or S

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