Patent application title: TREATMENT FOR DARK ADAPTATION
Daniel M. Schwartz (San Francisco, CA, US)
Keith G. Duncan (San Francisco, CA, US)
Kathy R. Bailey (San Francisco, CA, US)
John P. Kane (Hillsborough, CA, US)
Brian Y. Ishida (Walnut Creek, CA, US)
IPC8 Class: AA61K9127FI
Class name: Drug, bio-affecting and body treating compositions preparations characterized by special physical form liposomes
Publication date: 2010-02-25
Patent application number: 20100047330
The present invention addresses the treatment of age-related macular
degeneration using regulation of pathogenic mechanisms similar to
atherosclerosis. In further specific embodiments, compositions that
increase reverse cholesterol transport are utilized as therapeutic
targets for age-related macular degeneration. In a specific embodiment,
the lipid content of the retinal pigment epithelium, and/or Bruch's
membrane is reduced by delivering Apolipoprotein A1, particularly a
1. A method of treating macular degeneration in an individual, comprising
the step of increasing reverse cholesterol transport in an ocular tissue
of the individual, wherein said increasing reverse cholesterol transport
comprises increasing Apolipoprotein A-I (ApoA-I) level in the ocular
2. The method of claim 1, wherein said increasing ApoA-I level comprises administering to the individual a therapeutically effective amount of an ApoA-I composition.
3. The method of claim 2, wherein the ApoA-I composition comprises an ApoA-I polypeptide or peptide.
4. The method of claim 3, wherein the ApoA-I peptide comprises SEQ ID NO:15.
5. The method of claim 3, wherein the ApoA-I peptide comprises SEQ ID NO:16.
6. The method of claim 3, wherein the ApoA-I polypeptide or peptide comprises D-amino acids.
7. The method of claim 1, wherein increasing the ApoA-I level comprises upregulating expression of ApoA-I.
8. The method of claim 1, wherein increasing the ApoA-I level is further defined as administering an agent to the individual, such that said agent increases the level of circulating ApoA-I in the individual.
9. The method of claim 8, wherein the agent comprises 1,2 dimyristoyl-.alpha.-glycero-3-phosphocholine (DMPC).
10. The method of claim 1, wherein the composition is administered to the individual locally or systemically.
11. The method of claim 1, wherein the composition is administered orally, parenterally, topically, intradermally, subcutaneously, intramuscularly, intraperitoneally, or intravenously.
12. The method of claim 6, wherein said composition is administered to an individual orally, wherein said composition is further defined as comprising a liposome.
13. A method of increasing lipid efflux from an ocular tissue comprising the step of increasing the level of ApoA-I in said tissue.
14. A method of treating macular degeneration (AMD) in an individual, comprising the step of delivering a therapeutically effective amount of a liposome comprising ApoA-I to at least one tissue of the individual.
CROSS-REFERENCE TO RELATED APPLICATIONS
The present invention is a continuation application of U.S. Nonprovisional patent application Ser. No. 11/055,309, filed Feb. 10, 2005, which is a divisional application of U.S. Nonprovisional patent application Ser. No. 10/794,198, filed Mar. 5, 2004, which is a continuation-in-part of U.S. Nonprovisional patent application Ser. No. 10/428,551, filed May 2, 2003, which is a continuation-in-part of U.S. Nonprovisional patent application Ser. No. 10/313,641, filed Dec. 6, 2002, which claims the benefit of U.S. Provisional Patent Application 60/340,498, filed Dec. 7, 2001; and which also claims the benefit of U.S. Provisional Patent Application 60/415,864, filed Oct. 3, 2002; all priority applications are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
The present invention is directed to the fields of opthalmology and cell biology. Specifically, the invention regards increasing reverse cholesterol transport in the retinal pigment epithelium and Bruch's membrane. More specifically, the invention relates to treatment of age-related macular degeneration (AMD) utilizing regulation of reverse cholesterol transport.
BACKGROUND OF THE INVENTION
Age-related macular degeneration (AMD) is the leading cause of severe visual loss in the developed world (Taylor et al., 2001; VanNewkirk et al., 2000). In the early stages of the disease, before visual loss occurs from choroidal neovascularization, there is progressive accumulation of lipids in Bruch's membrane (Pauleikhoff et al., 1990; Holz et al., 1994; Sheraidah et al., 1993; Spaide et al., 1999). Bruch's membrane lies at the critical juncture between the outer retina and its blood supply, the choriocapillaris. Lipid deposition causes reduced hydraulic conductivity and macromolecular permeability in Bruch's membrane and is thought to impair retinal metabolism (Moore et al., 1995; Pauleikhoff et al., 1990; Starita et al., 1996). Retina and/or RPE may respond by elaboration of angiogenic factors (e.g. VEGF, vFGF) that promote growth of choroidal neovascularization.
Interestingly, lipid accumulation in Bruch's membrane similar to that in AMD has been observed in apolipoprotein E (apo E) null mice (Dithmar et al., 2000; Kliffen et al., 2000). Because of the additional association between apo E alleles and other age-related degenerations, Alzheimer's disease and atherosclerosis, there has been recent investigation into a potential role for apo E in AMD.
Several studies on apo E polymorphism in AMD have been conducted (Simonelli et al., 2001; Klayer et al., 1998; Souied et al., 1998). In contrast to Alzheimer's disease, the apo E-4 allele has been associated with reduced prevalence of AMD. Apo E-2 allele is slightly increased in patients with AMD. Further supporting a role in AMD pathogenesis, apo E has been detected in drusen, the Bruch's membrane deposits that are the hallmark of AMD (Klayer et al., 1998; Anderson et al., 2001). Immunohistochemistry on post-mortem eyes has demonstrated apo E in the basal aspect of the retinal pigment epithelium (RPE) (Anderson et al., 2001). Cultured RPE cells synthesize high levels of apo E mRNA, comparable to levels found in brain (Anderson et al., 2001).
While the role of apo E in AMD is not established, this apolipoprotein has several functions that may affect the course of this disease. Apo E has anti-angiogenic (Browning et al., 1994), anti-inflammatory (Michael et al., 1994), and anti-oxidative effects (Tangirala et al., 2001). These are all considered atheroprotective attributes of Apo E, but may also be important in protecting against progression of AMD. While atheroprotective effects of apo E were initially thought to stem from effects on plasma lipid levels, local effects on vascular macrophages are probably equally important. Thus, selective enhanced expression of macrophage apo E in the arterial wall reduces atherosclerosis in spite of hyperlipidemia (Shimano et al., 1995; Bellosta et al., 1995; Hasty et al., 1999). Conversely, reconstitution of apo E null macrophages in C57BL/6 wild type mice induces atherosclerosis (Fazio et al., 1994). Atheroprotective effects of arterial apo E expression are thought to derive in part from facilitation of reverse cholesterol transport (Mazzone et al., 1992; Lin et al., 1999). The mechanisms by which apo E facilitates reverse cholesterol transport are incompletely understood. Apo E expression increases cholesterol efflux to HDL3 in J774 macrophages (Mazzone and Reardon, 1994) and lipid free apolipoprotein A1 (Langer et al., 2000). Cell surface apo E is also hypothesized to induce efflux from the plasma membrane (Lin et al., 1999).
Reverse cholesterol transport may be important in the pathogenesis of AMD because of lipid efflux from RPE into Bruch's membrane. Very much like intimal macrophages, RPE cells progressively accumulate lipid deposits throughout life; however, unlike vessel wall macrophages, the source of RPE lipid is thought to be retinal photoreceptor outer segments (POS) (Kennedy et al., 1995). Every day, each RPE cell phagocytoses and degrades more than one thousand POS via lyzosmal enzymes. These POS are enriched in phospholipid and contain the photoreactive pigment, rhodopsin. Incompletely digested POS accumulate as lipofuscin in RPE. By age 80, approximately 20% of RPE cell volume is occupied by lipofuscin (Feeney-Burns et al., 1984).
Analysis of Bruch's membrane lipid reveals an age-related accumulation of phospholipid, triglyceride, cholesterol, and cholesterol ester (Holz et al., 1994; Curcio et al., 2001). The origin of these lipids also is thought to derive principally from POS rather than from the circulation (Holz et al., 1994; Spaide et al., 1999). POS lipids are hypothesized to efflux from the RPE into Bruch's membrane. Although cholesterol ester deposition in Bruch's suggests contribution from plasma lipids, biochemical analysis of these esters suggests esterification of intracellular cholesterol by RPE cell derived ACAT (Curcio et al., 2002). While trafficking of lipids from the retina to RPE cells has been studied extensively, mechanisms of lipid efflux from RPE to Bruch's membrane are not well understood. Furthermore, from a pathogenic standpoint, regulation of lipid efflux into Bruch's membrane may be important in determining the rate of lipid-induced thickening that occurs in aging.
In AS, similar to AMD, lipids accumulate in the extracellular matrix and within phagocytic cells, primarily macrophages. Mechanisms of lipid metabolism in AS have been investigated in detail. Similar investigations into lipid processing by RPE and subsequent lipid efflux into BM and the circulation have not been conducted with the same depth as those for AS. As a consequence, potential therapeutic approaches to dry AMD are wonting.
Navab et al. (2003) describe ApoA-I mimetic peptides comprising D-amino acids for oral delivery for the treatment of atherosclerosis.
U.S. Patent Application Publication US 2002/0142953 relates to human compositions encoding apolipoproteins that are related to lipid metabolism and cardiovascular disease.
Thus, the present invention provides a novel approach to reduce lipid content of ocular tissue, such as Bruch's membrane and further provides methods and compositions for the treatment of macular degeneration, such as AMD.
SUMMARY OF THE INVENTION
In the present invention, there are methods and compositions that relate to increasing reverse cholesterol transport in the retinal pigment epithelium (RPE). Particularly, the increase in reverse cholesterol transport is mediated, enhanced, facilitated, and/or triggered by administration of a composition. More particularly, one or more compositions promote efflux of lipids from Bruch's membrane and/or enhances binding of effluxed lipids from Bruch's membrane, thereby reducing accumulation of lipids in both retinal pigment epithelium and Bruch's membrane. This is beneficial in these regions, given that in aging Bruch's membrane, there is progressive accumulation of lipid and cross-linked protein that impedes hydraulic conductivity and macromolecular permeability. This abnormal deposition, in specific embodiments, also impairs the ability of some larger molecular weight species of HDL, a preferred cholesterol and phospholipids acceptor for lipids effluxed by cultured human RPE, to act as a lipoprotein acceptor. As HDL is unable to pass through BM and promote efflux and/or bind effluxed lipids, more lipids accumulate in both RPE and BM. A skilled artisan recognizes that such accumulations are a major finding in age-related macular degeneration (AMD), and, therefore, recognizes the need for novel compositions for the treatment of this debilitating disease.
Apolipoprotein A1 (ApoA-I) is the major lipoprotein component of HDL, and it has a mass of approximately 28 kDaltons. ApoA-I bound to phospholipids comprises nascent HDL particles that bind to ABCA1 on the RPE basal membrane and promote lipid efflux. Because of the low molecular weight of ApoA-I, it can penetrate an aged BM more easily than larger molecular weight species of HDL to bind to the RPE. In addition to its role in promoting reverse cholesterol transport from RPE, ApoA-I also is a potent anti-oxidant, which is known to reduce visual loss in patients with AMD.
In some embodiments, the present invention is directed to a system, method, and/or composition(s) related to treating AMD. Treatments for dry AMD have been lacking, because the pathogenesis of this common condition is poorly understood, and the inventors have demonstrated analogous biological behavior between human retinal pigment epithelial (RPE) cells and macrophages that point toward similar pathogenic mechanisms of AMD and atherosclerosis. Specifically, reverse cholesterol transport (RCT) is exploited in the present invention for the treatment of AMD. The present inventors provide the novel demonstration of RCT in RPE cells in the eye. More specifically, RCT is regulated through manipulation of levels of cholesterol and/or phospholipid transporters (ABCA-1, Apo E, SRB-1, SRB-2) by nuclear hormone receptor ligands such as agonists of thyroid hormone (TR), liver X receptor (LXR), and/or retinoid X receptor (RXR). A goal for the present invention is the reduction of lipid content of RPE Bruch's membrane to facilitate an improvement in visual function and/or, in some embodiments, prevent ocular disease, such as AMD. Reduction of the lipid content of Bruch's membrane preferably results in at least one or more of the following: reduction in development of CNV; improvement in dark adaptation; improvement in night vision; improved visual acuity; and/or improved recovery to bright flash stimulus.
In an additional embodiment of the present invention, there is a method of treating macular degeneration (AMD) in an individual, comprising the step of delivering to the individual a therapeutically effective amount of an ApoA-I composition. In a specific embodiment, the delivering occurs under conditions wherein reverse cholesterol transport is upregulated, wherein lipid accumulations in BM or RPE are reduced, wherein efflux of lipids from BM is increased, and/or wherein therapeutic anti-oxidant applications are achieved. In further specific embodiments, the administration of the ApoA-I composition results in effective treatment for AMD or any ocular disease, such as be ameliorating at least one symptom of the disease. Delivery of the ApoA-I composition may occur by any method in the art so long as it provides a therapeutically effective amount to the tissue or tissues in need thereof. The delivery may be local or systemic. In preferred embodiments, the delivery is intravenously, as has been done for ApoA-I in mouse models of atherosclerosis and in patients with coronary artery disease. In other embodiments, the delivery is oral.
In particular aspects of the invention, the ApoA-I composition may be any ApoA-I composition that upon delivery to an individual suffering from an ocular disease such as AMD, said disease has amelioration of at least one symptom. For example, the ApoA-I composition may be comprised of one or more L-amino acids or one or more D-amino acids, or mixtures thereof. In particular embodiments, there is an ApoA-I mimetic peptide comprised of D-amino acids, which is not recognized as readily by human proteases, and thus can be administered orally. In specific embodiments, this is more convenient than parenteral administration with an intravenous formulation comprising the L-amino acid ApoA-I or its mimetic peptide. In specific embodiments, exemplary mimetic ApoA-I peptides are used, such as are described by Navab et al. (2003), incorporated by reference herein in its entirety.
By way of example, patients with AMD (atrophic or exudative) are administered an ApoA-I composition, such as intravenous ApoA-I, ApoA-I mimetic peptide, or compositions that increase circulating ApoA-I, such as the exemplary oral synthetic phospholipid (1,2 Dimyristoyl-sn-glycero-3-phosphocholine) (DMPC). Administration could occur in any frequency so long as there is at least one therapeutic effect, and in preferred embodiments the effect is detectable. The administration, in specific but exemplary embodiments, is as frequent as daily or less frequently as in every other month depending on the method of administration and the clinical response.
In another embodiment of the present invention, there is a kit for the treatment of macular degeneration, housed in a suitable container, comprising a ApoA-I composition. In particular embodiments, the ApoA-I composition may be ApoA-I from any organism, but particularly human ApoA-I, a mimetic ApoA-I peptide, or an agent that increases ApoA-I circulating levels, such as DMPC. In a specific embodiment, the kit comprises a pharmaceutically acceptable excipient. In another specific embodiment, the ApoA-I composition is comprised in the pharmaceutically acceptable excipient. In other specific embodiments, the ApoA-I composition is comprised in a liposome and delivered orally to an individual.
The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which:
FIG. 1 shows that RPE cells express Apo E, ABCA1, and LXR α.
FIG. 2 shows RPE cell expression of SR-BI and SR-BII.
FIG. 3 illustrates SR-BI and SR-BII immunofluorescence in RPE cells.
FIG. 4 demonstrates ABCA1 immunofluorescence in RPE cells.
FIG. 5 demonstrates that basal Apo E expression is greater than apical Apo E expression in cultured human RPE cells.
FIG. 6 shows regulation of Apo E expression by nuclear hormone receptor ligands.
FIG. 7 provides a non-denatured polyacrylamide gel of lipoprotein fractions.
FIG. 8 shows 14C distribution of the fractions from FIG. 7.
FIG. 9 demonstrates thin layer chromatography illustrating the identification of six out of seventeen spots of an HDL fraction. Note: HDL is the high density lipoprotein fraction; POS is labeled POS starting material; PC is phophatidylcholine; PI is phosphatidylinisotol; PE is phosphatidylethanolamine; C is cholesterol; TRL is TG rich lipid, including triglycerides and cholesterol ester.
FIG. 10 demonstrates that 14C counts increase following drug treatments that increase RCT.
FIG. 11 illustrates ABCA1 regulation by RXR and LXR ligands.
FIG. 12 shows HDL, LDL and plasma stimulation of 14C-labeled lipid transport the identification of HDL from RPE cells.
FIG. 13 shows stimulation of CD36 expression by oxidized lipid.
FIG. 14 illustrates apical and basal secretion from RPE cells of apoe in the presence of T3 (T), 22(R) hydroxycholesterol, or cis retinoic acid (RA).
FIG. 15 shows that apoe secreted from RPE cells binds to HDL.
FIG. 16 demonstrates that HDL stimulates lipid efflux from RPE cells in culture.
FIG. 17 shows characterization of HDL and plasma bound POS lipids by thin layer chromatography.
FIG. 18 shows plasma and HDL levels of species identified in FIG. 17.
FIG. 19 shows measurement of 14C-labeled lipid efflux for no human high density lipoprotein (HDL) (Control); 100 μg/ml of human HDL; pure human apoA-I; or human apoA-I vesicles.
DETAILED DESCRIPTION OF THE INVENTION
As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.
The term "age-related macular degeneration" as used herein refers to macular degeneration in an individual over the age of about 50. In one specific embodiment, it is associated with destruction and loss of the photoreceptors in the macula region of the retina resulting in decreased central vision and, in advanced cases, legal blindness.
The term "Bruch's membrane" as used herein refers to a five-layered structure separating the choriocapillaris from the RPE.
The term "HDL or subspecies thereof" refers to the fact that high density lipoproteins (HDL) can be fractionated into particulate species defined in molecular size and composition. HDL as prepared by density ultracentrifugation and by native nondenaturing purification processes including anti-apolipoprotein A-I immunoaffinity chromatography have been characterized for its constituent species by two-dimensional nondenaturing polyacrylamide electrophoresis, immunoblotting, and mass spectroscopy. HDL has been resolved into more than twenty-five particle species that differ in charge and molecular size. Each particle is defined by a unique combination of protein (including apolipoproteins A-I, A-II, A-IV, A-V, C-III, D, E, J, L, lecithin:cholesterol acyltransferase, cholesterol ester transferase, phospholipid transfer protein, alpha-2 macroglobulin) and lipid (including phospholipid, triglyceride, cholesterol, cholesterol ester, fatty acids). A partial list of HDL species include HDL alpha-1, HDL alpha-2, HDL alpha-3, HDL prebeta-1, HDL prebeta-2 (and variants "a", "b", "c", "d"), HDL prebeta-3, HDL prebeta-4, and HDL prealpha-1.
The term "increase lipid efflux" or "increasing lipid efflux" as used herein refers to an increased level and/or rate of lipid efflux, promoting lipid efflux, enhancing lipid efflux, facilitating lipid efflux, upregulating lipid efflux, improving lipid efflux, and/or augmenting lipid efflux. In a specific embodiment, the efflux comprises efflux of phospholipid, triglyceride, cholesterol, and/or cholesterol ester.
A skilled artisan recognizes that the term "lipid transporter" as used herein refers to a lipoprotein that carries lipids away from peripheral cells into the circulation, and examples include HDL and subspecies thereof, or a mixture thereof. The term "lipid transporter" is also used in the art to refer to, for example, transmembrane proteins that transport cholesterol and phospholipids, for example, from inside a cell to outside the cell. Examples include ABCA1, SR-BI, SR-BII, ABCA4, ABCG5, ABCG8, or a mixture thereof.
The term "macula" as used herein refers to the light-sensing cells of the central region of the retina.
The term "macular degeneration" as used herein refers to deterioration of the central portion of the retina, the macula.
The term "reverse cholesterol transport" as used herein refers to transport of cholesterol from peripheral tissues to the liver. In a specific embodiment, it refers to efflux of lipid from RPE cells. In specific embodiments, it comprises efflux of cellular cholesterol and/or phospholipid to HDL, and, in further specific embodiments, it comprises HDL delivery of cholesterol ester to the liver, such as for biliary secretion.
The term "therapeutically effective" as used herein refers to the amount of a compound required to improve some symptom associated with a disease. For example, in the treatment of macular degeneration, a compound which improves sight to any degree or arrests any symptom of impaired sight would be therapeutically effective. A therapeutically effective amount of a compound is not required to cure a disease but will provide a treatment for a disease.
The term "upregulate" as used herein is defined as increasing the level and/or rate of an event, process, or mechanism, such as reverse cholesterol transport and/or the transcription and/or translation processes of a nucleic acid, such as a gene.
II. The Present Invention
The present inventors have shown that HDL is a preferred cholesterol and phospholipids acceptor for lipids effluxed by cultured human RPE. In aging BM, there is progressive accumulation of lipid and cross-linked protein that impedes hydraulic conductivity and macromolecular permeability. This abnormal deposition may also impair the ability of some larger molecular weight species of HDL to act as a lipoprotein acceptor. As HDL is unable to pass through BM and promote efflux and bind effluxed lipids, more lipids accumulate in both RPE and BM. Indeed, such accumulations are a major finding in age-related macular degeneration.
Apolipoprotein A1 (ApoA-I), having a mass of approximately 28 kDaltons, is the major lipoprotein component of HDL. When bound to phospholipids, it comprises nascent HDL particles that bind to ABCA1 on the RPE basal membrane and promote lipid efflux. Because of its low molecular weight, it can penetrate an aged BM more easily than larger molecular weight species of HDL to bind to the RPE. In addition to it role in promoting reverse cholesterol transport from RPE, ApoA-I also is a potent anti-oxidant. Anti-oxidants have been established to reduce visual loss in patients with AMD.
Several methods are used to increase ApoA-I delivery to RPE as a treatment for AMD, including administration intravenously as has been done in mouse models of atherosclerosis and in patients with coronary artery disease. ApoA-I, which is normally comprised of L-amino acids, can be administered as an ApoA-I mimetic peptide (e.g. amino acid sequence SEQ ID NO:15 and/or SEQ ID NO:16) comprising D-amino acids. The D-amino acid based ApoA-I mimentic peptide is not recognized as readily by human proteases, and thus can be administered orally. In some embodiments, this would be more convenient than parenteral administration with an intravenous formulation containing the L-amino acid ApoA-I or its mimetic peptide. Furthermore, oral synthetic phospholipid (1,2 Dimyristoyl-α-glycero-3-phosphocholine, DMPC) increases levels of circulating ApoA-I.
By way of example, patients with AMD (atrophic or exudative) are administered intravenously either ApoA-I, ApoA-I mimetic peptide, an agent to increase levels of circulating ApoA-I, such as DMPC, or a mixture thereof.
The histopathology of macula in patients with AMD shows diffuse thickening of Bruch's membrane, and the overlying RPE is attenuated and full of lipofuscin granules. Photoreceptors are shortened and atrophic, and much of the thickened Bruch's membrane consists of lipid deposition. It is known that following about 50 years of age, the rate of lipid accumulation accelerates (Holz et al, 1994).
Using cell culture methods to study lipid metabolism, the inventors have shown a number of analogous mechanisms for lipid metabolism that are shared by macrophages and human RPE cells. The shared biology of these two cell types indicates useful therapeutic approaches for treatment of AMD. Specifically, the present inventors are the first to show that RCT occurs in RPE cells, and enhancement of RCT is beneficial for removing undesired lipid from the RPE cells and/or Bruch's membrane to facilitate retinal metabolism. In a specific embodiment, the transporters in the RCT system are regulated to improve RCT. In a further specific embodiment, this regulatory aspect of the present invention provides a novel treatment for AMD.
Although there has been discussion in the field regarding mechanisms of lipid accumulation in macula of AMD individuals, the present invention regards efflux of lipid into the circulation, which reduces the amount of lipid in RPE and/or Bruch's membrane. Promotion of this efflux comprises one aspect of the invention and is an effective therapy for both early and late AMD. A skilled artisan recgonizes that early AMD comprises the presence of drusen and late stage AMD comprises visual loss from choroidal neovascularization or geographic atrophy.
Thus, the present invention provides the novel idea in the field in which reverse cholesterol transport occurs in RPE cells. In specific embodiments, the invention provides methods and compositions related to facilitating efflux of cholesterol and/or phospholipids from inside an RPE cell to the outside of the RPE cell, and further through Bruch's membrane. In another specific embodiment, following efflux from Bruch's membrane the cholesterol and/or phospholipids are transported by apolipoprotein E, apolipoprotein A1, and other transporters, or a combination thereof, to HDL for removal to the liver.
A skilled artisan recognizes the important role reverse cholesterol transport (RCT) plays in lipid homeostasis. HDL levels are inversely correlated with incidence of coronary artery disease (CAD). Tangier's disease, which comprises a mutation of ABCA1, leads to deposition of cholesterol in reticuloendothelial tissues and premature atherosclerosis. Furthermore, the Apo E null mouse is an excellent model of atherosclerosis and hyperlipidemia. Interestingly, supporting an important role of Apo E in RCT, reconstitution of Apo E positive macrophages via bone marrow transplant into an Apo E null mouse prevents atherosclerosis. This occurs in spite of persistent hyperlipidemia.
In one embodiment of the present invention a transporter of lipid from RPE cells is enhanced for the transport activity, such as by an increase in the level of the transporter. Examples of transporters include apo E, ABCA1, SR-BI, SR-BII, ABCA4, ABCG5, ABCG8; other proteins that might be involved are LCAT, CETP, PLTP, LRP receptor, LDL receptor, Lox-1, and lipases. In a specific embodiment, lox-1 and PLTP are expressed in RPE, as demonstrated by RT_PCR. In a specific embodiment of the present invention, ApoA-I is utilized to facilitate RCT from RPE cells. In an additional specific embodiment, ApoA-I is made by RPE cells.
In a specific embodiment of the present invention, strategies for intervention for treatment of AMD are provided in which reverse cholesterol transport is enhanced at the level of the RPE by upregulating ApoA-I, ABCA1, Apo E, SR-BI and/or SR-BII expression. SR-B has been reported to be upregulated by 17beta-Estradiol and testosterone. Additionally, or alone, HDL binding to effluxed lipids is enhanced, thereby increasing efflux of lipids from Bruch's membrane into the circulation and providing therapy for AMD. Although the present invention generally regards an increase in ApoA-I, a major lipoprotein component of HDL, in one embodiment, an increase in HDL levels overall is utilized to facilitate lipid efflux from RPE cells and/or Bruch's membrane, and in a specific embodiment, levels of specific subspecies of HDL are utilized to facilitate lipid efflux. For example, effluxed lipids could bind to preβ-HDL, HDL1, HDL2 or HDL3. Effluxed lipids could also bind prebeta-1, prebeta-2, prebeta-3, and/or prebeta-4 HDL. In a specific embodiment, the effluxed lipids bind preferentially to HDL2 that comprises apo E.
One skilled in the art recognizes particular RCT components are present in RPE cells (Mullins et al., 2000; Anderson et al., 2001). Nuclear hormone receptors known to regulate expression of reverse cholesterol transport proteins are also expressed in cultured human RPE. Thus, in a preferred embodiment of the present invention, ligands to at least one of the nuclear hormone receptors upregulates RCT. In further embodiments, following efflux from RPE cells, the lipids bind HDL, so in an embodiment of the present invention there is upregulation of HDL for AMD treatment, such as by statins and/or niacin.
In an alternative embodiment, treatment for AMD comprises reduction of RCT. For example, in individuals past a certain age, such as about 50, 55, 60, 65, 70, 75, 80, and so on, the transporters are preferentially inhibited. In one aspect of this embodiment, HDL is unable to enter Bruch's membrane to remove the lipids and the RPE continues to efflux lipids. In such cases where effluxed lipids from RPE cannot be removed by a lipoprotein acceptor, lipid efflux by RPE is inhibited to maintain macromolecular transport across Bruch's membrane. Inhibition of RCT by reducing levels of ABCA-1, apo E, and/or SRB-1, or SRB-2 would reduce accumulation of lipid in Bruch's membrane.
In embodiments of the present invention, ligands for nuclear hormone receptors are utilized as compounds for enhancing RCT for the reduction of lipid content of RPE and Bruch's membrane. In a specific embodiment, the nuclear hormone receptor ligands are utilized for treatment of AMD. In a further specific embodiment, the nuclear hormone receptors comprise TR, RXR, and/or LXR. In other specific embodiments, ligands of the nuclear hormone receptors are delivered to at least one RPE cell to facilitate efflux of lipids from the RPE cell and/or are delivered to Bruch's membrane for efflux from Bruch's membrane. Examples of ligands for TR include T3 (3,5,3'-L-triiodothyronine). Other examples of TR ligands include but are not limited to TRIAC (3-triiodothyroacetic acid); KB141 (Karo Bio); GC-1; and 3,5 dimethyl-3-isopropylthyronine. Examples of ligands for RXR include 9 cis-retinoic acid, and other RXR ligands also include but are not limited to: AGN 191659 [(E)-5-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-1-y- l]-2-thiophenecarboxylic acid]; AGN 191701 [(E) 2-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-1-yl]-4-- thiophene-carboxylic acid]; AGN 192849 [(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl) (5 carboxypyrid-2-yl)sulfide]; LGD346; LG100268; LG100754; BMS649; and bexaroteneR (Ligand Pharmaceuticals) (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)ethenyl]be- nzoic acid). Examples of ligands for LXR include 22 (R) hydroxycholesterol, acetyl-podocarpic dimer, T0901317, and GW3965.
In an embodiment of the present invention, expression of a sequence is monitored following administration of an upregulator of its expression or a compound suspected to be an upregulator. A skilled artisan recognizes how to obtain these sequences, such as commercially from Celera Genomics, Inc. (Rockville, Md.) or from the National Center for Biotechnology Information's GenBank database. Exemplary apo E polynucleotide sequences include the following, cited with their GenBank Accession number: SEQ ID NO:1 (K00396); SEQ ID NO:2 (M10065); and SEQ ID NO:3 (M12529). Some exemplary apo E polypeptide sequences include the following, cited with their GenBank Accession number: SEQ ID NO:4 (AAB59546); SEQ ID NO:5 (AAB59397); and SEQ ID NO:6 (AAB59518).
In other embodiments, sequences of ABCA-1 are utilized, such as to monitor ABCA-1 expression related to methods of the present invention. Some examples of ABCA1 polynucleotides include SEQ ID NO:7 (NM--005502); and SEQ ID NO:8 (AB055982). Some examples of ABCA1 polypeptides include SEQ ID NO:9 (NP--005493); and SEQ ID NO:10 (BAB63210).
In some methods of the present invention, expression levels of sequences of SR-BI and SR-B2 polynucleotides are monitored following administration of a nuclear hormone receptor ligand. An example of SR-BI polynucleotide is SEQ ID NO:11 (NM--005505) and an example of a SR-BI polypeptide is SEQ ID NO:12 (NP--005496).
III. Pharmaceutical Compositions and Routes of Administration
Compositions of the present invention may have an effective amount of a compound for therapeutic administration and, in some embodiments, in combination with an effective amount of a second compound that is also an anti-AMD agent. In a specific embodiment, the compound is a ligand/agonist of a nuclear hormone receptor. In other embodiments, compounds that upregulate expression of HDL are the compounds for therapeutic administration. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-AMD agents, can also be incorporated into the compositions.
In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like.
The delivery vehicles of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target ocular tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions. In some embodiments, the compositions are administered by sustained release intra- or extra-ocular devices.
The vehicles and therapeutic compounds therein of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection also may be prepared. These preparations also may be emulsified. A typical composition for such purposes comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as theyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well-known parameters.
Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray.
An effective amount of the therapeutic agent is determined based on the intended goal. The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
All of the essential materials and reagents required for AMD treatment, diagnosis and/or prevention may be assembled together in a kit. When the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
For in vivo use, an anti-AMD agent may be formulated into a single or separate pharmaceutically acceptable syringeable composition. In this case, the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.
The components of the kit may also be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. The kits of the invention may also include an instruction sheet defining administration of the anti-AMD composition.
The kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
The active compounds of the present invention will often be formulated for parenteral administration, e.g. formulated for injection via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. The preparation of an aqueous composition that contains a second agent(s) as active ingredients will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
The active compounds may be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
In certain cases, the therapeutic formulations of the invention could also be prepared in forms suitable for topical administration, such as in eye drops, cremes and lotions.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intraocular, intravenous, intramuscular, and subcutaneous administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
Targeting of ocular tissues may be accomplished in any one of a variety of ways. In one embodiment, there is the use of liposomes to target a compound of the present invention to the eye, and preferably to RPE cells and/or Bruch's membrane. For example, the compound may be complexed with liposomes in the manner described above, and this compound/liposome complex injected into patients with AMD, using intravenous injection to direct the compound to the desired ocular tissue or cell. Directly injecting the liposome complex into the proximity of the RPE or Bruch's membrane can also provide for targeting of the complex with some forms of AMD. In a specific embodiment, the compound is administered via intra-ocular sustained delivery (such as Vitrasert® or Envision® by Bauch and). In a specific embodiment, the compound is delivered by posterior subtenons injection. In another specific embodiment, microemulsion particles with apo E (such as, recombinant) are delivered to ocular tissue to take up lipid from Bruch's membrane, RPE cells, or both.
Those of skill in the art will recognize that the best treatment regimens for using compounds of the present invention to treat AMD can be straightforwardly determined. This is not a question of experimentation, but rather one of optimization, which is routinely conducted in the medical arts. In vivo studies in nude mice often provide a starting point from which to begin to optimize the dosage and delivery regimes. The frequency of injection will initially be once a week, as has been done in some mice studies. However, this frequency might be optimally adjusted from one day to every two weeks to monthly, depending upon the results obtained from the initial clinical trials and the needs of a particular patient. Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 1 mg compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight. In other embodiments this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 mg/Kg body weight. In other embodiments, it is envisaged that higher does may be used, such doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.
In some embodiments of the present invention, the ApoA-I composition is comprised as a polynucleotide, utilizing delivery vehicles well known in the art. In other embodiments of the present invention, ApoA-I composition comprises a polypeptide or peptide. Any form may be distributed in a delivery composition, such as a liposome, examples of which are known in the art.
Any of the compositions described herein may be comprised in a kit. In a non-limiting example, an ApoA-I composition, and in some embodiments, at least one additional agent, may be comprised in a kit. In other embodiments, a lipid transporter such as HDL or a subspecies thereof.
The kits may comprise a suitably aliquoted ApoA-I composition, and/or additional agent compositions of the present invention, whether labeled or unlabeled, as may be used to prepare a standard curve for treatment of macular degeneration, such as AMD. The components of the kits may be packaged in aqueous media or in lyophilized form. When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
Suitable ApoA-I compositions comprise those that are sufficient to upregulate reverse cholesterol transport, those that reduce lipid accumulations in BM or RPE, those that increase efflux of lipids from BM, and/or are sufficient to provide an anti-oxidant therapeutic effect. In preferred embodiments, the ApoA-I compositions are suitable to provide therapy for macular degeneration, such as by ameliorating and/or preventing at least one symptom.
The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the nuclear hormone receptor ligand, additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
V. Biological Functional Equivalents
As modifications and/or changes may be made in the structure of ApoA-I polypeptides or peptides according to the present invention, while obtaining molecules having similar or improved characteristics, such biologically functional equivalents are also encompassed within the present invention.
A. Modified Polynucleotides and Polypeptides
Although administration of ApoA-I peptides or polypeptides is preferable, in some embodiments the ApoA-I composition is a polynucleotide encoding the desired polypeptide or peptide. The biological functional equivalent may comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the "wild-type" or standard protein or other polypeptide or peptide of interest. This can be accomplished to the degeneracy of the genetic code, i.e., the presence of multiple codons, which encode for the same amino acids. In one example, one of skill in the art may wish to introduce a restriction enzyme recognition sequence into a polynucleotide while not disturbing the ability of that polynucleotide to encode a protein.
In another example, a polynucleotide may encode a biological functional equivalent with more significant changes. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like. So-called "conservative" changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges of the protein's ability to carry out its designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.
In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a "biologically functional equivalent" protein and/or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) in selected amino acids (or codons) may be substituted. Functional activity, such as the ability to bind lipids, is preferably retained in any natural or synthetic ApoA-I polypeptide or peptide.
In general, the shorter the length of the molecule, the fewer changes that can be made within the molecule while retaining function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification.
Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and/or the like. An analysis of the size, shape and/or type of the amino acid side-chain substituents reveals that arginine, lysine and/or histidine are all positively charged residues; that alanine, glycine and/or serine are all a similar size; and/or that phenylalanine, tryptophan and/or tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and/or histidine; alanine, glycine and/or serine; and/or phenylalanine, tryptophan and/or tyrosine; are defined herein as biologically functional equivalents.
To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and/or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and/or arginine (-4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index and/or score and/or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and/or those within ±0.5 are even more particularly preferred.
It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent protein and/or peptide thereby created is intended for use in immunological embodiments, as in certain embodiments of the present invention. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and/or antigenicity, i.e., with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and/or those within ±0.5 are even more particularly preferred.
B. Altered Amino Acids
The present invention, in some aspects, may rely on the synthesis of peptides and polypeptides in cyto, via transcription and translation of appropriate polynucleotides. In alternative embodiments, the polypeptide or peptide is synthesized outside a cell, such as chemically. These peptides and polypeptides may include the twenty "natural" amino acids, and, in some embodiments, post-translational modifications thereof. However, in vitro peptide synthesis permits the use of modified and/or unusual amino acids. A table of exemplary, but not limiting, modified and/or unusual amino acids is provided herein below.
TABLE-US-00001 TABLE 1 Modified and/or Unusual Amino Acids Abbr. Amino Acid Abbr. Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad 3-Aminoadipic acid Hyl Hydroxylysine BAla beta-alanine, beta-Amino- AHyl allo-Hydroxylysine propionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid Aile allo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine, sarcosine BAib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acid MeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2'-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGly N-Ethylglycine
In addition to the biological functional equivalents discussed above, the present inventors also contemplate that structurally similar compounds may be formulated to mimic the key portions of peptide or polypeptides of the present invention. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and, hence, also are functional equivalents. In a specific embodiment, the key portion comprises lipid binding activity.
Certain mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and/or antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
Some successful applications of the peptide mimetic concept have focused on mimetics of β-turns within proteins, which are known to be highly antigenic. Likely β-turn structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
Other approaches have focused on the use of small, multidisulfide-containing proteins as attractive structural templates for producing biologically active conformations that mimic the binding sites of large proteins. Vita et al. (1998). A structural motif that appears to be evolutionarily conserved in certain toxins is small (30-40 amino acids), stable, and high permissive for mutation. This motif is composed of a beta sheet and an alpha helix bridged in the interior core by three disulfides.
Beta II turns have been mimicked successfully using cyclic L-pentapeptides and those with D-amino acids. Weisshoff et al. (1999). Also, Johannesson et al. (1999) report on bicyclic tripeptides with reverse turn inducing properties.
Methods for generating specific structures have been disclosed in the art. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos. 5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures render the peptide or protein more thermally stable, also increase resistance to proteolytic degradation. Six, seven, eleven, twelve, thirteen and fourteen membered ring structures are disclosed.
Methods for generating conformationally restricted beta turns and beta bulges are described, for example, in U.S. Pat. Nos. 5,440,013; 5,618,914; and 5,670,155. Beta-turns permit changed side substituents without having changes in corresponding backbone conformation, and have appropriate termini for incorporation into peptides by standard synthesis procedures. Other types of mimetic turns include reverse and gamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S. Pat. Nos. 5,672,681 and 5,674,976.
The following is an illustration of preferred embodiments for practicing the present invention. However, they are not limiting examples. Other examples and methods are possible in practicing the present invention.
Materials and Methods
Cell Culture and Drug Treatments
Primary cultures of normal human RPE cells from passages 5 to 10 were used for the experiments described. RPE cells were grown to confluence on laminin-coated 6 well Transwell tissue culture plates (Costar) with DMEM-H21 containing 10% fetal bovine serum, 2 mM glutamine, 50 μg/ml gentamicin and 2.5 mg/ml fungizone in the top and bottom chambers. For immunofluorescent staining cells were grown on laminin coated slides in the same medium. Cells were grown for at least 1 week at confluence prior to drug treatment. Cells to be treated with drugs were incubated in serum free DMEM-H21 prior to drug addition. Drug treatments were in serum free DMEM-H21 with or without 10-7 M thyroid hormone (T3), 2.5×10-6 M 22 (R) hydroxycholesterol, or 10-7 M cis retinoic acid in both chambers for 36 hours.
Confluent cell cultures were harvested and total RNA was purified using RNAzol (Teltest, Inc., Friendswood, Tex.) according to the manufacturer's instructions. Equal amounts of purified RNA were used in each reaction as templates for cDNA synthesis using the 1st Strand Synthesis Kit for RT-PCR (AMV) (Boehringer, Indianapolis, Ind.). RT-PCR was carried out on 1 μg of cDNA with Amplitaq Taq polymerase (Perkin-Elmer, Branchburg, N.J.). In some experiments apo E RT-PCR products were quantified using the QuantumRNA assay kit according to the manufacturer's instructions (Ambion, Austin, Tex.). Briefly, 18S rRNA and apo E cDNAs are simultaneously amplified in each reaction. The RT-PCR products are resolved by electrophoresis on 1.4% agarose gels. The apo E mRNA expression is assessed relative to the internal 18S rRNA expression by densitometric analysis of photographed agarose gels.
RT-PCR primers specific to human apo E, ABCA1, SR-BI, SR-BII, and lxr α were used. The RT-PCR product of the predicted sizes for the apo E, ABCA1, SR-BI, and SR-BII RT-PCR products were excised form the gel and their identities were confirmed by DNA sequencing (not shown).
RPE cells, grown on slides, were στανεδ with either antisera to ABCA1, or with purified antibodies to SR-BI or SR-BII. Cells were fixed in ice cold 100% MeOH for 20 min. All subsequent steps were performed at room temperature. Cells were washed in phosphate buffered saline (PBS) and incubated for in 5% goat serum in PBS for 30 min. Cells were then washed in buffer A (150 mM NaCl, 10 mM phosphate, pH 7.8) and incubated with the primary antibody in buffer A for 45 min. After washing with buffer A the cells were incubated in Avidin Blocking Reagent (Vector Laboratories, Burlingame, Calif.) for 15 min, washed in buffer A again and incubated in Biotin Blocking Reagent (Vector Laboratories, Burlingame, Calif.) for 15 min. After washing in buffer A, cells were incubated in 10 μg/ml biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, Calif.) in buffer A for 30 min, washed in buffer A and incubated in 20 μg/ml fluorescein conjugated avidin D (Vector Laboratories, Burlingame, Calif.) in buffer B (150 mM NaCl, 100 mM sodium bicarbonate, pH 8.5) for 30 min. The cells were washed in buffer B and a cover slip was added to each slide, over a few drops of Vectashield (Vector Laboratories, Burlingame, Calif.). The slides were stored in the dark until ready for microscopic examination.
Apo E Western Blotting
Cells were treated with Media was concentrated 20-fold by centrifugal ultrafiltration (VIVA SPIN 20, MCO 5,000, Viva Sciences, Hannover, Germany), dialyzed against 0.15M NaCl, 1 mM sodium EDTA, 0.025% sodium azide (SalEN). Total protein content was determined by a modified Lowry assay (Bio-Rad DC kit, Richmond, Calif.). Concentrated media (50 μg protein) was made to Start Buffer (0.025 M NaCl, 0.010 M tris (pH 8.5), 5 mM MnCl2) and adsorbed onto a 0.1 ml column containing Heparin-Sepharose CL-4B (Pharmacia, Uppsala, Sweden). Following a 2 ml wash in Start Buffer, the apo E containing bound fraction was eluted with 0.5M NaCl in Start buffer. The eluate was concentrated to 20 μl and buffer-exchanged to SalEN by centrifugal ultrafiltration (Biomax, 5k MCO, Millipore, Bedford, Mass.). Apo E was resolved by tris-tricine buffered SDS-PAGE (5-25% linear acrylamide gradient) and proteins electrophoetically transferred (55V, 18 h) to nitrocelluose membrane filters (Schleicher and Shuell, Keen, N.H.). Membranes were blocked with 10% bovine serum albumin at room temperature and probed with 1% goat anti-human apo E antiserum (18 h, 3° C.) prepared in 0.15% NaCl, 1 mM EDTA (pH 7.4), 0.1% Triton X-100 (SalET). The primary-bound anti-apo E antibodies were detected calorimetrically with horseradish peroxidase conjugated rabbit anti-goat Ig (H+L) and NiCl2-enhanced diaminobenzine staining. Stained bands were compared densitometrically from the digitized scanned image (NIH Image, v. 1.62). Anti apo E antibodies were obtained by hyper-immunization of goats with purified apo E or obtained from Assay Designs (A299, Ann Arbor, Mich.).
Lipoprotein fractions were prepared from conditioned media that was adjusted with solid KBr to a density of 1.21 g/ml. Samples were ultracentrifuged in a Beckman 42.2 Ti rotor at 40,000 rpm for 18 h at 1° C. The lipoprotein and lipoprotein-free fractions, the top and bottom 50 μl, respectively, were dialysed against SalEN prior to analysis.
[14C] Docosohexanoic Acid (DHA) Labeled POS Uptake and Transport
Bovine outer photoreceptor outer segments (POS) were labeled by incubating for with Coenzyme A, ATP, Mg2+, and [14C]-DHA. Cells grown on laminin coated Transwell plates were incubated with 12 μg/ml labeled POS in the apical chamber for 36 hours in medium containing 10% lipoprotein deficient fetal bovine serum. The basal medium was subjected to scintillation counting to determine the amount of [14C] labeled lipids transported through the RPE cells.
Identification of Acceptors for Exported 14C Lipids
Bovine outer photoreceptor outer segments (POS) were labeled by incubating for with Coenzyme A, ATP, Mg2+, and [14C]-DHA. Cells grown on laminin coated Transwell plates were incubated with 12 μg/ml labeled POS in the apical chamber for 36 hours in medium containing 10% lipoprotein deficient fetal bovine serum. The basal chambers contained either 1 mg/ml human HDL, 1 mg/ml human LDL or 100% human plasma. The basal medium was collected and lipoproteins were repurified from by potassium bromide density gradient centrifugation at d=1.21 g/ml (Beckman 42.2 Ti rotor, 40,000 rpm, 18 h, 10° C.), dialyzed, and resolved by size in nondenaturing 0-35% PAGE. Gels were stained with coomassie blue R-250. Gel lanes were sectioned into thirty 2 mm slices that were digested (TS-1, Research Products International) and radioactivity quantified by liquid scintillation spectrometry.
Expression of Transporters in RPE Cells
One skilled in the art recognizes that certain RCT components in cultured human RPE cells have been demonstrated (Mullins et al., 2000; Anderson et al., 2001). Nuclear hormone receptors known to regulate expression of reverse cholesterol transport proteins are also expressed in cultured human RPE.
A skilled artisan recognizes that there is expression of TRs and RXRs in RPE cells in culture (Duncan et al., 1999). RT-PCR of human RPE cell cDNA revealed that these cells also express mRNAs for apo E, ABCA1, SR-BI, SR-BII and lxr α. As shown in FIG. 1 lane 1, FIG. 1 lane 2 and FIG. 1 lane 3, RPE cells express mRNAs for apo E, ABCA1 and lxr α, respectively.
As shown in FIG. 2, lane 1, and FIG. 2, lane 2, RPE cells express mRNA for SR-BI and SR-BII respectively.
Furthermore, in immunofluoresence microscopy experiments, RPE cells stain strongly for SR-BI (FIG. 3A) and SR-BII (FIG. 3B). Control non-specific IgG or antibody vehicle did not stain RPE cells (FIGS. 3C and 3D, respectively). Expression of SR-BI and SR-BII in these cells was confirmed by PCR.
Expression of ABCA1 protein was demonstrated by immunofluorescent staining of RPE cells with an antibody to ABCA1 (FIG. 4). Cell nuclei were stained with DAPI.
Regulation of Apo E Secretion in RPE Cells
In order to distinguish apical (A) from basally (B) secreted apo E, RPE cells were cultured on laminin-coated Transwell plates. Specifically, human cultured RPE (passage 2-10, 35 y.o. donor) were placed on laminin-coated Transwell plates, wherein the upper and lower wells both had serum-free media. Total protein and apo E-specific protein concentrations were measured from media pooled and concentrated from 3-6 replicate wells. To assess apo E-specific secretion, apo E was purified from conditioned media by heparin-sepharose affinity chromatography and visualized by western blotting. Apo E concentrations were consistently greater in the basolateral media (FIG. 5, lane 1 vs. lane 2). These data demonstrate that RPE cells display polarized secretion of cellular proteins, including apo E. Thus, this indicated that Apo E is preferentially secreted basally, supporting its role in RCT.
Since RPE cells express lxr α as well as thyroid hormone receptors (TRs) and retinoid-X-receptors (RXRs), the effect of 10-7 M T3, 2.5×10-6 M 22 (R) hydroxycholesterol (HC) (an lxr ααagonist), or 10-7 M cis retinoic acid (cRA) (an RXR agonist) on apo E secretion from RPE cells was tested. FIG. 6 illustrates the same experimental procedure as described above, but with basal and apical media both containing the following compounds for a 36 hour incubation: T3 (10-7) M (T); 9 cis-RA (10-6) M (RA); and 22 (R) hydroxycholesterol 2.5 (10-6) M (HC). The basal media was analyzed for Apo E expression with Western blot, and the results showed increased basal expression of Apo E with the compound treatments. Thus, as before, polarized apo E secretion was observed and, in this case, occurred in the presence of T3, HC or cRA, indicating that an increase in levels of basally secreted apo E is the result of administration of these compounds to RPE cells.
Assay of Efflux from RPE Cells
This example characterizes efflux of POS residues from RPE cells, particularly regarding binding to HDL. Giusto et al. (1986) describes a method of 14C decoshexanoic acid (DHA) labeling of bovine photoreceptor outer segment (POS) lipids. Generally, an approximately 36 hour incubation over human RPE cells wherein the basal medium contains plasma, HDL, or LDL is followed by centrifugation of the basal media to collect lipoprotein fraction, which is then analyzed to determine distribution of radioactivity.
Specifically, bovine photoreceptor outer segment (POS) are labeled with 14C decoshexanoic acid (DHA) and placed in lipoprotein deficient media. Following this, they are placed over cultured human RPE on Transwell plates for 36 hours, and the basal medium contained either 100% plasma, HDL (1 mg/cc) or LDL (1 mg/cc). After 36 hours, basal media was centrifuged to collect lipoprotein fraction (density 1.2). This fraction was then run on a non-denaturing gel and stained with Coomassie blue. FIG. 7 shows LDL and HDL fractions, both separately and together in plasma (PL). The PL fraction contains the same amount of HDL and LDL as each of the separated fractions (HDL, LDL).
The PA gel was cut into about 1 mm pieces, and the radioactivity distribution was determined (FIG. 8). With either LDL or HDL alone, counts were observed over respective lipoprotein fractions. When both LDL and HDL in plasma are present, counts localize preferentially over HDL fraction. This indicates that following phagocytosis of POS by RPE, POS residues are effluxed and preferentially bound by HDL. This is a novel demonstration illustrating that RCT to an HDL acceptor occurs in RPE cells.
To characterize the lipids in the lipoprotein fraction, thin layer chromatography was performed. Acid charring was used to identify lipid containing spots. The spots were scraped off of the plate and 14C was quantified by liquid scintillation counting. Six of 17 14C-containing spots were identified with standards shown (FIG. 9). Eleven 14C-containing spots bound to HDL remain unidentified and could be unique serum marker(s) for patients with early AMD.
Thus, in an embodiment of the present invention, a patient sample is obtained, such as by drawing blood, and the HDL is examined for bound POS residues. From this, a determination of their risk of visual loss from AMD is made. In a specific embodiment, the profile of bound POS residues is indicative of identifying an individual afflicted with ocular disease and/or of identifying an individual at risk for developing an ocular disease.
Modulation of RCT by Compound Administration
This experiment determines whether compound administration can upregulate efflux of labeled POS residues to HDL, particularly by showing regulation of 14C-DHA labeled POS efflux into basal media. An assay similar to that described in Example 4 is utilized; however, in this Example the cells were treated with T3, 9 cis-retinoic acid, and 22 (R) hydroxycholesterol in the concentrations described above for 36 hours. Total radioactivity (cpm) in the absence of HDL purification was determined by liquid scintillation counting of the basal media. FIG. 10 indicates that compound treatments increase RCT by cultured human RPE cells.
Specifically, cells were grown for 1 to 2 weeks at confluence on Transwell plates. 14C-labeled POS (30 mg/ml) were added to the apical medium. The apical and basal medium comprised either 10-7 M T3, 2.5×10-5 M 22 (R) hydroxycholesterol, or 10-7 M cis retinoic acid. The basal medium contained 1 mg/ml HDL. After 36 hours the basal medium was collected and 14C counts were determined by scintillation counting. As stated, all of the compound treatments increased transport of 14C-labeled POS to the basal medium.
The effect of T3 on Apo E mRNA levels was also assessed by RT-PCR. Treatment with 10-7 M T3 resulted in a 1.5 to 2-fold increase in apo E mRNA levels, suggesting that T3 is acting, at least in part, to increase apo E levels at the mRNA level. In specific embodiments, administration of 9 cis-retinoic acid and 22 (R) hydroxycholesterol similarly upregulates expression of apo E.
Thus, in a specific embodiment, RCT is regulated via nuclear hormone receptor ligands. For example, ABCA1 expression is upregulated by binding of LXR and RXR agonists to their respective nuclear hormone receptors (FIG. 11). Since these receptors form heterodimers bound to the ABCA1 promoter, ligand binding increases expression of ABCA1 and, hence, RCT.
Identification of HDL as Lipid Acceptor from RPE Cells
In the presence of added purified human LDL and HDL, radiolabeled lipid efflux is enhanced (FIG. 12). As shown graphically, efflux (bottoms in graph) was greatly enhanced by the presence of plasma (PL in graph), HDL or LDL, as compared to no addition to the bottom medium (left side of graph).
As shown in FIG. 8, when whole human EDTA-plasma is employed and lipoproteins are isolated, [14C]-labeled lipids are incorporated into LDL and HDL. However, radiolabel preferentially associated with HDL. Furthermore, the radiolabel in HDL was localized to the larger HDL 2 subspecies, which include the HDL particles enriched in apo E. This result suggests that lipid efflux from RPE is enhanced by the apo E--containing HDL.
Reduction of BM Lipids Via Scavenger Receptors (SRS)
Scavenger receptors in macrophages function to phagocytose oxLDL molecules. There are types of SRs previously described in macrophages including SR-A1, SR-A2, SR-B1, SR-B2, CD36, and LOX. SRs are distinct from LDL receptors in that levels of expression for SRs are upregulated by oxLDL. This upregulation by intracellular oxLDL levels is modulated by nuclear hormone receptors, peroxisome proliferator activated receptor (PPAR) and retinoic acid X receptor (RXR), that exert transcriptional control of CD36 expression. Because the earliest lesion of AS, the fatty streak, consists of macrophages engulfed with excessive oxLDL, and because RPE cells similarly become filled with lipid inclusions in AMD, SR expression was studied in RPE cells. Expression of the following SRs in RPE cells was identified: CD36 (confirmation of previous investigators), SR-A1, SR-A2 (both first time demonstrated in RPE), SR-B1, SR-B2 (both first time demonstrated in RPE).
The inventors have also shown that, like macrophages, oxLDL upregulates expression of CD36 in RPE cells (FIG. 13). Additionally, RPE cells express the nuclear hormone receptors, PPAR and RXR, indicating control mechanisms for SR expression are analogous between the cell types. Thus, in specific embodiments the expression of RPE SRs in patients is controlled with PPAR and RXR ligands (e.g. PG-J2, thiazolidinediones, cis-retinoic acid). This controls the rate at which RPE cells phagocytose oxidized photoreceptor outer segments, and hence slows the rate at which abnormal lipid inclusions accumulate in RPE and BM. In other specific embodiments, expression of CD36 is downregulated with a composition such as tamoxifen, TGF-beta or INF-gamma. Similarly, regulating expression of other RPE SRs would control levels of lipids in both RPE and BM. For example, for SR-A regulation IGF-1, TGF-beta, EGF, and/or PDGF is used, and for SR-B regulation cAMP and/or estradiol (for upregulation) or TNF-alpha, LPS, and/or INF-gamma (for downregulation) is used.
HDL Increases 14C Lipid Efflux from RPE Cells Preferentially to Other Lipoproteins
Transcription of the apo E gene is regulated by liver-X-receptor alpha (LXR α) that acts as heterodimers with retinoid-X-receptor alpha (RXR α) (Mak et al., 2002). The inventors have previously shown that RPE cells express T3 receptors (TRs) that also act as heterodimers with RXR α (Duncan et al., 1999). The inventors and others, have demonstrated that primary cultures of RPE cells express mRNA for lxr α, RXR α, apo E, and other proteins involved in regulation of lipid and cholesterol uptake, metabolism and efflux (summarized herein). In this Example, the inventors show that apo E secreted by primary cultures of RPE cells can be up-regulated by thyroid hormone (T3), 22(R) hydroxycholesterol (HC), and cis retinoic acid (RA). The inventors also demonstrated that a high density lipoprotein (HDL) fraction rich in apo E is a preferential acceptor for labeled POS lipids.
As shown in Table II, the present inventors and other investigators have identified mRNAs for the proteins involved in regulating lipid and cholesterol uptake, metabolism and efflux. The cells used in the experiments described below express only the apo E3 allele (E3/E3).
TABLE-US-00002 TABLE II Agents involved in regulating lipid and cholesterol uptake, metabolism, and efflux TRANSCRIPTION FACTORS LIGANDS TR α1 Thyroid hormone Receptor alpha1 T3 TR α2 Thyroid hormone Receptor alpha 2 T3 TR β 1 Thyroid hormone Receptor beta1 T3 RXR α Retinoid-X Receptor alpha Retinoic Acid RXR β Retinoid-X Receptor beta Retinoic Acid PPAR γ Peroxisome Proliferator Oxidized lipids Activator Receptor gamma Lxr α Liver-X Receptor alpha Oxysterols CELL SURFACE RECEPTORS LIGANDS SR-BI Scavenger Receptor BI Oxidized Lipids SR-BII Scavenger Receptor BII Oxidized Lipids SR-AI Scavenger Receptor AI Oxidized Lipoproteins SR-AII Scavenger Receptor AII Oxidized Lipoproteins Lox-1 Lectin-like Oxidized LDL receptor 1 Oxidized Lipoproteins CHOLESTEROL/LIPID TRANSPORT AND METABOLISM FUNCTIONS SR-BI Scavenger Receptor BI Reverse Cholesterol Transport SR-BII Scavenger Receptor BII Reverse Cholesterol Transport ABCA1 ATP Binding Cassette Protein A1 Reverse Cholesterol Transport ACAT1 Acyl-CoA Cholesterol Cholesterol Acylation Acyltransferase 1 Apo E Apolipoprotein E Cholesterol/Lipid Trafficking
As shown qualitatively in FIG. 6, T3 (TR agonist), RA (RXR agonist), HC (LXR agonist) stimulate basal apo E secretion. As previously indicated, RPE cells were treated for 36 hours on Transwell® plates with serum free DMEM in upper and lower chambers+/-the drugs indicated. Control (C) refers to no drug addition; T refers to 10-7 M T3; RA refers to 10-7 M cis retinoic acid; and HC refers to 2.5×10-6 M 22(R) hydroxycholesterol. Basal media from 3 wells were combined, concentrated, and apo E was detected by western blotting.
As shown quantitatively in FIG. 14, TR, LXR and RXR agonists upregulate apo E secretion alone and in combination, as assessed by ELISA assays. RPE cells were treated for 36 hours on Transwell® plates with serum free DMEM+/-the drugs indicated. Control refers to no drug addition, T refers to 10-7 M T3; HC refers to 2.5×10-6 M 22(R) hydroxycholesterol; RA refers to 10-7 M cis retinoic acid. N=6, * indicates p≦0.05 (two-tailed t-test) compared to Control.
As shown in FIG. 15, apo E secreted from RPE cells binds to HDL. RPE cells on Transwell® plates were grown in DMEM with 5% FBS for 36 hours (apical chamber). Basal chambers had serum free DMEM with either 200, 50, or 0 μg/ml mouse HDL (lanes 2, 3, and 4 respectively. Lane 1 illustrates molecular weight markers. HDL was purified by ultracentrifugation, resolved by polyacrylamide gel electrophoresis, and human apo E was identified by western blotting.
As shown in FIG. 16, HDL stimulates POS lipid efflux from RPE cells in culture. RPE cells on Transwell® plates were fed 14C labeled POS in DMEM with 5% FBS for 36 hours (apical chamber). Basal chambers had serum free DMEM. Both upper and lower media contained either no addition (Control), 10% human plasma, 100 μg/ml HDL, 1000 μg/ml LDL or 50 μg/ml HDL+500 μg/ml LDL as indicated. FIG. 16 left: Basal 14C cpm/130 μl. N=3, * indicates p≦0.05 (two-tailed t-test) compared to Control. FIG. 16 right: Lipoproteins were purified by ultracentrifugation, dialyzed to remove soluble 14C, and counted.
As shown in FIG. 8, 14C labeled POS lipids preferentially bind to apo E containing high molecular weight HDL (HDL3). 14C labeled lipoproteins from the lower chamber were purified by ultracentrifugation and resolved on native polyacrylamide gels.
Characterization of HDL and plasma bound POS lipids was made by thin layer chromatography, as shown in FIG. 17. 14C labeled lipoproteins from the lower chamber were purified by ultracentrifugation, and lipids were resolved by thin layer chromatography followed by acid charring.
As shown in FIG. 18, six spots in HDL and plasma were tentatively identified; at least 11 other spots are not yet identified. Spots identified by charring were cut out and 14C cpm determined by liquid scintillation counting.
Exemplary Methods and Materials for Example 8
Primary cultures of normal human RPE cells were prepared from a 35 year old donor eye as described (Song and Lui, 1990). Cells from passages 4 to 10 were used. RPE cells were grown on laminin-coated tissue culture plates, or on laminin coated 0.4 μM cellulose acetate Transwell® dishes (Costar) in DMEM H21 containing 5-10% fetal bovine serum (FBS), 2 mM glutamine, 5 μg/ml gentamycin, 100 IU/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml fungizone, 1 ng/ml bFGF, and 1 ng/ml EGF. Where indicated, FBS was substituted with: 6 g/L NEAA, 0.39 g/L methylcellulose (serum free medium). No differences in cell morphology or protein expression were observed in cultures from different passages. RPE cells were grown at confluence for at least 14 days prior to undergoing the experimental treatments described below.
RT-PCR was carried out on 1 μg of cDNA. The RT-PCR products are resolved by electrophoresis on 1.4% agarose gels. The RT-PCR primer sequences used are given followed by the predicted apo E RT-PCR product size. apo E forward: 5'-TAA GCT TGG CAC GGC TGT CCA AGG A (SEQ ID NO:13); apo E reverse: 5'-ACA GAA TTC GCC CCG GCC TGG TAC AC (SEQ ID NO:14); 241 bp product (detects both apo E3 and apo E4). PCR was conducted for 20-30 cycles at 55° C. in buffer containing 2.0-5.0 mM MgCl2. The RT-PCR product of the predicted size for apo E had its identity confirmed by DNA sequencing. Only the apo E3 mRNA sequence was detected. Messenger RNAs for the other proteins listed in the Table II were identified using similar strategies with primers specific to each cDNA.
Briefly, media was concentrated 20-fold by centrifugal ultrafiltration (VIVA SPIN 20, MCO 5,000; Viva Sciences). Concentrated media (20 μg protein) was purified over Heparin-Sepharose CL-4B. The apo E containing (bound) fraction was eluted and re-concentrated to 20 μl. Apo E was resolved by 5-25% linear gradient SDS polyacrylamide gel electrophoresis, and proteins were electrophoretically transferred to nitrocelluose. Membranes were blocked with 10% BSA and probed with 1% goat anti-human apo E antiserum. Apo E antibodies were detected colorimetrically with horseradish peroxidase conjugated rabbit anti-goat IgG and NiCl2-enhanced diaminobenzine staining.
Media samples treated with 0.1% Tween-20 containing 1% bovine serum albumin were incubated (37° C., 4 h) in 96-well plates previously coated with apo E-affinity purified goat anti-apo E antibody. Apo E was detected using a secondary antibody-peroxidase conjugate and 188.8.131.52'-tetramethylethylenediamine (TMB) substrate. Optical density was measured at 450 nm. The assay was calibrated with purified plasma apo E. The dynamic range of the assay was 1-40 ng/ml apo E with a CV<5%.
POS Lipid Transport and Lipoprotein Gel Analysis
Briefly, purified POS lipids were labeled with 14C docosohexanoic acid as described (Guisto et al., 1986). Twenty μg/ml (protein) of POS were added to the top chambers of 6 well Transwell® plates. The bottom chambers contained serum free medium with or without human high density lipoprotein (HDL), human low density lipoprotein (LDL), or human plasma in the amounts indicated. After 36 hours, cell culture medium was harvested from the bottom chambers, adjusted to a density of 1.25 g/ml with solid potassium bromide and underlayed over a KBr solution of d=1.21. Samples were ultracentrifuged (Beckman 50.2Ti, 45,000 rpm, 10° C.) for 20 hours. The top (lipoprotein) layer was removed, dialyzed, and subjected to non-denaturing gel electrophoresis. The gels were stained with Coomassie Blue and photographed, after which 2 mM slices were subjected to scintillation counting.
Thin Layer Chromatography
Lipoprotein samples were extracted for lipid by the method of Bligh-Dyer, which is well known in the art. Lipids were resolved by silica gel K6 thin layer chromtography using sequential developments in Solvent 1: chloroform/methanol/acetic acid/water (25:15:4:2) and Solvent 2: n-hexane/diethylether/acetic acid (65:35:2). Lipid species were detected by acid charring, plates were immersed in 7.5% copper acetate, 2.5% copper sulfate, 8% phosphoric acid and heated on a hot plate for 1 hour. 14C radioactivity was measured by liquid scintillation counting in standard methods known in the art.
APOA-I Delivery to Increase Reverse Cholesterol Transport
The present inventors have shown that HDL is a preferred cholesterol and phospholipids acceptor for lipids effluxed by cultured human RPE. In aging BM, there is progressive accumulation of lipid and cross-linked protein that impedes hydraulic conductivity and macromolecular permeability. This abnormal deposition may also impair the ability of some larger molecular weight species of HDL to act as a lipoprotein acceptor. As HDL is unable to pass through BM and promote efflux and bind effluxed lipids, more lipids would accumulate in both RPE and BM. Indeed such accumulations are a major finding in age-related macular degeneration (AMD).
Apolipoprotein A1 (ApoA-I) is the major lipoprotein component of HDL. It has a mass of approximately 28 kDaltons. ApoA-I bound to phospholipids comprises nascent HDL particles that bind to ABCA1 on the RPE basal membrane and promote lipid efflux. Because of ApoA-I's low molecular weight, it can penetrate an aged BM more easily than larger molecular weight species of HDL to bind to the RPE. In addition to it role in promoting reverse cholesterol transport from RPE, ApoA-I also in a potent anti-oxidant. Anti-oxidants have been established to reduce visual loss in patients with AMD.
Several methods are used to increase ApoA-I delivery to RPE as a treatment for AMD:
1. ApoA-I is administered intravenously as has been done in mouse models of atherosclerosis and in patients with coronary artery disease.
2. ApoA-I, which is normally comprised on L-amino acids, can be administered as an ApoA-I mimetic peptide consisting of D-amino acids. The D-amino acid based ApoA-I mimetic peptide is not recognized as readily by human proteases, and thus can be administered orally. This would be more convenient than parenteral administration with an intravenous formulation containing the L-amino acid ApoA-I or its mimetic peptide.
3. Oral synthetic phospholipid (1,2 Dimyristoyl-α-glycero-3-phosphocholine, DMPC) increases levels of circulating ApoA-I.
By way of example, patients with AMD (atrophic or exudative) are administered either intravenous ApoA-I, ApoA-I mimetic peptide, or DMPC to increase levels of circulating ApoA-I. Administration could occur as frequent as daily or less frequently as in every other month depending on the method of administration and the clinical response.
Apo A-I mimetic peptides may be synthesized according to standard methods in the art, and in some embodiments one or more amino acids in the peptide are the D-stereoisomer. Methods to synthesize mimetic peptides are known in the art, including those described in de Beer, M. C., et al. (2001) and Matz and Jonas, 1982.
The peptides are based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH2 (Ac-18A-NH2 or 2F) (SEQ ID NO:15) (Navab et al., 2003), where Ac symbolizes acetylated. Thus, in specific embodiments, the C-terminus is carboxylated. The 2F peptide or an analog of 2F with the primary amino acid sequence Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (4F) (SEQ ID NO:16) is used (Navab et al., 2003). An example of a human apolipoprotein A1 includes: LSPLGEEMRD RARAHVDALR THLAPYSDEL RQPLAARLEA LKENGGARLA EYHAKATEHL STLSEKA (SEQ ID NO:17). Other apolipoprotein A1 sequences, including those from organisms other than human, are available to the skilled artisan at the National Center for Biotechnology Information's GenBank database on the World Wide Web.
In some embodiments of the present invention, a mouse model is utilized to characterize administration of ApoA-I compositions. For example, the model generated by Dithmar et al. (2000) or an analogous model generated by similar methods in the art may be used in optimizing the present invention. In this model, ApoE.sup.- mice demonstrate ultrastructural changes in Bruch's membrane, such as accumulation of material similar to basal linear deposit and an increase in membrane-bound material.
APOA-I and RPE Cells
The present inventors have created liposomes comprising apoA-I (artificial preβ1 HDL). Artificial discoidal apoA-I liposomes comprising purified human plasma apoAI, the saturated phospholipid dimyristoyl-L-α-phoshatidylcholine (DMPC) and cholesterol were constructed by the sodium cholate dialysis method. Liposomes were prepared using a molar ratio of approximately Jan. 5, 1995, apoA-I/free cholesterol/DMPC. Human retinal pigment epithelial cells grown on 6 well laminin coated Transwell® plates were fed 14C-docosahexaenoic acid labeled photoreceptor outer segments in medium containing 5% lipoprotein free fetal bovine serum for 36 hours (apical chamber). Basal chambers contained serum free medium and either no human high density lipoprotein (HDL) (Control), 100 μg/ml of human HDL, pure human apoA-I, or human apoA-I vesicles. An aliquot of the basal medium was subjected to liquid scintillation counting. The results are shown in FIG. 19. Wells were treated in triplicate. HDL stimulated 14C-labeled lipid efflux by about 60%. ApoA-1 appeared to stimulate 14C-labeled lipid efflux. The apoA-I vesicles (apoA-I V) did not stimulate 14C-labeled lipid efflux.
All patents and publications mentioned in the specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.
1711156DNAHUMAN 1cgcagcggag gtgaaggacg tccttcccca ggagccgact ggccaatcac aggcaggaag 60atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 120gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 180ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 240ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 300ctgatggacg agaccatgaa ggagttgaag gcctacaaat cggaactgga ggaacaactg 360accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 420cggctgggcg cggacatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 480caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 540aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagcgcct ggcagtgtac 600caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 660cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 720ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 780agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 840ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 900agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 960gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca ctgaacgccg 1020aagcctgcag ccatgcgacc ccacgccacc ccgtgcctcc tgcctccgcg cagcctgcag 1080cgggagaccc tgtccccgcc ccagccgtcc tcctggggtg gaccctagtt taataaagat 1140tcaccaagtt tcacgc 115625515DNAHUMAN 2ggaacttgat gctcagagag gacaagtcat ttgcccaagg tcacacagct ggcaactggc 60agacgagatt cacgccctgg caatttgact ccagaatcct aaccttaacc cagaagcacg 120gcttcaagcc ctggaaacca caatacctgt ggcagccagg gggaggtgct ggaatctcat 180ttcacatgtg gggagggggc tcctgtgctc aaggtcacaa ccaaagagga agctgtgatt 240aaaacccagg tcccatttgc aaagcctcga cttttagcag gtgcatcata ctgttcccac 300ccctcccatc ccacttctgt ccagccgcct agccccactt tctttttttt ctttttttga 360gacagtctcc ctcttgctga ggctggagtg cagtggcgag atctcggctc actgtaacct 420ccgcctcccg ggttcaagcg attctcctgc ctcagcctcc caagtagcta ggattacagg 480cgcccgccac cacgcctggc taacttttgt atttttagta gagatggggt ttcaccatgt 540tggccaggct ggtctcaaac tcctgacctt aagtgattcg cccactgtgg cctcccaaag 600tgctgggatt acaggcgtga gctaccgccc ccagcccctc ccatcccact tctgtccagc 660cccctagccc tactttcttt ctgggatcca ggagtccaga tccccagccc cctctccaga 720ttacattcat ccaggcacag gaaaggacag ggtcaggaaa ggaggactct gggcggcagc 780ctccacattc cccttccacg cttggccccc agaatggagg agggtgtctg tattactggg 840cgaggtgtcc tcccttcctg gggactgtgg ggggtggtca aaagacctct atgccccacc 900tccttcctcc ctctgccctg ctgtgcctgg ggcaggggga gaacagccca cctcgtgact 960gggctgccca gcccgcccta tccctggggg agggggcggg acagggggag ccctataatt 1020ggacaagtct gggatccttg agtcctactc agccccagcg gaggtgaagg acgtccttcc 1080ccaggagccg gtgagaagcg cagtcggggg cacggggatg agctcagggg cctctagaaa 1140gagctgggac cctgggaagc cctggcctcc aggtagtctc aggagagcta ctcggggtcg 1200ggcttgggga gaggaggagc gggggtgagg caagcagcag gggactggac ctgggaaggg 1260ctgggcagca gagacgaccc gacccgctag aaggtggggt ggggagagca gctggactgg 1320gatgtaagcc atagcaggac tccacgagtt gtcactatca ttatcgagca cctactgggt 1380gtccccagtg tcctcagatc tccataactg gggagccagg ggcagcgaca cggtagctag 1440ccgtcgattg gagaacttta aaatgaggac tgaattagct cataaatgga acacggcgct 1500taactgtgag gttggagctt agaatgtgaa gggagaatga ggaatgcgag actgggactg 1560agatggaacc ggcggtgggg agggggtggg gggatggaat ttgaaccccg ggagaggaag 1620atggaatttt ctatggaggc cgacctgggg atggggagat aagagaagac caggagggag 1680ttaaataggg aatgggttgg gggcggcttg gtaaatgtgc tgggattagg ctgttgcaga 1740taatgcaaca aggcttggaa ggctaacctg gggtgaggcc gggttggggg cgctgggggt 1800gggaggagtc ctcactggcg gttgattgac agtttctcct tccccagact ggccaatcac 1860aggcaggaag atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggtatggg 1920ggcggggctt gctcggttcc ccccgctcct ccccctctca tcctcacctc aacctcctgg 1980ccccattcag acagaccctg ggccccctct tctgaggctt ctgtgctgct tcctggctct 2040gaacagcgat ttgacgctct ctgggcctcg gtttccccca tccttgagat aggagttaga 2100agttgttttg ttgttgttgt ttgttgttgt tgttttgttt ttttgagatg aagtctcgct 2160ctgtcgccca ggctggagtg cagtggcggg atctcggctc actgcaagct ccgcctccca 2220ggtccacgcc attctcctgc ctcagcctcc caagtagctg ggactacagg cacatgccac 2280cacacccgac taactttttt gtattttcag tagagacggg gtttcaccat gttggccagg 2340ctggtctgga actcctgacc tcaggtgatc tgcccgtttc gatctcccaa agtgctggga 2400ttacaggcgt gagccaccgc acctggctgg gagttagagg tttctaatgc attgcaggca 2460gatagtgaat accagacacg gggcagctgt gatctttatt ctccatcacc cccacacagc 2520cctgcctggg gcacacaagg acactcaata catgcttttc cgctgggccg gtggctcacc 2580cctgtaatcc cagcactttg ggaggccaag gtgggaggat cacttgagcc caggagttca 2640acaccagcct gggcaacata gtgagaccct gtctctacta aaaatacaaa aattagccag 2700gcatggtgcc acacacctgt gctctcagct actcaggagg ctgaggcagg aggatcgctt 2760gagcccagaa ggtcaaggtt gcagtgaacc atgttcaggc cgctgcactc cagcctgggt 2820gacagagcaa gaccctgttt ataaatacat aatgctttcc aagtgattaa accgactccc 2880ccctcaccct gcccaccatg gctccaaaga agcatttgtg gagcaccttc tgtgtgcccc 2940taggtagcta gatgcctgga cggggtcaga aggaccctga cccgaccttg aacttgttcc 3000acacaggatg ccaggccaag gtggagcaag cggtggagac agagccggag cccgagctgc 3060gccagcagac cgagtggcag agcggccagc gctgggaact ggcactgggt cgcttttggg 3120attacctgcg ctgggtgcag acactgtctg agcaggtgca ggaggagctg ctcagctccc 3180aggtcaccca ggaactgagg tgagtgtccc catcctggcc cttgaccctc ctggtgggcg 3240gctatacctc cccaggtcca ggtttcattc tgcccctgtc gctaagtctt ggggggcctg 3300ggtctctgct ggttctagct tcctcttccc atttctgact cctggcttta gctctctgga 3360attctctctc tcagctttgt ctctctctct tcccttctga ctcagtctct cacactcgtc 3420ctggctctgt ctctgtcctt ccctagctct tttatataga gacagagaga tggggtctca 3480ctgtgttgcc caggctggtc ttgaacttct gggctcaagc gatcctcccg cctcggcctc 3540ccaaagtgct gggattagag gcatgagcac cttgcccggc ctcctagctc cttcttcgtc 3600tctgcctctg ccctctgcat ctgctctctg catctgtctc tgtctccttc tctcggcctc 3660tgccccgttc cttctctccc tcttgggtct ctctggctca tccccatctc gcccgcccca 3720tcccagccct tctcccccgc ctccccactg tgcgacaccc tcccgccctc tcggccgcag 3780ggcgctgatg gacgagacca tgaaggagtt gaaggcctac aaatcggaac tggaggaaca 3840actgaccccg gtggcggagg agacgcgggc acggctgtcc aaggagctgc aggcggcgca 3900ggcccggctg ggcgcggaca tggaggacgt gcgcggccgc ctggtgcagt accgcggcga 3960ggtgcaggcc atgctcggcc agagcaccga ggagctgcgg gtgcgcctcg cctcccacct 4020gcgcaagctg cgtaagcggc tcctccgcga tgccgatgac ctgcagaagc gcctggcagt 4080gtaccaggcc ggggcccgcg agggcgccga gcgcggcctc agcgccatcc gcgagcgcct 4140ggggcccctg gtggaacagg gccgcgtgcg ggccgccact gtgggctccc tggccggcca 4200gccgctacag gagcgggccc aggcctgggg cgagcggctg cgcgcgcgga tggaggagat 4260gggcagccgg acccgcgacc gcctggacga ggtgaaggag caggtggcgg aggtgcgcgc 4320caagctggag gagcaggccc agcagatacg cctgcaggcc gaggccttcc aggcccgcct 4380caagagctgg ttcgagcccc tggtggaaga catgcagcgc cagtgggccg ggctggtgga 4440gaaggtgcag gctgccgtgg gcaccagcgc cgcccctgtg cccagcgaca atcactgaac 4500gccgaagcct gcagccatgc gaccccacgc caccccgtgc ctcctgcctc cgcgcagcct 4560gcagcgggag accctgtccc cgccccagcc gtcctcctgg ggtggaccct agtttaataa 4620agattcacca agtttcacgc atctgctggc ctccccctgt gatttcctct aagccccagc 4680ctcagtttct ctttctgccc acatactgcc acacaattct cagccccctc ctctccatct 4740gtgtctgtgt gtatctttct ctctgccctt tttttttttt tagacggagt ctggctctgt 4800cacccaggct agagtgcagt ggcacgatct tggctcactg caacctctgc ctcttgggtt 4860caagcgattc tgctgcctca gtagctggga ttacaggctc acaccaccac acccggctaa 4920tttttgtatt tttagtagag acgagctttc accatgttgg ccaggcaggt ctcaaactcc 4980tgaccaagtg atccacccgc cggcctccca aagtgctgag attacaggcc tgagccacca 5040tgcccggcct ctgcccctct ttctttttta gggggcaggg aaaggtctca ccctgtcacc 5100cgccatcaca gctcactgca gcctccacct cctggactca agtgataagt gatcctcccg 5160cctcagcctt tccagtagct gagactacag gcgcatacca ctaggattaa tttggggggg 5220ggtggtgtgt gtggagatgg ggtctggctt tgttggccag gctgatgtgg aattcctggg 5280ctcaagcgat actcccacct tggcctcctg agtagctgag actactggct agcaccacca 5340cacccagctt tttattatta tttgtagaga caaggtctca atatgttgcc caggctagtc 5400tcaaacccct ggctcaagag atcctccgcc atcggcctcc caaagtgctg ggattccagg 5460catgggctcc gagcggcctg cccaacttaa taatattgtt cctagagttg cactc 551531157DNAHUMAN 3ccccagcgga ggtgaaggac gtccttcccc aggagccgac tggccaatca caggcaggaa 60gatgaaggtt ctgtgggctg cgttgctggt cacattcctg gcaggatgcc aggccaaggt 120ggagcaagcg gtggagacag agccggagcc cgagctgcgc cagcagaccg agtggcagag 180cggccagcgc tgggaactgg cactgggtcg cttttgggat tacctgcgct gggtgcagac 240actgtctgag caggtgcagg aggagctgct cagctcccaa gtcacccaag aactgagggc 300gctgatggac gagaccatga aggagttgaa ggcctacaaa tcggaactgg aggaacaact 360gaccccggta gcggaggaga cgcgggcacg gctgtccaag gagctgcaga cggcgcaggc 420ccggctgggc gcggacatgg aggacgtgtg cggccgcctg gtgcagtacc gcggcgaggt 480gcaggccatg ctcggccaga gcaccgagga gctgcgggtg cgcctcgcct cccacctgcg 540caagctgcgt aagcggctcc tccgcgatcc cgatgacctg cagaagcgcc tggcagtgta 600ccaggccggg gcccgcgagg gcgccgagcg cggcctcagc gccatccgcg agcgcctggg 660gcccctggtg gaacagggcc gcgtgcgggc cgccactgtg ggctccctgg ccggccagcc 720gctacaggag cgggcccagg cctggggcga gcggctgcgc gcgcggatgg aggagatggg 780cagtcggacc cgcgaccgcc tggacgaggt gaaggagcag gtggcggagg tgcgcgccaa 840gctggaggag caggcccagc agatacgcct gcaggccgag gccttccagg cccgcctcaa 900gagctggttc gagcccctgg tggaagacat gcagcgccag tgggccgggc tggtggagaa 960ggtgcaggct gccgtgggca ccagcgccgc ccctgtgccc agcgacaatc actgaacgcc 1020gaagcctgca gccatgcgac cccacgccac cccgtgcctc ctgcctccgc gcagcctgca 1080gcgggagacc ctgtccccgc cccagccgtc ctcctggggt ggaccctagt ttaataaaga 1140ttcaccaagt ttcacgc 11574317PRTHUMAN 4Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys1 5 10 15Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu 20 25 30Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu 35 40 45Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln 50 55 60Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala65 70 75 80Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu 85 90 95Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser 100 105 110Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp 115 120 125Val Cys Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135 140Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg145 150 155 160Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg 165 170 175Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu 180 185 190Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val 195 200 205Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 210 215 220Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly225 230 235 240Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu 245 250 255Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala 260 265 270Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280 285Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala 290 295 300Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His305 310 3155317PRTHUMAN 5Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys1 5 10 15Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu 20 25 30Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu 35 40 45Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln 50 55 60Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala65 70 75 80Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu 85 90 95Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser 100 105 110Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp 115 120 125Val Arg Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135 140Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg145 150 155 160Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg 165 170 175Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu 180 185 190Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val 195 200 205Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 210 215 220Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly225 230 235 240Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu 245 250 255Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala 260 265 270Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280 285Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala 290 295 300Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His305 310 3156317PRTHUMAN 6Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys1 5 10 15Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu 20 25 30Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu 35 40 45Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln 50 55 60Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala65 70 75 80Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu 85 90 95Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser 100 105 110Lys Glu Leu Gln Thr Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp 115 120 125Val Cys Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135 140Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg145 150 155 160Lys Leu Arg Lys Arg Leu Leu Arg Asp Pro Asp Asp Leu Gln Lys Arg 165 170 175Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu 180 185 190Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val 195 200 205Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 210 215 220Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly225 230 235 240Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu 245 250 255Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala 260 265 270Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280 285Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala 290 295 300Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His305 310 315710412DNAHUMAN 7gtaattgcga gcgagagtga gtggggccgg gacccgcaga gccgagccga cccttctctc 60ccgggctgcg gcagggcagg gcggggagct ccgcgcacca acagagccgg ttctcagggc 120gctttgctcc ttgttttttc cccggttctg ttttctcccc ttctccggaa ggcttgtcaa 180ggggtaggag aaagagacgc aaacacaaaa gtggaaaaca gttaatgacc agccacggcg 240tccctgctgt gagctctggc cgctgccttc cagggctccc gagccacacg ctgggggtgc 300tggctgaggg aacatggctt gttggcctca gctgaggttg ctgctgtgga agaacctcac 360tttcagaaga agacaaacat gtcagctgct gctggaagtg gcctggcctc tatttatctt 420cctgatcctg atctctgttc ggctgagcta cccaccctat gaacaacatg aatgccattt 480tccaaataaa gccatgccct ctgcaggaac acttccttgg gttcagggga ttatctgtaa 540tgccaacaac ccctgtttcc gttacccgac tcctggggag gctcccggag ttgttggaaa 600ctttaacaaa tccattgtgg ctcgcctgtt ctcagatgct cggaggcttc ttttatacag 660ccagaaagac accagcatga aggacatgcg caaagttctg agaacattac agcagatcaa 720gaaatccagc tcaaacttga agcttcaaga tttcctggtg gacaatgaaa ccttctctgg 780gttcctgtat cacaacctct ctctcccaaa gtctactgtg gacaagatgc tgagggctga 840tgtcattctc cacaaggtat ttttgcaagg ctaccagtta catttgacaa gtctgtgcaa 900tggatcaaaa tcagaagaga tgattcaact tggtgaccaa gaagtttctg agctttgtgg 960cctaccaagg gagaaactgg ctgcagcaga gcgagtactt cgttccaaca tggacatcct 1020gaagccaatc ctgagaacac taaactctac atctcccttc ccgagcaagg agctggctga 1080agccacaaaa acattgctgc atagtcttgg gactctggcc caggagctgt tcagcatgag 1140aagctggagt gacatgcgac aggaggtgat gtttctgacc aatgtgaaca gctccagctc 1200ctccacccaa atctaccagg ctgtgtctcg tattgtctgc gggcatcccg agggaggggg 1260gctgaagatc aagtctctca actggtatga ggacaacaac tacaaagccc
tctttggagg 1320caatggcact gaggaagatg ctgaaacctt ctatgacaac tctacaactc cttactgcaa 1380tgatttgatg aagaatttgg agtctagtcc tctttcccgc attatctgga aagctctgaa 1440gccgctgctc gttgggaaga tcctgtatac acctgacact ccagccacaa ggcaggtcat 1500ggctgaggtg aacaagacct tccaggaact ggctgtgttc catgatctgg aaggcatgtg 1560ggaggaactc agccccaaga tctggacctt catggagaac agccaagaaa tggaccttgt 1620ccggatgctg ttggacagca gggacaatga ccacttttgg gaacagcagt tggatggctt 1680agattggaca gcccaagaca tcgtggcgtt tttggccaag cacccagagg atgtccagtc 1740cagtaatggt tctgtgtaca cctggagaga agctttcaac gagactaacc aggcaatccg 1800gaccatatct cgcttcatgg agtgtgtcaa cctgaacaag ctagaaccca tagcaacaga 1860agtctggctc atcaacaagt ccatggagct gctggatgag aggaagttct gggctggtat 1920tgtgttcact ggaattactc caggcagcat tgagctgccc catcatgtca agtacaagat 1980ccgaatggac attgacaatg tggagaggac aaataaaatc aaggatgggt actgggaccc 2040tggtcctcga gctgacccct ttgaggacat gcggtacgtc tgggggggct tcgcctactt 2100gcaggatgtg gtggagcagg caatcatcag ggtgctgacg ggcaccgaga agaaaactgg 2160tgtctatatg caacagatgc cctatccctg ttacgttgat gacatctttc tgcgggtgat 2220gagccggtca atgcccctct tcatgacgct ggcctggatt tactcagtgg ctgtgatcat 2280caagggcatc gtgtatgaga aggaggcacg gctgaaagag accatgcgga tcatgggcct 2340ggacaacagc atcctctggt ttagctggtt cattagtagc ctcattcctc ttcttgtgag 2400cgctggcctg ctagtggtca tcctgaagtt aggaaacctg ctgccctaca gtgatcccag 2460cgtggtgttt gtcttcctgt ccgtgtttgc tgtggtgaca atcctgcagt gcttcctgat 2520tagcacactc ttctccagag ccaacctggc agcagcctgt gggggcatca tctacttcac 2580gctgtacctg ccctacgtcc tgtgtgtggc atggcaggac tacgtgggct tcacactcaa 2640gatcttcgct agcctgctgt ctcctgtggc ttttgggttt ggctgtgagt actttgccct 2700ttttgaggag cagggcattg gagtgcagtg ggacaacctg tttgagagtc ctgtggagga 2760agatggcttc aatctcacca cttcggtctc catgatgctg tttgacacct tcctctatgg 2820ggtgatgacc tggtacattg aggctgtctt tccaggccag tacggaattc ccaggccctg 2880gtattttcct tgcaccaagt cctactggtt tggcgaggaa agtgatgaga agagccaccc 2940tggttccaac cagaagagaa tatcagaaat ctgcatggag gaggaaccca cccacttgaa 3000gctgggcgtg tccattcaga acctggtaaa agtctaccga gatgggatga aggtggctgt 3060cgatggcctg gcactgaatt tttatgaggg ccagatcacc tccttcctgg gccacaatgg 3120agcggggaag acgaccacca tgtcaatcct gaccgggttg ttccccccga cctcgggcac 3180cgcctacatc ctgggaaaag acattcgctc tgagatgagc accatccggc agaacctggg 3240ggtctgtccc cagcataacg tgctgtttga catgctgact gtcgaagaac acatctggtt 3300ctatgcccgc ttgaaagggc tctctgagaa gcacgtgaag gcggagatgg agcagatggc 3360cctggatgtt ggtttgccat caagcaagct gaaaagcaaa acaagccagc tgtcaggtgg 3420aatgcagaga aagctatctg tggccttggc ctttgtcggg ggatctaagg ttgtcattct 3480ggatgaaccc acagctggtg tggaccctta ctcccgcagg ggaatatggg agctgctgct 3540gaaataccga caaggccgca ccattattct ctctacacac cacatggatg aagcggacgt 3600cctgggggac aggattgcca tcatctccca tgggaagctg tgctgtgtgg gctcctccct 3660gtttctgaag aaccagctgg gaacaggcta ctacctgacc ttggtcaaga aagatgtgga 3720atcctccctc agttcctgca gaaacagtag tagcactgtg tcatacctga aaaaggagga 3780cagtgtttct cagagcagtt ctgatgctgg cctgggcagc gaccatgaga gtgacacgct 3840gaccatcgat gtctctgcta tctccaacct catcaggaag catgtgtctg aagcccggct 3900ggtggaagac atagggcatg agctgaccta tgtgctgcca tatgaagctg ctaaggaggg 3960agcctttgtg gaactctttc atgagattga tgaccggctc tcagacctgg gcatttctag 4020ttatggcatc tcagagacga ccctggaaga aatattcctc aaggtggccg aagagagtgg 4080ggtggatgct gagacctcag atggtacctt gccagcaaga cgaaacaggc gggccttcgg 4140ggacaagcag agctgtcttc gcccgttcac tgaagatgat gctgctgatc caaatgattc 4200tgacatagac ccagaatcca gagagacaga cttgctcagt gggatggatg gcaaagggtc 4260ctaccaggtg aaaggctgga aacttacaca gcaacagttt gtggcccttt tgtggaagag 4320actgctaatt gccagacgga gtcggaaagg attttttgct cagattgtct tgccagctgt 4380gtttgtctgc attgcccttg tgttcagcct gatcgtgcca ccctttggca agtaccccag 4440cctggaactt cagccctgga tgtacaacga acagtacaca tttgtcagca atgatgctcc 4500tgaggacacg ggaaccctgg aactcttaaa cgccctcacc aaagaccctg gcttcgggac 4560ccgctgtatg gaaggaaacc caatcccaga cacgccctgc caggcagggg aggaagagtg 4620gaccactgcc ccagttcccc agaccatcat ggacctcttc cagaatggga actggacaat 4680gcagaaccct tcacctgcat gccagtgtag cagcgacaaa atcaagaaga tgctgcctgt 4740gtgtccccca ggggcagggg ggctgcctcc tccacaaaga aaacaaaaca ctgcagatat 4800ccttcaggac ctgacaggaa gaaacatttc ggattatctg gtgaagacgt atgtgcagat 4860catagccaaa agcttaaaga acaagatctg ggtgaatgag tttaggtatg gcggcttttc 4920cctgggtgtc agtaatactc aagcacttcc tccgagtcaa gaagttaatg atgccatcaa 4980acaaatgaag aaacacctaa agctggccaa ggacagttct gcagatcgat ttctcaacag 5040cttgggaaga tttatgacag gactggacac caaaaataat gtcaaggtgt ggttcaataa 5100caagggctgg catgcaatca gctctttcct gaatgtcatc aacaatgcca ttctccgggc 5160caacctgcaa aagggagaga accctagcca ttatggaatt actgctttca atcatcccct 5220gaatctcacc aagcagcagc tctcagaggt ggctctgatg accacatcag tggatgtcct 5280tgtgtccatc tgtgtcatct ttgcaatgtc cttcgtccca gccagctttg tcgtattcct 5340gatccaggag cgggtcagca aagcaaaaca cctgcagttc atcagtggag tgaagcctgt 5400catctactgg ctctctaatt ttgtctggga tatgtgcaat tacgttgtcc ctgccacact 5460ggtcattatc atcttcatct gcttccagca gaagtcctat gtgtcctcca ccaatctgcc 5520tgtgctagcc cttctacttt tgctgtatgg gtggtcaatc acacctctca tgtacccagc 5580ctcctttgtg ttcaagatcc ccagcacagc ctatgtggtg ctcaccagcg tgaacctctt 5640cattggcatt aatggcagcg tggccacctt tgtgctggag ctgttcaccg acaataagct 5700gaataatatc aatgatatcc tgaagtccgt gttcttgatc ttcccacatt tttgcctggg 5760acgagggctc atcgacatgg tgaaaaacca ggcaatggct gatgccctgg aaaggtttgg 5820ggagaatcgc tttgtgtcac cattatcttg ggacttggtg ggacgaaacc tcttcgccat 5880ggccgtggaa ggggtggtgt tcttcctcat tactgttctg atccagtaca gattcttcat 5940caggcccaga cctgtaaatg caaagctatc tcctctgaat gatgaagatg aagatgtgag 6000gcgggaaaga cagagaattc ttgatggtgg aggccagaat gacatcttag aaatcaagga 6060gttgacgaag atatatagaa ggaagcggaa gcctgctgtt gacaggattt gcgtgggcat 6120tcctcctggt gagtgctttg ggctcctggg agttaatggg gctggaaaat catcaacttt 6180caagatgtta acaggagata ccactgttac cagaggagat gctttcctta acaaaaatag 6240tatcttatca aacatccatg aagtacatca gaacatgggc tactgccctc agtttgatgc 6300catcacagag ctgttgactg ggagagaaca cgtggagttc tttgcccttt tgagaggagt 6360cccagagaaa gaagttggca aggttggtga gtgggcgatt cggaaactgg gcctcgtgaa 6420gtatggagaa aaatatgctg gtaactatag tggaggcaac aaacgcaagc tctctacagc 6480catggctttg atcggcgggc ctcctgtggt gtttctggat gaacccacca caggcatgga 6540tcccaaagcc cggcggttct tgtggaattg tgccctaagt gttgtcaagg aggggagatc 6600agtagtgctt acatctcata gtatggaaga atgtgaagct ctttgcacta ggatggcaat 6660catggtcaat ggaaggttca ggtgccttgg cagtgtccag catctaaaaa ataggtttgg 6720agatggttat acaatagttg tacgaatagc agggtccaac ccggacctga agcctgtcca 6780ggatttcttt ggacttgcat ttcctggaag tgttctaaaa gagaaacacc ggaacatgct 6840acaataccag cttccatctt cattatcttc tctggccagg atattcagca tcctctccca 6900gagcaaaaag cgactccaca tagaagacta ctctgtttct cagacaacac ttgaccaagt 6960atttgtgaac tttgccaagg accaaagtga tgatgaccac ttaaaagacc tctcattaca 7020caaaaaccag acagtagtgg acgttgcagt tctcacatct tttctacagg atgagaaagt 7080gaaagaaagc tatgtatgaa gaatcctgtt catacggggt ggctgaaagt aaagaggaac 7140tagactttcc tttgcaccat gtgaagtgtt gtggagaaaa gagccagaag ttgatgtggg 7200aagaagtaaa ctggatactg tactgatact attcaatgca atgcaattca atgcaatgaa 7260aacaaaattc cattacaggg gcagtgcctt tgtagcctat gtcttgtatg gctctcaagt 7320gaaagacttg aatttagttt tttacctata cctatgtgaa actctattat ggaacccaat 7380ggacatatgg gtttgaactc acactttttt tttttttttt gttcctgtgt attctcattg 7440gggttgcaac aataattcat caagtaatca tggccagcga ttattgatca aaatcaaaag 7500gtaatgcaca tcctcattca ctaagccatg ccatgcccag gagactggtt tcccggtgac 7560acatccattg ctggcaatga gtgtgccaga gttattagtg ccaagttttt cagaaagttt 7620gaagcaccat ggtgtgtcat gctcactttt gtgaaagctg ctctgctcag agtctatcaa 7680cattgaatat cagttgacag aatggtgcca tgcgtggcta acatcctgct ttgattccct 7740ctgataagct gttctggtgg cagtaacatg caacaaaaat gtgggtgtct ccaggcacgg 7800gaaacttggt tccattgtta tattgtccta tgcttcgagc catgggtcta cagggtcatc 7860cttatgagac tcttaaatat acttagatcc tggtaagagg caaagaatca acagccaaac 7920tgctggggct gcaagctgct gaagccaggg catgggatta aagagattgt gcgttcaaac 7980ctagggaagc ctgtgcccat ttgtcctgac tgtctgctaa catggtacac tgcatctcaa 8040gatgtttatc tgacacaagt gtattatttc tggctttttg aattaatcta gaaaatgaaa 8100agatggagtt gtattttgac aaaaatgttt gtacttttta atgttatttg gaattttaag 8160ttctatcagt gacttctgaa tccttagaat ggcctctttg tagaaccctg tggtatagag 8220gagtatggcc actgccccac tatttttatt ttcttatgta agtttgcata tcagtcatga 8280ctagtgccta gaaagcaatg tgatggtcag gatctcatga cattatattt gagtttcttt 8340cagatcattt aggatactct taatctcact tcatcaatca aatatttttt gagtgtatgc 8400tgtagctgaa agagtatgta cgtacgtata agactagaga gatattaagt ctcagtacac 8460ttcctgtgcc atgttattca gctcactggt ttacaaatat aggttgtctt gtggttgtag 8520gagcccactg taacaatact gggcagcctt tttttttttt tttttaattg caacaatgca 8580aaagccaaga aagtataagg gtcacaagtc taaacaatga attcttcaac agggaaaaca 8640gctagcttga aaacttgctg aaaaacacaa cttgtgttta tggcatttag taccttcaaa 8700taattggctt tgcagatatt ggatacccca ttaaatctga cagtctcaaa tttttcatct 8760cttcaatcac tagtcaagaa aaatataaaa acaacaaata cttccatatg gagcattttt 8820cagagttttc taacccagtc ttatttttct agtcagtaaa catttgtaaa aatactgttt 8880cactaatact tactgttaac tgtcttgaga gaaaagaaaa atatgagaga actattgttt 8940ggggaagttc aagtgatctt tcaatatcat tactaacttc ttccactttt tccagaattt 9000gaatattaac gctaaaggtg taagacttca gatttcaaat taatctttct atatttttta 9060aatttacaga atattatata acccactgct gaaaaagaaa aaaatgattg ttttagaagt 9120taaagtcaat attgatttta aatataagta atgaaggcat atttccaata actagtgata 9180tggcatcgtt gcattttaca gtatcttcaa aaatacagaa tttatagaat aatttctcct 9240catttaatat ttttcaaaat caaagttatg gtttcctcat tttactaaaa tcgtattcta 9300attcttcatt atagtaaatc tatgagcaac tccttacttc ggttcctctg atttcaaggc 9360catattttaa aaaatcaaaa ggcactgtga actattttga agaaaacaca acattttaat 9420acagattgaa aggacctctt ctgaagctag aaacaatcta tagttataca tcttcattaa 9480tactgtgtta ccttttaaaa tagtaatttt ttacattttc ctgtgtaaac ctaattgtgg 9540tagaaatttt taccaactct atactcaatc aagcaaaatt tctgtatatt ccctgtggaa 9600tgtacctatg tgagtttcag aaattctcaa aatacgtgtt caaaaatttc tgcttttgca 9660tctttgggac acctcagaaa acttattaac aactgtgaat atgagaaata cagaagaaaa 9720taataagccc tctatacata aatgcccagc acaattcatt gttaaaaaac aaccaaacct 9780cacactactg tatttcatta tctgtactga aagcaaatgc tttgtgacta ttaaatgttg 9840cacatcattc attcactgta tagtaatcat tgactaaagc catttgtctg tgttttcttc 9900ttgtggttgt atatatcagg taaaatattt tccaaagagc catgtgtcat gtaatactga 9960accactttga tattgagaca ttaatttgta cccttgttat tatctactag taataatgta 10020atactgtaga aatattgctc taattctttt caaaattgtt gcatccccct tagaatgttt 10080ctatttccat aaggatttag gtatgctatt atcccttctt ataccctaag atgaagctgt 10140ttttgtgctc tttgttcatc attggccctc attccaagca ctttacgctg tctgtaatgg 10200gatctatttt tgcactggaa tatctgagaa ttgcaaaact agacaaaagt ttcacaacag 10260atttctaagt taaatcattt tcattaaaag gaaaaaagaa aaaaaatttt gtatgtcaat 10320aactttatat gaagtattaa aatgcatatt tctatgttgt aatataatga gtcacaaaat 10380aaagctgtga cagttctgtt ggtctacaga aa 1041286786DNAHUMAN 8atggcttgtt ggcctcagct gaggttgctg ctgtggaaga acctcacttt cagaagaaga 60caaacatgtc agctgctgct ggaagtggcc tggcctctat ttatcttcct gatcctgatc 120tctgttcggc tgagctaccc accctatgaa caacatgaat gccattttcc aaataaagcc 180atgccctctg caggaacact tccttgggtt caggggatta tctgtaatgc caacaacccc 240tgtttccgtt acccgactcc tggggaggct cccggagttg ttggaaactt taacaaatcc 300attgtggctc gcctgttctc agatgctcgg aggcttcttt tatacagcca gaaagacacc 360agcatgaagg acatgcgcaa agttctgaga acattacagc agatcaagaa atccagctca 420aacttgaagc ttcaagattt cctggtggac aatgaaacct tctctgggtt cctgtatcac 480aacctctctc tcccaaagtc tactgtggac aagatgctga gggctgatgt cattctccac 540aaggtatttt tgcaaggcta ccagttacat ttgacaagtc tgtgcaatgg atcaaaatca 600gaagagatga ttcaacttgg tgaccaagaa gtttctgagc tttgtggcct accaagggag 660aaactggctg cagcagagcg agtacttcgt tccaacatgg acatcctgaa gccaatcctg 720agaacactaa actctacatc tcccttcccg agcaaggagc tggctgaagc cacaaaaaca 780ttgctgcata gtcttgggac tctggcccag gagctgttca gcatgagaag ctggagtgac 840atgcgacagg aggtgatgtt tctgaccaat gtgaacagct ccagctcctc cacccaaatc 900taccaggctg tgtctcgtat tgtctgcggg catcccgagg gaggggggct gaagatcaag 960tctctcaact ggtatgagga caacaactac aaagccctct ttggaggcaa tggcactgag 1020gaagatgctg aaaccttcta tgacaactct acaactcctt actgcaatga tttgatgaag 1080aatttggagt ctagtcctct ttcccgcatt atctggaaag ctctgaagcc gctgctcgtt 1140gggaagatcc tgtatacacc tgacactcca gccacaaggc aggtcatggc tgaggtgaac 1200aagaccttcc aggaactggc tgtgttccat gatctggaag gcatgtggga ggaactcagc 1260cccaagatct ggaccttcat ggagaacagc caagaaatgg accttgtccg gatgctgttg 1320gacagcaggg acaatgacca cttttgggaa cagcagttgg atggcttaga ttggacagcc 1380caagacatcg tggcgttttt ggccaagcac ccagaggatg tccagtccag taatggttct 1440gtgtacacct ggagagaagc tttcaacgag actaaccagg caatccggac catatctcgc 1500ttcatggagt gtgtcaacct gaacaagcta gaacccatag caacagaagt ctggctcatc 1560aacaagtcca tggagctgct ggatgagagg aagttctggg ctggtattgt gttcactgga 1620attactccag gcagcattga gctgccccat catgtcaagt acaagatccg aatggacatt 1680gacaatgtgg agaggacaaa taaaatcaag gatgggtact gggaccctgg tcctcgagct 1740gacccctttg aggacatgcg gtacgtctgg gggggcttcg cctacttgca ggatgtggtg 1800gagcaggcaa tcatcagggt gctgacgggc accgagaaga aaactggtgt ctatatgcaa 1860cagatgccct atccctgtta cgttgatgac atctttctgc gggtgatgag ccggtcaatg 1920cccctcttca tgacgctggc ctggatttac tcagtggctg tgatcatcaa gggcatcgtg 1980tatgagaagg aggcacggct gaaagagacc atgcggatca tgggcctgga caacagcatc 2040ctctggttta gctggttcat tagtagcctc attcctcttc ttgtgagcgc tggcctgcta 2100gtggtcatcc tgaagttagg aaacctgctg ccctacagtg atcccagcgt ggtgtttgtc 2160ttcctgtccg tgtttgctgt ggtgacaatc ctgcagtgct tcctgattag cacactcttc 2220tccagagcca acctggcagc agcctgtggg ggcatcatct acttcacgct gtacctgccc 2280tacgtcctgt gtgtggcatg gcaggactac gtgggcttca cactcaagat cttcgctagc 2340ctgctgtctc ctgtggcttt tgggtttggc tgtgagtact ttgccctttt tgaggagcag 2400ggcattggag tgcagtggga caacctgttt gagagtcctg tggaggaaga tggcttcaat 2460ctcaccactt cggtctccat gatgctgttt gacaccttcc tctatggggt gatgacctgg 2520tacattgagg ctgtctttcc aggccagtac ggaattccca ggccctggta ttttccttgc 2580accaagtcct actggtttgg cgaggaaagt gatgagaaga gccaccctgg ttccaaccag 2640aagagaatat cagaaatctg catggaggag gaacccaccc acttgaagct gggcgtgtcc 2700attcagaacc tggtaaaagt ctaccgagat gggatgaagg tggctgtcga tggcctggca 2760ctgaattttt atgagggcca gatcacctcc ttcctgggcc acaatggagc ggggaagacg 2820accaccatgt caatcctgac cgggttgttc cccccgacct cgggcaccgc ctacatcctg 2880ggaaaagaca ttcgctctga gatgagcacc atccggcaga acctgggggt ctgtccccag 2940cataacgtgc tgtttgacat gctgactgtc gaagaacaca tctggttcta tgcccgcttg 3000aaagggctct ctgagaagca cgtgaaggcg gagatggagc agatggccct ggatgttggt 3060ttgccatcaa gcaagctgaa aagcaaaaca agccagctgt caggtggaat gcagagaaag 3120ctatctgtgg ccttggcctt tgtcggggga tctaaggttg tcattctgga tgaacccaca 3180gctggtgtgg acccttactc ccgcagggga atatgggagc tgctgctgaa ataccgacaa 3240ggccgcacca ttattctctc tacacaccac atggatgaag cggacgtcct gggggacagg 3300attgccatca tctcccatgg gaagctgtgc tgtgtgggct cctccctgtt tctgaagaac 3360cagctgggaa caggctacta cctgaccttg gtcaagaaag atgtggaatc ctccctcagt 3420tcctgcagaa acagtagtag cactgtgtca tacctgaaaa aggaggacag tgtttctcag 3480agcagttctg atgctggcct gggcagcgac catgagagtg acacgctgac catcgatgtc 3540tctgctatct ccaacctcat caggaagcat gtgtctgaag cccggctggt ggaagacata 3600gggcatgagc tgacctatgt gctgccatat gaagctgcta aggagggagc ctttgtggaa 3660ctctttcatg agattgatga ccggctctca gacctgggca tttctagtta tggcatctca 3720gagacgaccc tggaagaaat attcctcaag gtggccgaag agagtggggt ggatgctgag 3780acctcagatg gtaccttgcc agcaagacga aacaggcggg ccttcgggga caagcagagc 3840tgtcttcgcc cgttcactga agatgatgct gctgatccaa atgattctga catagaccca 3900gaatccagag agacagactt gctcagtggg atggatggca aagggtccta ccaggtgaaa 3960ggctggaaac ttacacagca acagtttgtg gcccttttgt ggaagagact gctaattgcc 4020agacggagtc ggaaaggatt ttttgctcag attgtcttgc cagctgtgtt tgtctgcatt 4080gcccttgtgt tcagcctgat cgtgccaccc tttggcaagt accccagcct ggaacttcag 4140ccctggatgt acaacgaaca gtacacattt gtcagcaatg atgctcctga ggacacggga 4200accctggaac tcttaaacgc cctcaccaaa gaccctggct tcgggacccg ctgtatggaa 4260ggaaacccaa tcccagacac gccctgccag gcaggggagg aagagtggac cactgcccca 4320gttccccaga ccatcatgga cctcttccag aatgggaact ggacaatgca gaacccttca 4380cctgcatgcc agtgtagcag cgacaaaatc aagaagatgc tgcctgtgtg tcccccaggg 4440gcaggggggc tgcctcctcc acaaagaaaa caaaacactg cagatatcct tcaggacctg 4500acaggaagaa acatttcgga ttatctggtg aagacgtatg tgcagatcat agccaaaagc 4560ttaaagaaca agatctgggt gaatgagttt aggtatggcg gcttttccct gggtgtcagt 4620aatactcaag cacttcctcc gagtcaagaa gttaatgatg ccatcaaaca aatgaagaaa 4680cacctaaagc tggccaagga cagttctgca gatcgatttc tcaacagctt gggaagattt 4740atgacaggac tggacaccaa aaataatgtc aaggtgtggt tcaataacaa gggctggcat 4800gcaatcagct ctttcctgaa tgtcatcaac aatgccattc tccgggccaa cctgcaaaag 4860ggagagaacc ctagccatta tggaattact gctttcaatc atcccctgaa tctcaccaag 4920cagcagctct cagaggtggc tctgatgacc acatcagtgg atgtccttgt gtccatctgt 4980gtcatctttg caatgtcctt cgtcccagcc agctttgtcg tattcctgat ccaggagcgg 5040gtcagcaaag caaaacacct gcagttcatc agtggagtga agcctgtcat ctactggctc 5100tctaattttg tctgggatat gtgcaattac gttgtccctg ccacactggt cattatcatc 5160ttcatctgct tccagcagaa gtcctatgtg tcctccacca atctgcctgt gctagccctt 5220ctacttttgc tgtatgggtg gtcaatcaca cctctcatgt acccagcctc ctttgtgttc 5280aagatcccca gcacagccta tgtggtgctc accagcgtga acctcttcat tggcattaat 5340ggcagcgtgg ccacctttgt gctggagctg ttcaccgaca ataagctgaa taatatcaat 5400gatatcctga agtccgtgtt cttgatcttc ccacattttt gcctgggacg agggctcatc 5460gacatggtga aaaaccaggc aatggctgat gccctggaaa ggtttgggga gaatcgcttt 5520gtgtcaccat tatcttggga cttggtggga cgaaacctct tcgccatggc cgtggaaggg 5580gtggtgttct tcctcattac tgttctgatc cagtacagat tcttcatcag gcccagacct 5640gtaaatgcaa agctatctcc tctgaatgat gaagatgaag atgtgaggcg ggaaagacag 5700agaattcttg atggtggagg ccagaatgac atcttagaaa tcaaggagtt gacgaagata 5760tatagaagga agcggaagcc tgctgttgac aggatttgcg tgggcattcc tcctggtgag 5820tgctttgggc tcctgggagt taatggggct ggaaaatcat caactttcaa gatgttaaca 5880ggagatacca ctgttaccag aggagatgct ttccttaaca
aaaatagtat cttatcaaac 5940atccatgaag tacatcagaa catgggctac tgccctcagt ttgatgccat cacagagctg 6000ttgactggga gagaacacgt ggagttcttt gcccttttga gaggagtccc agagaaagaa 6060gttggcaagg ttggtgagtg ggcgattcgg aaactgggcc tcgtgaagta tggagaaaaa 6120tatgctggta actatagtgg aggcaacaaa cgcaagctct ctacagccat ggctttgatc 6180ggcgggcctc ctgtggtgtt tctggatgaa cccaccacag gcatggatcc caaagcccgg 6240cggttcttgt ggaattgtgc cctaagtgtt gtcaaggagg ggagatcagt agtgcttaca 6300tctcatagta tggaagagtg tgaagctctt tgcactagga tggcaatcat ggtcaatgga 6360aggttcaggt gccttggcag tgtccagcat ctaaaaaata ggtttggaga tggttataca 6420atagttgtac gaatagcagg gtccaacccg gacctgaagc ctgtccagga tttctttgga 6480cttgcatttc ctggaagtgt tctaaaagag aaacaccgga acatgctaca ataccagctt 6540ccatcttcat tatcttctct ggccaggata ttcagcatcc tctcccagag caaaaagcga 6600ctccacatag aagactactc tgtttctcag acaacacttg accaagtatt tgtgaacttt 6660gccaaggacc aaagtgatga tgaccactta aaagacctct cattacacaa aaaccagaca 6720gtagtggacg ttgcagttct cacatctttt ctacaggatg agaaagtgaa agaaagctat 6780gtatga 678692261PRTHUMAN 9Met Ala Cys Trp Pro Gln Leu Arg Leu Leu Leu Trp Lys Asn Leu Thr1 5 10 15Phe Arg Arg Arg Gln Thr Cys Gln Leu Leu Leu Glu Val Ala Trp Pro 20 25 30Leu Phe Ile Phe Leu Ile Leu Ile Ser Val Arg Leu Ser Tyr Pro Pro 35 40 45Tyr Glu Gln His Glu Cys His Phe Pro Asn Lys Ala Met Pro Ser Ala 50 55 60Gly Thr Leu Pro Trp Val Gln Gly Ile Ile Cys Asn Ala Asn Asn Pro65 70 75 80Cys Phe Arg Tyr Pro Thr Pro Gly Glu Ala Pro Gly Val Val Gly Asn 85 90 95Phe Asn Lys Ser Ile Val Ala Arg Leu Phe Ser Asp Ala Arg Arg Leu 100 105 110Leu Leu Tyr Ser Gln Lys Asp Thr Ser Met Lys Asp Met Arg Lys Val 115 120 125Leu Arg Thr Leu Gln Gln Ile Lys Lys Ser Ser Ser Asn Leu Lys Leu 130 135 140Gln Asp Phe Leu Val Asp Asn Glu Thr Phe Ser Gly Phe Leu Tyr His145 150 155 160Asn Leu Ser Leu Pro Lys Ser Thr Val Asp Lys Met Leu Arg Ala Asp 165 170 175Val Ile Leu His Lys Val Phe Leu Gln Gly Tyr Gln Leu His Leu Thr 180 185 190Ser Leu Cys Asn Gly Ser Lys Ser Glu Glu Met Ile Gln Leu Gly Asp 195 200 205Gln Glu Val Ser Glu Leu Cys Gly Leu Pro Arg Glu Lys Leu Ala Ala 210 215 220Ala Glu Arg Val Leu Arg Ser Asn Met Asp Ile Leu Lys Pro Ile Leu225 230 235 240Arg Thr Leu Asn Ser Thr Ser Pro Phe Pro Ser Lys Glu Leu Ala Glu 245 250 255Ala Thr Lys Thr Leu Leu His Ser Leu Gly Thr Leu Ala Gln Glu Leu 260 265 270Phe Ser Met Arg Ser Trp Ser Asp Met Arg Gln Glu Val Met Phe Leu 275 280 285Thr Asn Val Asn Ser Ser Ser Ser Ser Thr Gln Ile Tyr Gln Ala Val 290 295 300Ser Arg Ile Val Cys Gly His Pro Glu Gly Gly Gly Leu Lys Ile Lys305 310 315 320Ser Leu Asn Trp Tyr Glu Asp Asn Asn Tyr Lys Ala Leu Phe Gly Gly 325 330 335Asn Gly Thr Glu Glu Asp Ala Glu Thr Phe Tyr Asp Asn Ser Thr Thr 340 345 350Pro Tyr Cys Asn Asp Leu Met Lys Asn Leu Glu Ser Ser Pro Leu Ser 355 360 365Arg Ile Ile Trp Lys Ala Leu Lys Pro Leu Leu Val Gly Lys Ile Leu 370 375 380Tyr Thr Pro Asp Thr Pro Ala Thr Arg Gln Val Met Ala Glu Val Asn385 390 395 400Lys Thr Phe Gln Glu Leu Ala Val Phe His Asp Leu Glu Gly Met Trp 405 410 415Glu Glu Leu Ser Pro Lys Ile Trp Thr Phe Met Glu Asn Ser Gln Glu 420 425 430Met Asp Leu Val Arg Met Leu Leu Asp Ser Arg Asp Asn Asp His Phe 435 440 445Trp Glu Gln Gln Leu Asp Gly Leu Asp Trp Thr Ala Gln Asp Ile Val 450 455 460Ala Phe Leu Ala Lys His Pro Glu Asp Val Gln Ser Ser Asn Gly Ser465 470 475 480Val Tyr Thr Trp Arg Glu Ala Phe Asn Glu Thr Asn Gln Ala Ile Arg 485 490 495Thr Ile Ser Arg Phe Met Glu Cys Val Asn Leu Asn Lys Leu Glu Pro 500 505 510Ile Ala Thr Glu Val Trp Leu Ile Asn Lys Ser Met Glu Leu Leu Asp 515 520 525Glu Arg Lys Phe Trp Ala Gly Ile Val Phe Thr Gly Ile Thr Pro Gly 530 535 540Ser Ile Glu Leu Pro His His Val Lys Tyr Lys Ile Arg Met Asp Ile545 550 555 560Asp Asn Val Glu Arg Thr Asn Lys Ile Lys Asp Gly Tyr Trp Asp Pro 565 570 575Gly Pro Arg Ala Asp Pro Phe Glu Asp Met Arg Tyr Val Trp Gly Gly 580 585 590Phe Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile Ile Arg Val Leu 595 600 605Thr Gly Thr Glu Lys Lys Thr Gly Val Tyr Met Gln Gln Met Pro Tyr 610 615 620Pro Cys Tyr Val Asp Asp Ile Phe Leu Arg Val Met Ser Arg Ser Met625 630 635 640Pro Leu Phe Met Thr Leu Ala Trp Ile Tyr Ser Val Ala Val Ile Ile 645 650 655Lys Gly Ile Val Tyr Glu Lys Glu Ala Arg Leu Lys Glu Thr Met Arg 660 665 670Ile Met Gly Leu Asp Asn Ser Ile Leu Trp Phe Ser Trp Phe Ile Ser 675 680 685Ser Leu Ile Pro Leu Leu Val Ser Ala Gly Leu Leu Val Val Ile Leu 690 695 700Lys Leu Gly Asn Leu Leu Pro Tyr Ser Asp Pro Ser Val Val Phe Val705 710 715 720Phe Leu Ser Val Phe Ala Val Val Thr Ile Leu Gln Cys Phe Leu Ile 725 730 735Ser Thr Leu Phe Ser Arg Ala Asn Leu Ala Ala Ala Cys Gly Gly Ile 740 745 750Ile Tyr Phe Thr Leu Tyr Leu Pro Tyr Val Leu Cys Val Ala Trp Gln 755 760 765Asp Tyr Val Gly Phe Thr Leu Lys Ile Phe Ala Ser Leu Leu Ser Pro 770 775 780Val Ala Phe Gly Phe Gly Cys Glu Tyr Phe Ala Leu Phe Glu Glu Gln785 790 795 800Gly Ile Gly Val Gln Trp Asp Asn Leu Phe Glu Ser Pro Val Glu Glu 805 810 815Asp Gly Phe Asn Leu Thr Thr Ser Val Ser Met Met Leu Phe Asp Thr 820 825 830Phe Leu Tyr Gly Val Met Thr Trp Tyr Ile Glu Ala Val Phe Pro Gly 835 840 845Gln Tyr Gly Ile Pro Arg Pro Trp Tyr Phe Pro Cys Thr Lys Ser Tyr 850 855 860Trp Phe Gly Glu Glu Ser Asp Glu Lys Ser His Pro Gly Ser Asn Gln865 870 875 880Lys Arg Ile Ser Glu Ile Cys Met Glu Glu Glu Pro Thr His Leu Lys 885 890 895Leu Gly Val Ser Ile Gln Asn Leu Val Lys Val Tyr Arg Asp Gly Met 900 905 910Lys Val Ala Val Asp Gly Leu Ala Leu Asn Phe Tyr Glu Gly Gln Ile 915 920 925Thr Ser Phe Leu Gly His Asn Gly Ala Gly Lys Thr Thr Thr Met Ser 930 935 940Ile Leu Thr Gly Leu Phe Pro Pro Thr Ser Gly Thr Ala Tyr Ile Leu945 950 955 960Gly Lys Asp Ile Arg Ser Glu Met Ser Thr Ile Arg Gln Asn Leu Gly 965 970 975Val Cys Pro Gln His Asn Val Leu Phe Asp Met Leu Thr Val Glu Glu 980 985 990His Ile Trp Phe Tyr Ala Arg Leu Lys Gly Leu Ser Glu Lys His Val 995 1000 1005Lys Ala Glu Met Glu Gln Met Ala Leu Asp Val Gly Leu Pro Ser 1010 1015 1020Ser Lys Leu Lys Ser Lys Thr Ser Gln Leu Ser Gly Gly Met Gln 1025 1030 1035Arg Lys Leu Ser Val Ala Leu Ala Phe Val Gly Gly Ser Lys Val 1040 1045 1050Val Ile Leu Asp Glu Pro Thr Ala Gly Val Asp Pro Tyr Ser Arg 1055 1060 1065Arg Gly Ile Trp Glu Leu Leu Leu Lys Tyr Arg Gln Gly Arg Thr 1070 1075 1080Ile Ile Leu Ser Thr His His Met Asp Glu Ala Asp Val Leu Gly 1085 1090 1095Asp Arg Ile Ala Ile Ile Ser His Gly Lys Leu Cys Cys Val Gly 1100 1105 1110Ser Ser Leu Phe Leu Lys Asn Gln Leu Gly Thr Gly Tyr Tyr Leu 1115 1120 1125Thr Leu Val Lys Lys Asp Val Glu Ser Ser Leu Ser Ser Cys Arg 1130 1135 1140Asn Ser Ser Ser Thr Val Ser Tyr Leu Lys Lys Glu Asp Ser Val 1145 1150 1155Ser Gln Ser Ser Ser Asp Ala Gly Leu Gly Ser Asp His Glu Ser 1160 1165 1170Asp Thr Leu Thr Ile Asp Val Ser Ala Ile Ser Asn Leu Ile Arg 1175 1180 1185Lys His Val Ser Glu Ala Arg Leu Val Glu Asp Ile Gly His Glu 1190 1195 1200Leu Thr Tyr Val Leu Pro Tyr Glu Ala Ala Lys Glu Gly Ala Phe 1205 1210 1215Val Glu Leu Phe His Glu Ile Asp Asp Arg Leu Ser Asp Leu Gly 1220 1225 1230Ile Ser Ser Tyr Gly Ile Ser Glu Thr Thr Leu Glu Glu Ile Phe 1235 1240 1245Leu Lys Val Ala Glu Glu Ser Gly Val Asp Ala Glu Thr Ser Asp 1250 1255 1260Gly Thr Leu Pro Ala Arg Arg Asn Arg Arg Ala Phe Gly Asp Lys 1265 1270 1275Gln Ser Cys Leu Arg Pro Phe Thr Glu Asp Asp Ala Ala Asp Pro 1280 1285 1290Asn Asp Ser Asp Ile Asp Pro Glu Ser Arg Glu Thr Asp Leu Leu 1295 1300 1305Ser Gly Met Asp Gly Lys Gly Ser Tyr Gln Val Lys Gly Trp Lys 1310 1315 1320Leu Thr Gln Gln Gln Phe Val Ala Leu Leu Trp Lys Arg Leu Leu 1325 1330 1335Ile Ala Arg Arg Ser Arg Lys Gly Phe Phe Ala Gln Ile Val Leu 1340 1345 1350Pro Ala Val Phe Val Cys Ile Ala Leu Val Phe Ser Leu Ile Val 1355 1360 1365Pro Pro Phe Gly Lys Tyr Pro Ser Leu Glu Leu Gln Pro Trp Met 1370 1375 1380Tyr Asn Glu Gln Tyr Thr Phe Val Ser Asn Asp Ala Pro Glu Asp 1385 1390 1395Thr Gly Thr Leu Glu Leu Leu Asn Ala Leu Thr Lys Asp Pro Gly 1400 1405 1410Phe Gly Thr Arg Cys Met Glu Gly Asn Pro Ile Pro Asp Thr Pro 1415 1420 1425Cys Gln Ala Gly Glu Glu Glu Trp Thr Thr Ala Pro Val Pro Gln 1430 1435 1440Thr Ile Met Asp Leu Phe Gln Asn Gly Asn Trp Thr Met Gln Asn 1445 1450 1455Pro Ser Pro Ala Cys Gln Cys Ser Ser Asp Lys Ile Lys Lys Met 1460 1465 1470Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu Pro Pro Pro Gln 1475 1480 1485Arg Lys Gln Asn Thr Ala Asp Ile Leu Gln Asp Leu Thr Gly Arg 1490 1495 1500Asn Ile Ser Asp Tyr Leu Val Lys Thr Tyr Val Gln Ile Ile Ala 1505 1510 1515Lys Ser Leu Lys Asn Lys Ile Trp Val Asn Glu Phe Arg Tyr Gly 1520 1525 1530Gly Phe Ser Leu Gly Val Ser Asn Thr Gln Ala Leu Pro Pro Ser 1535 1540 1545Gln Glu Val Asn Asp Ala Ile Lys Gln Met Lys Lys His Leu Lys 1550 1555 1560Leu Ala Lys Asp Ser Ser Ala Asp Arg Phe Leu Asn Ser Leu Gly 1565 1570 1575Arg Phe Met Thr Gly Leu Asp Thr Lys Asn Asn Val Lys Val Trp 1580 1585 1590Phe Asn Asn Lys Gly Trp His Ala Ile Ser Ser Phe Leu Asn Val 1595 1600 1605Ile Asn Asn Ala Ile Leu Arg Ala Asn Leu Gln Lys Gly Glu Asn 1610 1615 1620Pro Ser His Tyr Gly Ile Thr Ala Phe Asn His Pro Leu Asn Leu 1625 1630 1635Thr Lys Gln Gln Leu Ser Glu Val Ala Leu Met Thr Thr Ser Val 1640 1645 1650Asp Val Leu Val Ser Ile Cys Val Ile Phe Ala Met Ser Phe Val 1655 1660 1665Pro Ala Ser Phe Val Val Phe Leu Ile Gln Glu Arg Val Ser Lys 1670 1675 1680Ala Lys His Leu Gln Phe Ile Ser Gly Val Lys Pro Val Ile Tyr 1685 1690 1695Trp Leu Ser Asn Phe Val Trp Asp Met Cys Asn Tyr Val Val Pro 1700 1705 1710Ala Thr Leu Val Ile Ile Ile Phe Ile Cys Phe Gln Gln Lys Ser 1715 1720 1725Tyr Val Ser Ser Thr Asn Leu Pro Val Leu Ala Leu Leu Leu Leu 1730 1735 1740Leu Tyr Gly Trp Ser Ile Thr Pro Leu Met Tyr Pro Ala Ser Phe 1745 1750 1755Val Phe Lys Ile Pro Ser Thr Ala Tyr Val Val Leu Thr Ser Val 1760 1765 1770Asn Leu Phe Ile Gly Ile Asn Gly Ser Val Ala Thr Phe Val Leu 1775 1780 1785Glu Leu Phe Thr Asp Asn Lys Leu Asn Asn Ile Asn Asp Ile Leu 1790 1795 1800Lys Ser Val Phe Leu Ile Phe Pro His Phe Cys Leu Gly Arg Gly 1805 1810 1815Leu Ile Asp Met Val Lys Asn Gln Ala Met Ala Asp Ala Leu Glu 1820 1825 1830Arg Phe Gly Glu Asn Arg Phe Val Ser Pro Leu Ser Trp Asp Leu 1835 1840 1845Val Gly Arg Asn Leu Phe Ala Met Ala Val Glu Gly Val Val Phe 1850 1855 1860Phe Leu Ile Thr Val Leu Ile Gln Tyr Arg Phe Phe Ile Arg Pro 1865 1870 1875Arg Pro Val Asn Ala Lys Leu Ser Pro Leu Asn Asp Glu Asp Glu 1880 1885 1890Asp Val Arg Arg Glu Arg Gln Arg Ile Leu Asp Gly Gly Gly Gln 1895 1900 1905Asn Asp Ile Leu Glu Ile Lys Glu Leu Thr Lys Ile Tyr Arg Arg 1910 1915 1920Lys Arg Lys Pro Ala Val Asp Arg Ile Cys Val Gly Ile Pro Pro 1925 1930 1935Gly Glu Cys Phe Gly Leu Leu Gly Val Asn Gly Ala Gly Lys Ser 1940 1945 1950Ser Thr Phe Lys Met Leu Thr Gly Asp Thr Thr Val Thr Arg Gly 1955 1960 1965Asp Ala Phe Leu Asn Lys Asn Ser Ile Leu Ser Asn Ile His Glu 1970 1975 1980Val His Gln Asn Met Gly Tyr Cys Pro Gln Phe Asp Ala Ile Thr 1985 1990 1995Glu Leu Leu Thr Gly Arg Glu His Val Glu Phe Phe Ala Leu Leu 2000 2005 2010Arg Gly Val Pro Glu Lys Glu Val Gly Lys Val Gly Glu Trp Ala 2015 2020 2025Ile Arg Lys Leu Gly Leu Val Lys Tyr Gly Glu Lys Tyr Ala Gly 2030 2035 2040Asn Tyr Ser Gly Gly Asn Lys Arg Lys Leu Ser Thr Ala Met Ala 2045 2050 2055Leu Ile Gly Gly Pro Pro Val Val Phe Leu Asp Glu Pro Thr Thr 2060 2065 2070Gly Met Asp Pro Lys Ala Arg Arg Phe Leu Trp Asn Cys Ala Leu 2075 2080 2085Ser Val Val Lys Glu Gly Arg Ser Val Val Leu Thr Ser His Ser 2090 2095 2100Met Glu Glu Cys Glu Ala Leu Cys Thr Arg Met Ala Ile Met Val 2105 2110 2115Asn Gly Arg Phe Arg Cys Leu Gly Ser Val Gln His Leu Lys Asn 2120 2125 2130Arg Phe Gly Asp Gly Tyr Thr Ile Val Val Arg Ile Ala Gly Ser 2135 2140 2145Asn Pro Asp Leu Lys Pro Val Gln Asp Phe Phe Gly Leu Ala Phe 2150 2155 2160Pro Gly Ser Val Leu Lys Glu Lys His Arg Asn Met Leu Gln Tyr 2165 2170 2175Gln Leu Pro Ser Ser Leu Ser Ser Leu Ala Arg Ile Phe Ser Ile 2180 2185 2190Leu Ser Gln Ser Lys Lys Arg Leu His Ile Glu Asp Tyr Ser Val 2195 2200 2205Ser Gln Thr Thr Leu Asp Gln Val Phe Val Asn Phe Ala Lys Asp 2210 2215 2220Gln Ser Asp Asp Asp His Leu Lys Asp Leu Ser Leu His Lys Asn 2225 2230 2235Gln Thr Val Val Asp Val Ala Val Leu Thr Ser Phe Leu Gln Asp 2240 2245 2250Glu Lys Val Lys Glu Ser Tyr Val 2255 2260102261PRTHUMAN 10Met Ala Cys Trp Pro Gln Leu Arg Leu Leu Leu Trp Lys Asn Leu Thr1 5 10 15Phe Arg Arg Arg Gln Thr Cys Gln Leu Leu Leu Glu Val Ala Trp Pro 20 25 30Leu Phe Ile Phe Leu Ile Leu Ile
Ser Val Arg Leu Ser Tyr Pro Pro 35 40 45Tyr Glu Gln His Glu Cys His Phe Pro Asn Lys Ala Met Pro Ser Ala 50 55 60Gly Thr Leu Pro Trp Val Gln Gly Ile Ile Cys Asn Ala Asn Asn Pro65 70 75 80Cys Phe Arg Tyr Pro Thr Pro Gly Glu Ala Pro Gly Val Val Gly Asn 85 90 95Phe Asn Lys Ser Ile Val Ala Arg Leu Phe Ser Asp Ala Arg Arg Leu 100 105 110Leu Leu Tyr Ser Gln Lys Asp Thr Ser Met Lys Asp Met Arg Lys Val 115 120 125Leu Arg Thr Leu Gln Gln Ile Lys Lys Ser Ser Ser Asn Leu Lys Leu 130 135 140Gln Asp Phe Leu Val Asp Asn Glu Thr Phe Ser Gly Phe Leu Tyr His145 150 155 160Asn Leu Ser Leu Pro Lys Ser Thr Val Asp Lys Met Leu Arg Ala Asp 165 170 175Val Ile Leu His Lys Val Phe Leu Gln Gly Tyr Gln Leu His Leu Thr 180 185 190Ser Leu Cys Asn Gly Ser Lys Ser Glu Glu Met Ile Gln Leu Gly Asp 195 200 205Gln Glu Val Ser Glu Leu Cys Gly Leu Pro Arg Glu Lys Leu Ala Ala 210 215 220Ala Glu Arg Val Leu Arg Ser Asn Met Asp Ile Leu Lys Pro Ile Leu225 230 235 240Arg Thr Leu Asn Ser Thr Ser Pro Phe Pro Ser Lys Glu Leu Ala Glu 245 250 255Ala Thr Lys Thr Leu Leu His Ser Leu Gly Thr Leu Ala Gln Glu Leu 260 265 270Phe Ser Met Arg Ser Trp Ser Asp Met Arg Gln Glu Val Met Phe Leu 275 280 285Thr Asn Val Asn Ser Ser Ser Ser Ser Thr Gln Ile Tyr Gln Ala Val 290 295 300Ser Arg Ile Val Cys Gly His Pro Glu Gly Gly Gly Leu Lys Ile Lys305 310 315 320Ser Leu Asn Trp Tyr Glu Asp Asn Asn Tyr Lys Ala Leu Phe Gly Gly 325 330 335Asn Gly Thr Glu Glu Asp Ala Glu Thr Phe Tyr Asp Asn Ser Thr Thr 340 345 350Pro Tyr Cys Asn Asp Leu Met Lys Asn Leu Glu Ser Ser Pro Leu Ser 355 360 365Arg Ile Ile Trp Lys Ala Leu Lys Pro Leu Leu Val Gly Lys Ile Leu 370 375 380Tyr Thr Pro Asp Thr Pro Ala Thr Arg Gln Val Met Ala Glu Val Asn385 390 395 400Lys Thr Phe Gln Glu Leu Ala Val Phe His Asp Leu Glu Gly Met Trp 405 410 415Glu Glu Leu Ser Pro Lys Ile Trp Thr Phe Met Glu Asn Ser Gln Glu 420 425 430Met Asp Leu Val Arg Met Leu Leu Asp Ser Arg Asp Asn Asp His Phe 435 440 445Trp Glu Gln Gln Leu Asp Gly Leu Asp Trp Thr Ala Gln Asp Ile Val 450 455 460Ala Phe Leu Ala Lys His Pro Glu Asp Val Gln Ser Ser Asn Gly Ser465 470 475 480Val Tyr Thr Trp Arg Glu Ala Phe Asn Glu Thr Asn Gln Ala Ile Arg 485 490 495Thr Ile Ser Arg Phe Met Glu Cys Val Asn Leu Asn Lys Leu Glu Pro 500 505 510Ile Ala Thr Glu Val Trp Leu Ile Asn Lys Ser Met Glu Leu Leu Asp 515 520 525Glu Arg Lys Phe Trp Ala Gly Ile Val Phe Thr Gly Ile Thr Pro Gly 530 535 540Ser Ile Glu Leu Pro His His Val Lys Tyr Lys Ile Arg Met Asp Ile545 550 555 560Asp Asn Val Glu Arg Thr Asn Lys Ile Lys Asp Gly Tyr Trp Asp Pro 565 570 575Gly Pro Arg Ala Asp Pro Phe Glu Asp Met Arg Tyr Val Trp Gly Gly 580 585 590Phe Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile Ile Arg Val Leu 595 600 605Thr Gly Thr Glu Lys Lys Thr Gly Val Tyr Met Gln Gln Met Pro Tyr 610 615 620Pro Cys Tyr Val Asp Asp Ile Phe Leu Arg Val Met Ser Arg Ser Met625 630 635 640Pro Leu Phe Met Thr Leu Ala Trp Ile Tyr Ser Val Ala Val Ile Ile 645 650 655Lys Gly Ile Val Tyr Glu Lys Glu Ala Arg Leu Lys Glu Thr Met Arg 660 665 670Ile Met Gly Leu Asp Asn Ser Ile Leu Trp Phe Ser Trp Phe Ile Ser 675 680 685Ser Leu Ile Pro Leu Leu Val Ser Ala Gly Leu Leu Val Val Ile Leu 690 695 700Lys Leu Gly Asn Leu Leu Pro Tyr Ser Asp Pro Ser Val Val Phe Val705 710 715 720Phe Leu Ser Val Phe Ala Val Val Thr Ile Leu Gln Cys Phe Leu Ile 725 730 735Ser Thr Leu Phe Ser Arg Ala Asn Leu Ala Ala Ala Cys Gly Gly Ile 740 745 750Ile Tyr Phe Thr Leu Tyr Leu Pro Tyr Val Leu Cys Val Ala Trp Gln 755 760 765Asp Tyr Val Gly Phe Thr Leu Lys Ile Phe Ala Ser Leu Leu Ser Pro 770 775 780Val Ala Phe Gly Phe Gly Cys Glu Tyr Phe Ala Leu Phe Glu Glu Gln785 790 795 800Gly Ile Gly Val Gln Trp Asp Asn Leu Phe Glu Ser Pro Val Glu Glu 805 810 815Asp Gly Phe Asn Leu Thr Thr Ser Val Ser Met Met Leu Phe Asp Thr 820 825 830Phe Leu Tyr Gly Val Met Thr Trp Tyr Ile Glu Ala Val Phe Pro Gly 835 840 845Gln Tyr Gly Ile Pro Arg Pro Trp Tyr Phe Pro Cys Thr Lys Ser Tyr 850 855 860Trp Phe Gly Glu Glu Ser Asp Glu Lys Ser His Pro Gly Ser Asn Gln865 870 875 880Lys Arg Ile Ser Glu Ile Cys Met Glu Glu Glu Pro Thr His Leu Lys 885 890 895Leu Gly Val Ser Ile Gln Asn Leu Val Lys Val Tyr Arg Asp Gly Met 900 905 910Lys Val Ala Val Asp Gly Leu Ala Leu Asn Phe Tyr Glu Gly Gln Ile 915 920 925Thr Ser Phe Leu Gly His Asn Gly Ala Gly Lys Thr Thr Thr Met Ser 930 935 940Ile Leu Thr Gly Leu Phe Pro Pro Thr Ser Gly Thr Ala Tyr Ile Leu945 950 955 960Gly Lys Asp Ile Arg Ser Glu Met Ser Thr Ile Arg Gln Asn Leu Gly 965 970 975Val Cys Pro Gln His Asn Val Leu Phe Asp Met Leu Thr Val Glu Glu 980 985 990His Ile Trp Phe Tyr Ala Arg Leu Lys Gly Leu Ser Glu Lys His Val 995 1000 1005Lys Ala Glu Met Glu Gln Met Ala Leu Asp Val Gly Leu Pro Ser 1010 1015 1020Ser Lys Leu Lys Ser Lys Thr Ser Gln Leu Ser Gly Gly Met Gln 1025 1030 1035Arg Lys Leu Ser Val Ala Leu Ala Phe Val Gly Gly Ser Lys Val 1040 1045 1050Val Ile Leu Asp Glu Pro Thr Ala Gly Val Asp Pro Tyr Ser Arg 1055 1060 1065Arg Gly Ile Trp Glu Leu Leu Leu Lys Tyr Arg Gln Gly Arg Thr 1070 1075 1080Ile Ile Leu Ser Thr His His Met Asp Glu Ala Asp Val Leu Gly 1085 1090 1095Asp Arg Ile Ala Ile Ile Ser His Gly Lys Leu Cys Cys Val Gly 1100 1105 1110Ser Ser Leu Phe Leu Lys Asn Gln Leu Gly Thr Gly Tyr Tyr Leu 1115 1120 1125Thr Leu Val Lys Lys Asp Val Glu Ser Ser Leu Ser Ser Cys Arg 1130 1135 1140Asn Ser Ser Ser Thr Val Ser Tyr Leu Lys Lys Glu Asp Ser Val 1145 1150 1155Ser Gln Ser Ser Ser Asp Ala Gly Leu Gly Ser Asp His Glu Ser 1160 1165 1170Asp Thr Leu Thr Ile Asp Val Ser Ala Ile Ser Asn Leu Ile Arg 1175 1180 1185Lys His Val Ser Glu Ala Arg Leu Val Glu Asp Ile Gly His Glu 1190 1195 1200Leu Thr Tyr Val Leu Pro Tyr Glu Ala Ala Lys Glu Gly Ala Phe 1205 1210 1215Val Glu Leu Phe His Glu Ile Asp Asp Arg Leu Ser Asp Leu Gly 1220 1225 1230Ile Ser Ser Tyr Gly Ile Ser Glu Thr Thr Leu Glu Glu Ile Phe 1235 1240 1245Leu Lys Val Ala Glu Glu Ser Gly Val Asp Ala Glu Thr Ser Asp 1250 1255 1260Gly Thr Leu Pro Ala Arg Arg Asn Arg Arg Ala Phe Gly Asp Lys 1265 1270 1275Gln Ser Cys Leu Arg Pro Phe Thr Glu Asp Asp Ala Ala Asp Pro 1280 1285 1290Asn Asp Ser Asp Ile Asp Pro Glu Ser Arg Glu Thr Asp Leu Leu 1295 1300 1305Ser Gly Met Asp Gly Lys Gly Ser Tyr Gln Val Lys Gly Trp Lys 1310 1315 1320Leu Thr Gln Gln Gln Phe Val Ala Leu Leu Trp Lys Arg Leu Leu 1325 1330 1335Ile Ala Arg Arg Ser Arg Lys Gly Phe Phe Ala Gln Ile Val Leu 1340 1345 1350Pro Ala Val Phe Val Cys Ile Ala Leu Val Phe Ser Leu Ile Val 1355 1360 1365Pro Pro Phe Gly Lys Tyr Pro Ser Leu Glu Leu Gln Pro Trp Met 1370 1375 1380Tyr Asn Glu Gln Tyr Thr Phe Val Ser Asn Asp Ala Pro Glu Asp 1385 1390 1395Thr Gly Thr Leu Glu Leu Leu Asn Ala Leu Thr Lys Asp Pro Gly 1400 1405 1410Phe Gly Thr Arg Cys Met Glu Gly Asn Pro Ile Pro Asp Thr Pro 1415 1420 1425Cys Gln Ala Gly Glu Glu Glu Trp Thr Thr Ala Pro Val Pro Gln 1430 1435 1440Thr Ile Met Asp Leu Phe Gln Asn Gly Asn Trp Thr Met Gln Asn 1445 1450 1455Pro Ser Pro Ala Cys Gln Cys Ser Ser Asp Lys Ile Lys Lys Met 1460 1465 1470Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu Pro Pro Pro Gln 1475 1480 1485Arg Lys Gln Asn Thr Ala Asp Ile Leu Gln Asp Leu Thr Gly Arg 1490 1495 1500Asn Ile Ser Asp Tyr Leu Val Lys Thr Tyr Val Gln Ile Ile Ala 1505 1510 1515Lys Ser Leu Lys Asn Lys Ile Trp Val Asn Glu Phe Arg Tyr Gly 1520 1525 1530Gly Phe Ser Leu Gly Val Ser Asn Thr Gln Ala Leu Pro Pro Ser 1535 1540 1545Gln Glu Val Asn Asp Ala Ile Lys Gln Met Lys Lys His Leu Lys 1550 1555 1560Leu Ala Lys Asp Ser Ser Ala Asp Arg Phe Leu Asn Ser Leu Gly 1565 1570 1575Arg Phe Met Thr Gly Leu Asp Thr Lys Asn Asn Val Lys Val Trp 1580 1585 1590Phe Asn Asn Lys Gly Trp His Ala Ile Ser Ser Phe Leu Asn Val 1595 1600 1605Ile Asn Asn Ala Ile Leu Arg Ala Asn Leu Gln Lys Gly Glu Asn 1610 1615 1620Pro Ser His Tyr Gly Ile Thr Ala Phe Asn His Pro Leu Asn Leu 1625 1630 1635Thr Lys Gln Gln Leu Ser Glu Val Ala Leu Met Thr Thr Ser Val 1640 1645 1650Asp Val Leu Val Ser Ile Cys Val Ile Phe Ala Met Ser Phe Val 1655 1660 1665Pro Ala Ser Phe Val Val Phe Leu Ile Gln Glu Arg Val Ser Lys 1670 1675 1680Ala Lys His Leu Gln Phe Ile Ser Gly Val Lys Pro Val Ile Tyr 1685 1690 1695Trp Leu Ser Asn Phe Val Trp Asp Met Cys Asn Tyr Val Val Pro 1700 1705 1710Ala Thr Leu Val Ile Ile Ile Phe Ile Cys Phe Gln Gln Lys Ser 1715 1720 1725Tyr Val Ser Ser Thr Asn Leu Pro Val Leu Ala Leu Leu Leu Leu 1730 1735 1740Leu Tyr Gly Trp Ser Ile Thr Pro Leu Met Tyr Pro Ala Ser Phe 1745 1750 1755Val Phe Lys Ile Pro Ser Thr Ala Tyr Val Val Leu Thr Ser Val 1760 1765 1770Asn Leu Phe Ile Gly Ile Asn Gly Ser Val Ala Thr Phe Val Leu 1775 1780 1785Glu Leu Phe Thr Asp Asn Lys Leu Asn Asn Ile Asn Asp Ile Leu 1790 1795 1800Lys Ser Val Phe Leu Ile Phe Pro His Phe Cys Leu Gly Arg Gly 1805 1810 1815Leu Ile Asp Met Val Lys Asn Gln Ala Met Ala Asp Ala Leu Glu 1820 1825 1830Arg Phe Gly Glu Asn Arg Phe Val Ser Pro Leu Ser Trp Asp Leu 1835 1840 1845Val Gly Arg Asn Leu Phe Ala Met Ala Val Glu Gly Val Val Phe 1850 1855 1860Phe Leu Ile Thr Val Leu Ile Gln Tyr Arg Phe Phe Ile Arg Pro 1865 1870 1875Arg Pro Val Asn Ala Lys Leu Ser Pro Leu Asn Asp Glu Asp Glu 1880 1885 1890Asp Val Arg Arg Glu Arg Gln Arg Ile Leu Asp Gly Gly Gly Gln 1895 1900 1905Asn Asp Ile Leu Glu Ile Lys Glu Leu Thr Lys Ile Tyr Arg Arg 1910 1915 1920Lys Arg Lys Pro Ala Val Asp Arg Ile Cys Val Gly Ile Pro Pro 1925 1930 1935Gly Glu Cys Phe Gly Leu Leu Gly Val Asn Gly Ala Gly Lys Ser 1940 1945 1950Ser Thr Phe Lys Met Leu Thr Gly Asp Thr Thr Val Thr Arg Gly 1955 1960 1965Asp Ala Phe Leu Asn Lys Asn Ser Ile Leu Ser Asn Ile His Glu 1970 1975 1980Val His Gln Asn Met Gly Tyr Cys Pro Gln Phe Asp Ala Ile Thr 1985 1990 1995Glu Leu Leu Thr Gly Arg Glu His Val Glu Phe Phe Ala Leu Leu 2000 2005 2010Arg Gly Val Pro Glu Lys Glu Val Gly Lys Val Gly Glu Trp Ala 2015 2020 2025Ile Arg Lys Leu Gly Leu Val Lys Tyr Gly Glu Lys Tyr Ala Gly 2030 2035 2040Asn Tyr Ser Gly Gly Asn Lys Arg Lys Leu Ser Thr Ala Met Ala 2045 2050 2055Leu Ile Gly Gly Pro Pro Val Val Phe Leu Asp Glu Pro Thr Thr 2060 2065 2070Gly Met Asp Pro Lys Ala Arg Arg Phe Leu Trp Asn Cys Ala Leu 2075 2080 2085Ser Val Val Lys Glu Gly Arg Ser Val Val Leu Thr Ser His Ser 2090 2095 2100Met Glu Glu Cys Glu Ala Leu Cys Thr Arg Met Ala Ile Met Val 2105 2110 2115Asn Gly Arg Phe Arg Cys Leu Gly Ser Val Gln His Leu Lys Asn 2120 2125 2130Arg Phe Gly Asp Gly Tyr Thr Ile Val Val Arg Ile Ala Gly Ser 2135 2140 2145Asn Pro Asp Leu Lys Pro Val Gln Asp Phe Phe Gly Leu Ala Phe 2150 2155 2160Pro Gly Ser Val Leu Lys Glu Lys His Arg Asn Met Leu Gln Tyr 2165 2170 2175Gln Leu Pro Ser Ser Leu Ser Ser Leu Ala Arg Ile Phe Ser Ile 2180 2185 2190Leu Ser Gln Ser Lys Lys Arg Leu His Ile Glu Asp Tyr Ser Val 2195 2200 2205Ser Gln Thr Thr Leu Asp Gln Val Phe Val Asn Phe Ala Lys Asp 2210 2215 2220Gln Ser Asp Asp Asp His Leu Lys Asp Leu Ser Leu His Lys Asn 2225 2230 2235Gln Thr Val Val Asp Val Ala Val Leu Thr Ser Phe Leu Gln Asp 2240 2245 2250Glu Lys Val Lys Glu Ser Tyr Val 2255 2260112566DNAHUMAN 11cgtcgccgtc cccgtctcct gccaggcgcg gagccctgcg agccgcgggt gggccccagg 60cgcgcagaca tgggctgctc cgccaaagcg cgctgggctg ccggggcgct gggcgtcgcg 120gggctactgt gcgctgtgct gggcgctgtc atgatcgtga tggtgccgtc gctcatcaag 180cagcaggtcc ttaagaacgt gcgcatcgac cccagtagcc tgtccttcaa catgtggaag 240gagatcccta tccccttcta tctctccgtc tacttctttg acgtcatgaa ccccagcgag 300atcctgaagg gcgagaagcc gcaggtgcgg gagcgcgggc cctacgtgta cagggagtcc 360aggcacaaaa gcaacatcac cttcaacaac aacgacaccg tgtccttcct cgagtaccgc 420accttccagt tccagccctc caagtcccac ggctcggaga gcgactacat cgtcatgccc 480aacatcctgg tcttgggtgc ggcggtgatg atggagaata agcccatgac cctgaagctc 540atcatgacct tggcattcac caccctcggc gaacgtgcct tcatgaaccg cactgtgggt 600gagatcatgt ggggctacaa ggaccccctt gtgaatctca tcaacaagta ctttccaggc 660atgttcccct tcaaggacaa gttcggatta tttgctgagc tcaacaactc cgactctggg 720ctcttcacgg tgttcacggg ggtccagaac atcagcagga tccacctcgt ggacaagtgg 780aacgggctga gcaaggttga cttctggcat tccgatcagt gcaacatgat caatggaact 840tctgggcaaa tgtggccgcc cttcatgact cctgagtcct cgctggagtt ctacagcccg 900gaggcctgcc gatccatgaa gctaatgtac aaggagtcag gggtgtttga aggcatcccc 960acctatcgct tcgtggctcc caaaaccctg tttgccaacg ggtccatcta cccacccaac 1020gaaggcttct gcccgtgcct ggagtctgga attcagaacg tcagcacctg caggttcagt 1080gcccccttgt ttctctccca tcctcacttc ctcaacgccg acccggttct ggcagaagcg 1140gtgactggcc tgcaccctaa ccaggaggca cactccttgt tcctggacat ccacccggtc 1200acgggaatcc ccatgaactg ctctgtgaaa ctgcagctga gcctctacat gaaatctgtc 1260gcaggcattg gacaaactgg gaagattgag cctgtggtcc tgccgctgct ctggtttgca 1320gagagcgggg ccatggaggg ggagactctt cacacattct acactcagct ggtgttgatg 1380cccaaggtga
tgcactatgc ccagtacgtc ctcctggcgc tgggctgcgt cctgctgctg 1440gtccctgtca tctgccaaat ccggagccaa gagaaatgct atttattttg gagtagtagt 1500aaaaagggct caaaggataa ggaggccatt caggcctatt ctgaatccct gatgacatca 1560gctcccaagg gctctgtgct gcaggaagca aaactgtagg gtcctgagga caccgtgagc 1620cagccaggcc tggccgctgg gcctgaccgg ccccccagcc cctacacccc gcttctcccg 1680gactctccca gcagacagcc ccccagcccc acagcctgag cctcccagct gccatgtgcc 1740tgttgcacac ctgcacacac gccctggcac acatacacac atgcgtgcag gcttgtgcag 1800acactcaggg atggagctgc tgctgaaggg acttgtaggg agaggctcgt caacaagcac 1860tgttctggaa ccttctctcc acgtggccca caggctgacc acaggggctg tgggtcctgc 1920gtccccttcc tcgggtgagc ctggcctgtc ccgttcagcc gttgggccag gcttcctccc 1980ctccaaggtg aaacactgca gtcccggtgt ggtggctccc catgcaggac gggccaggct 2040gggagtgccg ccttcctgtg ccaaattcag tggggactca gtgcccaggc cctggcacga 2100gctttggcct tggtctacct gccaggccag gcaaagcgcc tttacacagg cctcggaaaa 2160caatggagtg agcacaagat gccctgtgca gctgcccgag ggtctccgcc caccccggcc 2220ggactttgat ccccccgaag tcttcacagg cactgcatcg ggttgtctgg cgcccttttc 2280ctccagccta aactgacatc atcctatgga ctgagccggc cactctctgg ccgaagtggc 2340gcaggctgtg cccccgagct gcccccaccc cctcacaggg tccctcagat tataggtgcc 2400caggctgagg tgaagaggcc tgggggccct gccttccggg cgctcctgga ccctggggca 2460aacctgtgac ccttttctac tggaatagaa atgagtttta tcatctttga aaaataattc 2520actcttgaag taataaacgt ttaaaaaaat ggaaaaaaaa aaaaaa 256612509PRTHUMAN 12Met Gly Cys Ser Ala Lys Ala Arg Trp Ala Ala Gly Ala Leu Gly Val1 5 10 15Ala Gly Leu Leu Cys Ala Val Leu Gly Ala Val Met Ile Val Met Val 20 25 30Pro Ser Leu Ile Lys Gln Gln Val Leu Lys Asn Val Arg Ile Asp Pro 35 40 45Ser Ser Leu Ser Phe Asn Met Trp Lys Glu Ile Pro Ile Pro Phe Tyr 50 55 60Leu Ser Val Tyr Phe Phe Asp Val Met Asn Pro Ser Glu Ile Leu Lys65 70 75 80Gly Glu Lys Pro Gln Val Arg Glu Arg Gly Pro Tyr Val Tyr Arg Glu 85 90 95Ser Arg His Lys Ser Asn Ile Thr Phe Asn Asn Asn Asp Thr Val Ser 100 105 110Phe Leu Glu Tyr Arg Thr Phe Gln Phe Gln Pro Ser Lys Ser His Gly 115 120 125Ser Glu Ser Asp Tyr Ile Val Met Pro Asn Ile Leu Val Leu Gly Ala 130 135 140Ala Val Met Met Glu Asn Lys Pro Met Thr Leu Lys Leu Ile Met Thr145 150 155 160Leu Ala Phe Thr Thr Leu Gly Glu Arg Ala Phe Met Asn Arg Thr Val 165 170 175Gly Glu Ile Met Trp Gly Tyr Lys Asp Pro Leu Val Asn Leu Ile Asn 180 185 190Lys Tyr Phe Pro Gly Met Phe Pro Phe Lys Asp Lys Phe Gly Leu Phe 195 200 205Ala Glu Leu Asn Asn Ser Asp Ser Gly Leu Phe Thr Val Phe Thr Gly 210 215 220Val Gln Asn Ile Ser Arg Ile His Leu Val Asp Lys Trp Asn Gly Leu225 230 235 240Ser Lys Val Asp Phe Trp His Ser Asp Gln Cys Asn Met Ile Asn Gly 245 250 255Thr Ser Gly Gln Met Trp Pro Pro Phe Met Thr Pro Glu Ser Ser Leu 260 265 270Glu Phe Tyr Ser Pro Glu Ala Cys Arg Ser Met Lys Leu Met Tyr Lys 275 280 285Glu Ser Gly Val Phe Glu Gly Ile Pro Thr Tyr Arg Phe Val Ala Pro 290 295 300Lys Thr Leu Phe Ala Asn Gly Ser Ile Tyr Pro Pro Asn Glu Gly Phe305 310 315 320Cys Pro Cys Leu Glu Ser Gly Ile Gln Asn Val Ser Thr Cys Arg Phe 325 330 335Ser Ala Pro Leu Phe Leu Ser His Pro His Phe Leu Asn Ala Asp Pro 340 345 350Val Leu Ala Glu Ala Val Thr Gly Leu His Pro Asn Gln Glu Ala His 355 360 365Ser Leu Phe Leu Asp Ile His Pro Val Thr Gly Ile Pro Met Asn Cys 370 375 380Ser Val Lys Leu Gln Leu Ser Leu Tyr Met Lys Ser Val Ala Gly Ile385 390 395 400Gly Gln Thr Gly Lys Ile Glu Pro Val Val Leu Pro Leu Leu Trp Phe 405 410 415Ala Glu Ser Gly Ala Met Glu Gly Glu Thr Leu His Thr Phe Tyr Thr 420 425 430Gln Leu Val Leu Met Pro Lys Val Met His Tyr Ala Gln Tyr Val Leu 435 440 445Leu Ala Leu Gly Cys Val Leu Leu Leu Val Pro Val Ile Cys Gln Ile 450 455 460Arg Ser Gln Glu Lys Cys Tyr Leu Phe Trp Ser Ser Ser Lys Lys Gly465 470 475 480Ser Lys Asp Lys Glu Ala Ile Gln Ala Tyr Ser Glu Ser Leu Met Thr 485 490 495Ser Ala Pro Lys Gly Ser Val Leu Gln Glu Ala Lys Leu 500 5051325DNAARTIFICIAL SEQUENCEPrimer 13taagcttggc acggctgtcc aagga 251426DNAARTIFICIAL SEQUENCEPrimer 14acagaattcg ccccggcctg gtacac 261518PRTARTIFICIAL SEQUENCESynthetic Peptide 15Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu1 5 10 15Ala Phe1618PRTARTIFICIAL SEQUENCESynthetic Peptide 16Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu1 5 10 15Ala Phe1767PRTARTIFICIAL SEQUENCESynthetic Peptide 17Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val1 5 10 15Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln 20 25 30Pro Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg 35 40 45Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser 50 55 60Glu Lys Ala65
Patent applications by Brian Y. Ishida, Walnut Creek, CA US
Patent applications by Daniel M. Schwartz, San Francisco, CA US
Patent applications by John P. Kane, Hillsborough, CA US
Patent applications by Kathy R. Bailey, San Francisco, CA US
Patent applications by Keith G. Duncan, San Francisco, CA US
Patent applications in class Liposomes
Patent applications in all subclasses Liposomes