FGF21 COMPOSITIONS FOR TREATMENT OR PREVENTION OF NEOVASCULARIZATION OF THE EYE AND METHODS THEREFOR

Abstract

The instant disclosure provides methods and compositions related to discovery of a long-acting FGF21 as a therapeutic target for treatment or prevention of neovascular eye diseases or disorders that are characterized by angiogenesis, or of vascular diseases of the eye. Therapeutic and/or prophylactic uses and compositions of long-acting FGF21 are described.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U.S. Provisional Application 62/453,352 filed on Feb. 1, 2017 and U.S. Provisional Application 62/595,917 filed on Dec. 7, 2017. The entire contents of these applications are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

[0003] Pathological neovascularization, a leading cause of blindness, is seen in retinopathy of prematurity, diabetic retinopathy and age related macular degeneration. There is currently no cure for retinopathy of prematurity, diabetic retinopathy, and age related macular degeneration. Therefore, there is an unmet need for treatments of retinopathy of prematurity, diabetic retinopathy, and age related macular degeneration that ameliorate and/or prevent pathological neovascularization.

SUMMARY OF THE INVENTION

[0004] The instant disclosure relates, at least in part, to the discovery of FGF21 as a therapeutic target for pathologic vessel growth in patients with neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. In certain aspects, stable and/or stabilized forms of FGF21 (i.e., long-acting FGF21) are used to treat or prevent neovascular eye diseases. In certain aspects of the disclosure, it is also identified that targeting of the FGF21 pathway as described herein can exert a therapeutic effect for neurovascular diseases of the eye such as diabetic retinopathy, retinopathy of prematurity (ROP), retinitis pigmentosa (RP), age-related macular degeneration (AMD) and macular telangiectasia (MacTel). The disclosure provides a method for treating or preventing neovascular eye diseases in a subject, the method involving (a) identifying a subject having or at risk of neovascular eye disease; and (b) administering a long-acting FGF21 composition to the subject, thereby treating or preventing neovascular eye disease in the subject.

[0005] In one aspect, the disclosure provides a method for treating or preventing neovascularization and/or angiogenesis in the eye of a subject, the method involving identifying a subject having or at risk of neovascularization and/or angiogenesis in the eye; and administering a pharmaceutical composition including a stabilized FGF21 agent to the subject, thereby treating or preventing neovascularization and/or angiogenesis in the eye of the subject.

[0006] In one embodiment, the subject has or is at risk of developing neovascular retinopathy. In another embodiment, the subject has or is at risk of developing diabetic retinopathy in Type I or Type II diabetes, retinopathy of prematurity (ROP), retinitis pigmentosa (RP) and/or macular telangiectasia (MacTel).

[0007] In another embodiment, choroidal neovascularization and/or angiogenesis is treated or prevented.

[0008] Optionally, retinal neovascularization and/or angiogenesis in the retinal cells of the eye is treated or prevented.

[0009] In one embodiment, the stabilized FGF21 agent comprises an FGF21 polypeptide conjugated to an antibody scaffold. Optionally, the FGF21 polypeptide is a modified FGF21. In certain embodiments, the modified FGF21 is dHis/Ala129Cys, optionally the modified FGF21 is conjugated at Cys 129 to the antibody scaffold. Optionally, two or more FGF21 polypeptide molecules are conjugated to one antibody scaffold the two or more FGF21 polypeptide molecules can be the same or different from each other. In some embodiments, the antibody scaffold is a CovX-2000 scaffold.

[0010] In certain embodiments, the stabilized FGF21 agent is a long acting FGF21 analog, optionally PF-05231023.

[0011] In one embodiment, the stabilized FGF21 agent possesses a half-life of at least 1.5.times. the half-life of a native FGF21 peptide when assayed for stability under identical conditions, optionally at least 2.times. the half-life of a native FGF21 peptide, optionally at least 3.times. the half-life of a native FGF21 peptide, optionally at least 4.times. the half-life of a native FGF21 peptide, optionally at least 5.times. the half-life of a native FGF21 peptide, optionally at least 8.times. the half-life of a native FGF21 peptide, optionally at least 10.times. the half-life of a native FGF21 peptide, optionally at least 12.times. the half-life of a native FGF21 peptide, optionally at least 15.times. the half-life of a native FGF21 peptide, optionally at least 20.times. the half-life of a native FGF21 peptide, optionally at least 30.times. the half-life of a native FGF21 peptide, optionally at least 40.times. the half-life of a native FGF21 peptide, optionally at least 50.times. the half-life of a native FGF21 peptide, optionally at least 60.times. the half-life of a native FGF21 peptide or optionally at least 70.times. the half-life of a native FGF21 peptide.

[0012] In certain embodiments, the stabilized FGF21 agent possesses a half-life of at least 0.8 h in the circulation of a mammal, optionally at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 7 h, at least 10 h, at least 15 h, at least 20 h, at least 25 h, at least 28 h or at least 30 h in the circulation of a mammal, optionally wherein the mammal is human.

[0013] In one embodiment, the pharmaceutical composition is administered to the eye of the subject, optionally by intravitreal injection.

[0014] In some embodiments, the pharmaceutical composition is administered in an amount sufficient to increase retinal levels of APN in the subject.

[0015] Another aspect of the disclosure provides a pharmaceutical composition for use in treating or preventing neovascularization and/or angiogenesis in the eye of a subject that includes a stabilized FGF21 agent and a pharmaceutically acceptable carrier.

[0016] An additional aspect of the disclosure provides for use of a stabilized FGF21 agent in the preparation of a medicament for treatment or prevention of neovascular retinopathy, diabetic retinopathy in type I diabetes, retinopathy of prematurity (ROP), retinitis pigmentosa (RP) or macular telangiectasia (MacTel) in a subject.

[0017] A further aspect of the disclosure provides a method of treating a subject suffering from diabetic retinopathy, retinitis pigmentosa or age-related macular degeneration involving administering an effective amount of a long acting FGF-21 agent to the subject.

[0018] In one embodiment, the age-related macular degeneration is wet macular degeneration or dry macular degeneration.

[0019] In another embodiment, the diabetic retinopathy is proliferative diabetic retinopathy or non-proliferative diabetic retinopathy.

[0020] In an additional embodiment, the retinitis pigmentosa is autosomal recessive, autosomal dominant or X-linked.

[0021] In certain embodiments, the long acting FGF-21 agent is two .DELTA.His1/A129C modified FGF-21 peptides each conjugated to a Fab region of a humanized IgG1k mAb.

Definitions

[0022] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0023] Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0024] An "agent" is meant any small compound, antibody, nucleic acid molecule, or peptide or fragment thereof.

[0025] As used herein, "Age-related macular degeneration," or "AMD" refers to an eye condition which causes a deterioration or breakdown of the macula, a small spot near the center of the retina and the part of the eye needed for sharp central vision. More specifically, the photoreceptor cells within the macula die off slowly, thus accounting for the progressive loss of vision.

[0026] By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0027] An "agonist" as used herein is a molecule which enhances the biological function of a protein. The agonist may thereby bind to the target protein to elicit its functions. However, agonists which do not bind the protein are also envisioned. The agonist may enhance the biological function of the protein directly or indirectly. Agonists which increase expression of certain genes are envisioned within the scope of particular embodiments of the disclosure. Suitable agonists will be evident to those of skill in the art. For the present disclosure it is not necessary that the agonist enhances the function of the target protein directly. Rather, agonists are also envisioned which stabilize or enhance the function of one or more proteins upstream in a pathway that eventually leads to activation of targeted protein. Alternatively, the agonist may inhibit the function of a negative transcriptional regulator of the target protein, wherein the transcriptional regulator acts upstream in a pathway that eventually represses transcription of the target protein.

[0028] An "antagonist" may refer to a molecule that interferes with the activity or binding of another molecule, for example, by competing for the one or more binding sites of an agonist, but does not induce an active response.

[0029] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0030] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0031] By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0032] The phrase "longer half-life" has its ordinary meaning as understood by persons of skill in the art. Merely by way of example, and by no means as a limitation on the meaning of the term, the following description of the term is informative: any appreciable increase in the length of time in which FGF21 may be circulating and able to bind beta klotho and FGF21 receptors either in vivo or in vitro as compared to the half-life of the native FGF21 alone either in vivo or in vitro.

[0033] The term "macula" refers to a small area within the retina. The macula is the part of the retina that is responsible for central vision, allowing things to be seen clearly. Although only a small part of the retina, the macula is more sensitive to detail than the rest of the retina. Many older people develop macular degeneration as part of the body's natural aging process. Symptoms of macular degeneration include blurriness, dark areas or distortion in central vision, or even permanent loss in central vision. It usually does not affect side or peripheral vision.

[0034] The term "neovascularization" refers to the formation of functional microvascular networks with red blood cell perfusion. Neovascularization differs from angiogenesis in that angiogenesis is mainly characterized by the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels. Choroidal neovascularization is the formation of new microvasculature within the innermost layer of the choroid of the eye.

[0035] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

[0036] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0037] By "photoreceptor cells" refers to the bulk of neurons in the retina. The photoreceptor cells capture light energy units (photons) and register the events as electrical signals of the central nervous system. The signals are then relayed to intermediary layers of neurons in the retina that process and organize the information before it is transmitted along the optic nerve fibers to the brain.

[0038] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0039] By "reference" is meant a standard or control, e.g., a standard or control condition.

[0040] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0041] By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0042] By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

[0043] As used herein, a "stable FGF21" or "stabilized FGF21" refers to a composition that includes an active FGF21 polypeptide and a modification (as compared to a WT, unmodified form of FGF21) that has the effect of producing a form of FGF21 agent that is more protease stable than a corresponding WT, unmodified form of FGF21 (i.e., than native FGF21). A stabilized FGF21 composition of the instant disclosure therefore possess a greater half-life than native FGF21, when assessed for stability in an art-recognized stability assay (e.g., an in vitro stability assay, e.g., in the presence of one or more proteases and/or and in vivo stability assay, e.g., when administered to the plasma, blood, saliva, etc. of a subject). Samples are analyzed for the levels of FGF21 using an ELISA in 96-well microtiter plate coated with an anti-FGF21 mAb.

[0044] In certain embodiments, a "stable FGF21" or "stabilized FGF21" of the instant disclosure can exhibit biological activity over an extended period of time, as compared to a WT, unmodified form of FGF21. For example, a "stable FGF21" or "stabilized FGF21" of the instant disclosure can exhibit biological activity (e.g., a therapeutic and/or prophylactic effect) in a subject to which such stable FGF21 is administered over a period of an hour or more, two hours or more, three hours or more, four hours or more, five hours or more, ten hours or more, twenty hours or more, a day or more, two days or more, three days or more, four days or more, five days or more, a week or more, two weeks or more, or even a month or more after a single administration of the "stable FGF21" or "stabilized FGF21". Optionally, a single dose or multiple doses of the "stable FGF21" or "stabilized FGF21" can be administered to a subject, optionally to achieve dosage to a subject within a desired (e.g., therapeutically effective) range over an extended duration of time.

[0045] A "therapeutically effective amount" is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations.

[0046] As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms (e.g., AMD, MacTel or other angiogenesis-associated disease or disorder of the eye, or of tumors in general) associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0047] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0048] Other features and advantages of the disclosure will be apparent to those skilled in the art from the following detailed description and claims

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIGS. 1A and 1B depict micrographs and charts that demonstrate FGF21 treatment (particularly stabilized/long-acting FGF21 treatment) decreased hypoxia-induced retinal neovascularization, whereas FGF21 deficiency increased hypoxia-induced retinal neovascularization. Quantification of neovascularization in P17 retinal whole mounts was performed in mouse oxygen-induced retinopathy (OIR). FIG. 1A shows micrographs and an accompanying chart demonstrating the extent of neovascularization in retinas treated with native FGF21 (nFGF21), or a long-acting FGF21 analog (PF-05231023), or with vehicle control. n=14-19 retinas per group. One-way ANOVA was performed upon raw results, followed by Bonferroni's multiple comparisons post test. n.s.=no significance. FIG. 1B shows micrographs demonstrating observed neovascularization and quantifying fold changes observed for such neovascularization in retinas of Fgf21.sup.+/+ and Fgf21.sup.-/- mice. n=12-13 retinas per group. Unpaired t test. Whole mount vessels were stained with isolectin (red), and neovascularization was pseudo-colored white. Representative images are shown. Scale bar, 1 mm Data were presented as mean.+-.SEM. Fold-changes were calculated in comparison to the control group.

[0050] FIGS. 2A-2F depict micrographs, charts, and schematics demonstrating that FGF21 suppressed retinal neovascularization via adiponectin (APN) to reduce TNF.alpha.. Quantification of neovascularization in P17 retinal whole mounts was performed after oxygen-induced retinopathy. In whole mounts, vessels were stained with isolectin (red) and neovascularization was pseudo-colored white. Representative images are shown. Scale bar, 1 mm Data was presented as mean.+-.SEM. Unpaired t test. n.s., no significance. Fold of change was calculated. FIG. 2A depicts a graph showing relative mRNA levels in WT P17 normal retinas for FGF21 receptors Fgfr1, Fgfr2, Fgfr3, Fgfr4 and co-receptor .beta.-klotho (Klb) (n=6-8 pooled retinas per group). FIG. 2B depicts a graph demonstrating that in WT P17 hypoxic retinas, long-acting FGF21 agent PF-05231023 exerted an inducing effect on retinal APN (n=6 pooled retinas per group). PF-05231023 was administered from P12-16. FIG. 2C depicts micrographs and charts demonstrating that Apn.sup.-/- mice exhibited increased levels of neovascularization, as compared to WT mice (n=17-20 retinas per group). FIG. 2D depicts micrographs and charts demonstrating that Apn.sup.-/- mice treated with PF-05231023 did not show a significant response to PF-05231023 treatment (n=11-14 retinas per group). FIG. 2E depicts graphs demonstrating the effect of PF-05231023 treatment on Tnf.alpha. levels in WT or Apn.sup.-/- retinas (n=6-8 pooled retinas per group), where WT mice were clearly responsive to PF-05231023 treatment and Apn.sup.-/- retinas exhibited an ablated response. FIG. 2F depicts a schematic of the perceived role of FGF21 modulation in neovascular retinas.

[0051] FIGS. 3A-3D depict micrographs and charts demonstrating that exogenous FGF21 decreased retinal neovascularization in Vldlr.sup.-/- mice. FIG. 3A depicts 3 dimensional (3D) reconstruction of retinal neovessels extending from the outer plexiform layer (OPL) towards the retinal pigment epithelium (RPE). PF-05231023 (i.p. injected daily from P8-15) decreased retinal neovessel extension towards RPE seen with isolectin (red) stained vessels. FIG. 3B depicts representative images of isolectin-stained vessels in retinal whole mounts, and neovessels (lesions) are highlighted in white (bottom) (with photoreceptor layer facing up). Scale bar, 1 mm FIG. 3C shows the result of quantifying number and area (size) of vascular lesions (n=11-18 retinas per group). FIG. 3D demonstrates that in Vldlr.sup.-/- retinas, PF-0523102 exerted significant effects on both Apn and Tnf.alpha. levels (n=4-6 pooled retinas per group). Data was presented as mean.+-.SEM. Unpaired t test. Fold-changes were calculated.

[0052] FIGS. 4A-4D depicts micrographs, charts, and schematics demonstrating that FGF21 administration decreased choroidal neovascularization in mice. FIG. 4A depicts a schematic showing an approach for laser-induced choroidal neovascularization in mice.

[0053] FIG. 4B depicts representative images of isolectin-stained choroidal neovessels, in both vehicle- and PF-05231023-treated mice. The area of induced lesions was quantified (n=6-8 mice per group). Scale bar: 200 .mu.m (top); 50 .mu.m (bottom). FIG. 4C depicts charts that demonstrate the effect of PF-05231023 on Apn and Tnf.alpha. levels in neovascular choroid-retina complexes (n=4-6 pooled retinas per group). FIG. 4D depicts charts that demonstrate the effect of PF-05231023 on Vegfa in the three mouse models of neovascular eye diseases examined (n=6-8 pooled retinas per group). Data were presented as mean.+-.SEM. Unpaired t test. n.s., no significance. Fold-changes were calculated.

[0054] FIG. 5 depicts micrographs and charts demonstrating that intra-vitreal PF-05231023 administration induced a trend of reduction in neovascularization in OIR. In WT mice after P12 intra-vitreal PF-05231023, a trend of reduction of neovascularization was observed. The contralateral eye was injected with vehicle. (n=7 retinas per group). Unpaired t test. See also FIGS. 1A-1B.

[0055] FIGS. 6A-6D depict graphs demonstrating that FGF21 promoted normal retinal revascularization. Quantification of revascularization in P17 retinal whole mounts after oxygen-induced retinopathy was performed. FIG. 6A depicts WT retinas treated with PF-05231023 or vehicle control from P12-P16 (n=14-19 retinas per group). Unpaired t test. FIG. 6B depicts Fgf21.sup.+/+ and Fgf21.sup.-/- retinas (n=12-13 retinas per group). Unpaired t test. FIG. 6C depicts WT and Apn.sup.-/- mice (n=17-20 retinas per group). Unpaired t test. FIG. 6D depicts Apn.sup.-/- mice treated with PF-05231023 (n=11-14 retinas per group). Unpaired t test.

[0056] FIGS. 7A-7D depict micrographs and charts demonstrating that FGF21 receptors and adiponectin colocalized in retinal neovessels. FIG. 7A depicts laser-capture microdissected retinal layers and vessels from normal (N) and oxygen-induced retinopathy (OIR) P17 retinas; localization and quantitation of Fgfr1 and Fgfr3 was performed using qPCR (n=6-8 pooled retinas). Unpaired t test. Scale bar: 20 .mu.m. FIG. 7B depicts immunohistochemistry for adiponectin (green) and lectin (red) in P17 OIR retinal whole mounts. Scale bar: 40 .mu.m. FIG. 7C depicts the effect of PF-05231023 (left) and adipoRon (right) on migrated area in HRMEC would healing assays. One-way ANOVA. FIG. 7D depicts effect of PF-05231023 (left) and adipoRon (right) on HRMEC cell viablity. One-way ANOVA.

[0057] FIGS. 8A-8B depict micrographs and graphs demonstrating the observed impacts of FGF21 deficiency. FIG. 8A depicts that FGF21 deficiency delayed normal retinal vascular development in neonatal mice. FIG. 8B depicts that FGF21 deficiency worsened hyperglycemic retinopathy in neonatal mice.

[0058] FIGS. 9A-9B show micrographs and graphs demonstrating that FGF21 promoted retinal vascular development in hyperglycemic mouse neonates. FIG. 9A shows the effects observed following administration of native FGF21 (nFGF21). FIG. 9B depicts the effects observed after administration of long-acting FGF21 (PF-05231023). As shown, administration of long-acting FGF21 improved retinal vascular growth. Unpaired t test.

[0059] FIGS. 10A-10B depict micrographs and graphs demonstrating that long-acting FGF21 protection against hyperglycemic retinopathy was dependent on adiponectin (APN). FIG. 10A demonstrates that APN deficiency completely abolished long-acting FGF21 effects that would otherwise have been observed upon treatment of hyperglycemic retinopathy. FIG. 10B shows that FGF21 administration increased high-molecular-weight (HMW) and hexamer APN levels in serum. Unpaired t test.

[0060] FIG. 11 demonstrates that long-acting FGF21 protection against the decrease in cone function in retinal degenerating mice (Rd10) modeling retinitis pigmentosa.

[0061] FIG. 12 depicts a polypeptide sequence and structure of an exemplary long-acting FGF21 polypeptide, as depicted in Huang et al., 2013 and described elsewhere herein (SED ID NO.: 1). The sequence of recombinant human .DELTA.His FGF21 (A129C) and a schematic of the bivalent FGF21 CovX-Body, CVX-343, is shown. The underlined C residue corresponds to position 129, where the FGF21 protein is conjugated to the antibody scaffold.

[0062] FIGS. 13A-13D depict graphs showing that serum FGF21 levels were decreased in Akita mice. FIG. 13A shows serum FGF21 levels measured by ELISA. n=6-8 mice per group. FIGS. 13B-13D shows results from qPCR for Fgf21, Fgfr1, .beta.-klotho expression in diabetic WT and Akita retinas. n=3-4 mice per group. Unpaired t test.

[0063] FIGS. 14A-14E depict a series of schematic illustrations and graphs showing that PF-05231023 administration improved retinal function in diabetic Akita mice. FIG. 14A is a schematic illustration of PF-05231023 treatment in 7-to-8-month-old Akita mice. 10 mg/kg PF-05231023 was i.p. injected twice a week for a month. ERG was measured before and after treatment. ERG plots with `white` (for maximal a-wave) and "green" (for maximal b-wave) light stimulation are shown to demonstrate the parameters: a-wave (photoreceptors), b-wave (bipolar cells), oscillitatory potentials (OPs, inner retinal neurons). Photoreceptor amplitude (Rm.sub.P3) and sensitivity (S), bipolar cell response amplitude (Rm.sub.P2) and sensitivity (1/K.sub.P2), inner retinal neuronal saturating energy (Em) and sensitivity (1/i.sub.1/2Em), as well as total retinal sensitivity (Sm) were measured and calculated. FIG. 14B shows representative ERG plots in 7-to-8-month-old WT mice (black), age-matched Akita mice before (blue) and after (orange) PF-05231023 administration. FIG. 14C shows overall changes in different ERG parameters in WT mice, Akita mice before and after PF-05231023 administration. FIG. 14D shows a comparison of retinal sensitivity (Sm) in WT mice, Akita mice before and after PF-05231023 administration. n=5 to 10 mice per group. ANOVA followed by Tukey''s test. FIG. 18E shows plots of ERG parameters in 7-to-8-month-old Akita mice before and after PF-05231023 administration. n=5 mice per group. Paired t test. Data was presented as Mean.+-.SEM.

[0064] FIGS. 15A-15E depict a series of plots photographs and graphs showing that PF-05231023 administration restored the retinal morphology in Akita mice. FIG. 15A shows the correlation of post-receptor cell sensitivity (1/K.sub.P2) with the sum of changes in photoreceptor sensitivity (S.sub.Rod) and saturated amplitude (R.sub.Rod) in Akita mice. n=10 eyes per group. Pearson r test. FIG. 15B shows results from qPCR of Arrestin4 and Rhodopsin in age-matched WT mice and Akita mice treated with either vehicle or PF-05231023. n=3-4 mice per group. ANOVA. FIGS. 15C-15D are photographs showing the immunohistochemistry of cones (cone arrestin, red), rods (rhodopsin, green) and nuclei (DAPI, blue) in age-matched WT mice and Akita mice treated with either vehicle or PF-05231023. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 50 .mu.m. FIG. 19E shows an optical coherence tomography (OCT) for photoreceptor inner and outer segment in age-matched wild-type (WT) mice (black line) and Akita mice treated with either vehicle (blue line) or PF-05231023 (orange line). n=4-10 mice per group. *P<0.05, **P<0.01. ANOVA.

[0065] FIGS. 16A-16B depict graphs showing that PF-05231023 administration decreased IL-1.beta. expression in diabetic retinas. FIG. 16A shows results from qPCR for pro-inflammatory markers (IL-1.beta., Vegfa, Tnf.alpha., IL-6) and anti-inflammatory markers (IL-10, Apn) in WT and Akita mouse retinas. n=3-4 mice per group. FIG. 16B shows results from Akita mouse retinas treated with vehicle (PBS) or PF-05231023. n=3-4 mice per group. Unpaired t test. Data is presented as Mean.+-.SEM.

[0066] FIGS. 17A-17E depict a series of graphs and photographs of Western blots showing that PF-05231023 administration induced NRF2 levels by activating the AKT pathway and decreased photoreceptor-derived IL-1.beta.. FIG. 17A is a Western blot of p-AKT, AKT, NRF2 in non-diabetic WT, diabetic Akita with vehicle or PF-05231023 administration for one month. .beta.-ACTIN was used as internal control. n=3 mice per group. ANOVA. FIG. 17B show results from qPCR of IL-1.beta. in cone photoreceptors in vitro (661W). Oxidative stress was induced with 0.5 mM paraquat (PQ) for 1 hour. The culture medium was changed and cells were treated with 500 ng/ml PF-05231023 or vehicle for 24 hours. n=4 independent replicates. ANOVA followed by Bonferroni's multiple comparisons test. FIG. 17C shows results from qPCR of Nrf2 and Nf.kappa.b in 661W with PQ-induced oxidative stress followed by treatment with 500 ng/ml PF-05231023 or vehicle for 24 hours. n=4 independent replicates. ANOVA followed by Bonferroni's multiple comparisons test. FIG. 17D shows results from qPCR of Nrf2 in 661W with PQ-induced oxidative stress followed by co-treatment with 500 ng/ml PF-05231023 and perifosine at 5 or 10 .mu.M. ANOVA. FIG. 17E is a Western blot of NRF2, p-NF.kappa.B and NF.kappa.B in 661W with PQ-induced oxidative stress followed by treatment with 500 ng/ml PF-05231023 or vehicle for 24 hours. Protein lysate was isolated from 661W. .beta.-ACTIN was used as internal control. Lane only with loading dye was negative control. n=4 independent replicates. ANOVA followed by Bonferroni's multiple comparisons test.

[0067] FIGS. 18A-18I depict a series of plots and graphs showing that PF-05231023 administration protected the retinal function in STZ-induced diabetic mice, independent of APN. FIG. 18A is a schematic of STZ-induced type 1 diabetes in C57BL/6J (WT) mice. STZ was i.p. injected in 6-to-8-week-old WT mice (Day1-2: 60 mg/kg; Day3-5: 55 mg/kg). ERG was compared in diabetic mice before and after PF-05231023 treatment (10 mg/kg i.p., twice a week). FIG. 18B depicts representative ERG plots of 7-to-8-month-old WT control mice, and WT diabetic mice before and after PF-05231023 administration. FIG. 18C depicts the overall changes in different ERG parameters in WT normal mice, WT diabetic mice before and after PF-05231023 administration. FIG. 18D depicts a comparison of retinal sensitivity (Sm) in WT normal mice, WT diabetic mice before and after PF-05231023 administration. n=3 to 5 mice per group. ANOVA followed by Tukey's test. FIG. 18E are plots of retinal sensitivity (Sm) in 7-to-8-month-old WT diabetic mice before and after PF-05231023 administration. n=3 mice per group. Paired t test. FIG. 18F show representative ERG plots of 7-to-8-month-old Apn diabetic mice before and after PF-05231023 administration. FIG. 18G shows the overall changes in different ERG parameters in Apn diabetic mice before and after PF-05231023 administration. FIG. 18H show plots of retinal sensitivity (Sm) in Apn.sup.-/- diabetic mice before and after PF-05231023 administration. n=3 mice per group. Paired t test. FIG. 18I are graphs showing results from qPCR for IL-1.beta. in diabetic WT and Apn.sup.-/- mouse retinas treated with vehicle (PBS) or PF-05231023. n=3 mice per group. Unpaired t test. ERG plots with `white" and "green" light stimulation are shown. Data was presented as Mean.+-.SEM.

[0068] FIG. 19 is a schematic of a flow chart for PF-05231023 protection against DR in type 1 diabetic mice. Schematic of signaling pathway that PF-05231023 may improve retinal neurovascular activity in diabetic mice by activating the AKT pathway and inducing anti-oxidative NRF2, which in turn decreases photoreceptor-derived pro-inflammatory marker IL-1.beta..

[0069] FIG. 20 depicts a graph showing FGF21 receptor Fgfr1 expression in retinal neuronal layers. RGC (retinal ganglion cells), INL (inner nuclear layer), and ONL (outer nuclear layer) were isolated using laser-captured microdissection and RNA was extracted. qPCR of Fgfr1 was conducted.

[0070] FIGS. 21A-21C depict graphs showing that PF-05231023 administration did not change body weight (FIG. 21A), blood glucose levels (FIG. 21B) or serum triglycerides (FIG. 21C) in Akita mice. Body weight was decreased, blood glucose levels and serum triglyceride (TG) levels were induced in diabetic Akita mice versus normal WT mice; PF-05231023 administration lowered blood glucose levels, but did not change the body weight and serum TG levels in Akita mice. Data is presented as Mean.+-.SEM. n=5-12 mice per group. ANOVA followed by Bonferroni's multiple comparisons test. n.s., no significance.

[0071] FIG. 22 is a series of plots and Comparison of ERG parameters in Akita mice measured at 7-month and 8-months of age. n=3 mice per group. ANOVA followed by Tukey's test. Representative ERG plot is shown. Data was presented as Mean.+-.SEM.

[0072] FIGS. 23A-23C are graphs showing that blood glucose levels (FIG. 23B) were increased, body weight (FIG. 23A) and serum TG levels (FIG. 23C) were not changed in STZ-induced diabetic WT mice versus normal WT mice; PF-05231023 administration did not change the body weight, blood glucose levels or serum TG levels in STZ-induced diabetic WT mice. Data is presented as Mean.+-.SEM. n=3-5 mice per group. ANOVA followed by Bonferroni's multiple comparisons test. n.s., no significance.

[0073] FIGS. 24A-24C are graphs showing results from a qPCR of retinal Fgf21 (FIG. 24A), Fgfr1 (FIG. 24B) and Klb (FIG. 24C) in Akita mice with PF-05231023 or vehicle treatment. n=3-4 mice per group. Unpaired t test.

DETAILED DESCRIPTION OF THE INVENTION

[0074] The disclosure is based, at least in part, upon the discovery of FGF21 as a therapeutic target for pathologic vessel growth in patients with neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. In certain aspects of the disclosure, stable forms of FGF21 (i.e., long-acting FGF21) are used to treat neovascular eye diseases. In certain aspects of the disclosure, it is also identified that targeting of the FGF21 pathway as described herein can exert a therapeutic effect for neurovascular diseases of the eye such as diabetic retinopathy, retinopathy of prematurity, retinitis pigmentosa, age-related macular degeneration (AMD) and macular telangiectasia (MacTel). The disclosure provides a method for treating or preventing neovascular eye diseases in a subject, the method involving (a) identifying a subject having or at risk of neovascular eye disease; and (b) administering a long-acting FGF21 composition to the subject, thereby treating or preventing neovascular eye disease in the subject.

[0075] Pathological neovascularization, a leading cause of blindness, is seen in retinopathy of prematurity, diabetic retinopathy and age related macular degeneration. Using a mouse model of hypoxia-driven retinal neovascularization, oxygen-induced retinopathy, it was discovered that fibroblast growth factor 21 (FGF21) administration suppressed, and FGF21 deficiency worsened, retinal neovessel growth. The protective effect of long-acting FGF21 against neovessel growth was abolished in adiponectin (APN)-deficient mice, and long-acting FGF21 administration also decreased neovascular lesions in two models of neovascular age-related macular degeneration, very-low-density-lipoprotein-receptor-deficient mice having retinal angiomatous proliferation and laser-induced choroidal neovascularization. Long-acting FGF21 inhibited Tnf.alpha. expression but did not alter Vegfa expression in neovascular eyes. As described herein, FGF21 (particularly long-acting FGF21) is thereby described as a therapeutic modality for pathologic vessel growth in patients having neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration.

[0076] Additional aspects and embodiments of the invention are described below.

Mechanism of Action

[0077] Without wishing to be bound by theory, the instant disclosure is believed to function in the following manner Pathological retinal neovessel growth is a major cause of vision loss in retinopathy of prematurity in premature infants, in macular telangiectasia, diabetic retinopathy and age-related macular degeneration in adults. Uncontrolled neovessel growth is driven by the need for oxygen and for energy substrates (Hellstrom et al., 2013; Joyal et al., 2016). Current treatments for retinal neovascularization have limitations. Although laser photocoagulation helps preserve central vision, it causes peripheral visual loss (Ciulla et al., 2003). In addition, anti-vascular endothelial growth factor (VEGF) drugs effectively treat neovascularization in some, but not all patients and there are safety concerns about the long-term effects including degeneration of normal blood vessels, neural retina and choroid (Arevalo, 2013; Cheung et al., 2012; Fernando Arevalo, 2013; Osaadon et al., 2014; Sato et al., 2012). Therefore, better therapeutic agents are needed for the effective treatment of vision-threatening neovascularization.

[0078] As described herein, FGF21 suppresses retinal and choroidal ocular pathologic angiogenesis in three different mouse models of disease. In humans, long-acting FGF21 analog administration increases circulating adiponectin (APN) in a dose-dependent manner (Gaich et al., 2013; Talukdar et al., 2016). In mice, FGF21 administration increases APN production to modulate glucose and lipid metabolism (Holland et al., 2013; Lin et al., 2013). Low circulating APN levels may contribute to the development of neovascular eye diseases in humans (Fu et al., 2016; Fu et al., 2015; Kaarniranta et al., 2012; Mao et al., 2012; Omae et al., 2015). APN administration inhibits retinal and choroidal neovascularization in rodents (Higuchi et al., 2009; Lyzogubov et al., 2012). As described herein, FGF21 inhibits pathological retinal and choroidal angiogenesis in the eye and FGF21 administration improves neovascular eye diseases. As described herein, FGF21 has roles in 1) hypoxia-induced neovascular retinopathy (Smith et al., 1994); 2) retinal neovascularization driven by energy deficiency (Joyal et al., 2016); and 3) laser-induced choroidal neovascularization (Gong et al., 2015).

[0079] The compositions and methods of the instant disclosure are expressly contemplated for treatment and/or prevention of at least the following disorders and/or diseases of the eye, either alone or in combination.

Choroidal Neovascularization (CNV)

[0080] Choroidal neovascularization (CNV) is the creation of new blood vessels in the choroid layer of the eye. CNV can occur rapidly in individuals with defects in Bruch's membrane, the innermost layer of the choroid. It is also associated with excessive amounts of vascular endothelial growth factor (VEGF). In addition to wet macular degeneration, CNV can also occur frequently with the rare genetic disease pseudoxanthoma elasticum and rarely with the more common optic disc drusen. CNV has also been associated with extreme myopia or malignant myopic degeneration, where in choroidal neovascularization occurs primarily in the presence of cracks within the retinal (specifically) macular tissue known as lacquer cracks. CNV can create a sudden deterioration of central vision, noticeable within a few weeks. Other symptoms which can occur include colour disturbances, and metamorphopsia (distortions in which straight lines appears wavy). Hemorrhaging of the new blood vessels can accelerate the onset of symptoms of CNV. CNV may also include the feeling of pressure behind a subject's eye.

Age-Related Macular Degeneration (AMD)

[0081] AMD is a common eye condition and a leading cause of vision loss among people age 50 and older. It causes damage to the macula, a small spot near the center of the retina and the part of the eye needed for sharp, central vision, which lets us see objects that are straight ahead. In some people, AMD advances so slowly that vision loss does not occur for a long time. In others, the disease progresses faster and may lead to a loss of vision in one or both eyes. As AMD progresses, a blurred area near the center of vision is a common symptom. Over time, the blurred area may grow larger or you may develop blank spots in your central vision. Objects also may not appear to be as bright as they used to be.

[0082] AMD by itself does not lead to complete blindness, with no ability to see. However, the loss of central vision in AMD can interfere with simple everyday activities, such as the ability to see faces, drive, read, write, or do close work, such as cooking or fixing things around the house.

Macula

[0083] The macula is made up of millions of light-sensing cells that provide sharp, central vision. It is the most sensitive part of the retina, which is located at the back of the eye. The retina turns light into electrical signals and then sends these electrical signals through the optic nerve to the brain, where they are translated into the images we see. When the macula is damaged, the center of your field of view may appear blurry, distorted, or dark.

MacTel (macular telangiectasia)

[0084] Macular telangiectasia is a disease in which the macula is affected, causing a loss of central vision. The macula is a small area in the retina (the light-sensitive tissue lining the back of the eye) that is responsible for central vision, allowing fine details to be seen clearly. Macular telangiectasia develops when there are problems with the tiny blood vessels around the fovea, the center of the macula. There are two types of macular telangiectasia (Type 1 and Type 2), and each affects the blood vessels differently. Macular telangiectasia may occur as a result of a retinal vascular disease or a systemic disease such as diabetes or hypertension, but in many cases, clinical findings reveal no known cause.

[0085] One serious complication of macular telangiectasia is the development of abnormal blood vessels under the retina. This is called choroidal neovascularization, and may call for injections of a drug called vascular endothelial growth factor inhibitors (anti-VEGF). Anti-VEGF medication targets a specific chemical in the eye that causes abnormal blood vessels to grow under the retina. That chemical is called vascular endothelial growth factor, or VEGF. Blocking VEGF with medication injections reduces the growth of abnormal blood vessels, slows their leakage, helps to reduce swelling of the retina, and in some cases may improve vision.

Type 1 Macular Telangiectasia

[0086] In Type 1 macular telangiectasia, the blood vessels become dilated forming tiny aneurysms, causing swelling and damaging macular cells. The disease almost always occurs in one eye, which differentiates it from Type 2.

Type 2 Macular Telangiectasia

[0087] The most common form of macular telangiectasia is Type 2 macular telangiectasia, in which the tiny blood vessels around the fovea leak, become dilated (widen), or both. It is a bilateral disease of unknown cause, which characteristic alterations of macular capillary network and neurosensory atrophy. In some cases, new blood vessels form under the retina and they can also break or leak. Fluid from leaking blood vessels causes the macula to swell or thicken, a condition called macular edema, which affects central vision. Also, scar tissue can sometimes form over the macula and the fovea, causing loss of detail vision. Type 2 affects both eyes but not necessarily with the same severity.

Retinopathy of Prematurity (ROP)

[0088] Retinopathy of prematurity (ROP) is a potentially blinding eye disorder that primarily affects premature infants weighing about 23/4 pounds (1250 grams) or less that are born before 31 weeks of gestation (A full-term pregnancy has a gestation of 38-42 weeks). The smaller a baby is at birth, the more likely that baby is to develop ROP. This disorder--which usually develops in both eyes--is one of the most common causes of visual loss in childhood and can lead to lifelong vision impairment and blindness. ROP was first diagnosed in 1942.

[0089] With advances in neonatal care, smaller and more premature infants are being saved. These infants are at a much higher risk for ROP. Not all babies who are premature develop ROP. There are approximately 3.9 million infants born in the U.S. each year; of those, about 28,000 weigh 23/4 pounds or less. About 14,000-16,000 of these infants are affected by some degree of ROP. The disease improves and leaves no permanent damage in milder cases of ROP. About 90 percent of all infants with ROP are in the milder category and do not need treatment. However, infants with more severe disease can develop impaired vision or even blindness. About 1,100-1,500 infants annually develop ROP that is severe enough to require medical treatment. About 400-600 infants each year in the US become legally blind from ROP.

[0090] ROP is classified in five stages, ranging from mild (stage I) to severe (stage V):

Stage I--Mildly abnormal blood vessel growth. Many children who develop stage I improve with no treatment and eventually develop normal vision. The disease resolves on its own without further progression. Stage II--Moderately abnormal blood vessel growth. Many children who develop stage II improve with no treatment and eventually develop normal vision. The disease resolves on its own without further progression. Stage III--Severely abnormal blood vessel growth. The abnormal blood vessels grow toward the center of the eye instead of following their normal growth pattern along the surface of the retina. Some infants who develop stage III improve with no treatment and eventually develop normal vision. However, when infants have a certain degree of Stage III and "plus disease" develops, treatment is considered. "Plus disease" means that the blood vessels of the retina have become enlarged and twisted, indicating a worsening of the disease. Treatment at this point has a good chance of preventing retinal detachment. Stage IV--Partially detached retina. Traction from the scar produced by bleeding, abnormal vessels pulls the retina away from the wall of the eye. Stage V--Completely detached retina and the end stage of the disease. If the eye is left alone at this stage, the baby can have severe visual impairment and even blindness. Most babies who develop ROP have stages I or II. However, in a small number of babies, ROP worsens, sometimes very rapidly. Untreated ROP threatens to destroy vision.

[0091] Infants with ROP are considered to be at higher risk for developing certain eye problems later in life, such as retinal detachment, myopia (nearsightedness), strabismus (crossed eyes), amblyopia (lazy eye), and glaucoma. In many cases, these eye problems can be treated or controlled.

[0092] ROP occurs when abnormal blood vessels grow and spread throughout the retina, the tissue that lines the back of the eye. These abnormal blood vessels are fragile and can leak, scarring the retina and pulling it out of position. This causes a retinal detachment. Retinal detachment is the main cause of visual impairment and blindness in ROP.

[0093] Several complex factors may be responsible for the development of ROP. The eye starts to develop at about 16 weeks of pregnancy, when the blood vessels of the retina begin to form at the optic nerve in the back of the eye. The blood vessels grow gradually toward the edges of the developing retina, supplying oxygen and nutrients. During the last 12 weeks of a pregnancy, the eye develops rapidly. When a baby is born full-term, the retinal blood vessel growth is mostly complete (The retina usually finishes growing a few weeks to a month after birth). But if a baby is born prematurely, before these blood vessels have reached the edges of the retina, normal vessel growth may stop. The edges of the retina--the periphery--may not get enough oxygen and nutrients.

[0094] It is believed that the periphery of the retina then sends out signals to other areas of the retina for nourishment. As a result, new abnormal vessels begin to grow. These new blood vessels are fragile and weak and can bleed, leading to retinal scarring. When these scars shrink, they pull on the retina, causing it to detach from the back of the eye.

[0095] To date, the most effective proven treatments for ROP are laser therapy or cryotherapy. Laser therapy "burns away" the periphery of the retina, which has no normal blood vessels. With cryotherapy, physicians use an instrument that generates freezing temperatures to briefly touch spots on the surface of the eye that overlie the periphery of the retina. Both laser treatment and cryotherapy destroy the peripheral areas of the retina, slowing or reversing the abnormal growth of blood vessels. Unfortunately, the treatments also destroy some side vision. This is done to save the most important part of our sight--the sharp, central vision we need for "straight ahead" activities such as reading, sewing, and driving.

[0096] Both laser treatments and cryotherapy are performed only on infants with advanced ROP, particularly stage III with "plus disease." Both treatments are considered invasive surgeries on the eye, and doctors don't know the long-term side effects of each.

[0097] In the later stages of ROP, other treatment options include:

[0098] Scleral buckle. This involves placing a silicone band around the eye and tightening it. This keeps the vitreous gel from pulling on the scar tissue and allows the retina to flatten back down onto the wall of the eye. Infants who have had a sclera buckle need to have the band removed months or years later, since the eye continues to grow; otherwise they will become nearsighted. Sclera buckles are usually performed on infants with stage IV or V.

[0099] Vitrectomy. Vitrectomy involves removing the vitreous and replacing it with a saline solution. After the vitreous has been removed, the scar tissue on the retina can be peeled back or cut away, allowing the retina to relax and lay back down against the eye wall. Vitrectomy is performed only at stage V.

[0100] While ROP treatment decreases the chances for vision loss, it does not always prevent it. Not all babies respond to ROP treatment, and the disease may get worse. If treatment for ROP does not work, a retinal detachment may develop. Often, only part of the retina detaches (stage IV). When this happens, no further treatments may be needed, since a partial detachment may remain the same or go away without treatment. However, in some instances, physicians may recommend treatment to try to prevent further advancement of the retinal detachment (stage V). If the center of the retina or the entire retina detaches, central vision is threatened, and surgery may be recommended to reattach the retina.

Diabetic Retinopathy

[0101] Diabetic retinopathy describes a diabetic eye disease that affects blood vessels in the retina and is both the most common cause of vision loss among people with diabetes and the leading cause of vision impairment and blindness among working-age adults. Chronically high blood sugar from diabetes is associated with damage to the blood vessels in the retina, thereby leading to diabetic retinopathy. This changes the curvature of the lens and results in the development of symptoms of blurred vision. The blurring of distance vision as a result of lens swelling will subside once the blood sugar levels are brought under control. Better control of blood sugar levels in patients with diabetes also slows the onset and progression of diabetic retinopathy. Symptoms of diabetic retinopathy may include seeing spots or floaters in a subject's field of vision, blurred vision, having a dark or empty spot in the center of a subject's vision, and difficulty seeing well at night. Diabetic retinopathy may progress through four stages:

[0102] I. Mild nonproliferative retinopathy: small areas of swelling in the retinal blood vessels causing tiny bulges, called microaneurysms to protrude from their walls may occur.

[0103] II. Moderate nonproliferative retinopathy: progression of the disease may lead to blood vessels swelling and distorting, therefore affecting their ability to transport blood.

[0104] III. Severe nonproliferative retinopathy: more blood vessels become blocked, depriving the blood supply to areas of the retina.

[0105] IV. Proliferative diabetic retinopathy: advanced stage of the disease where growth factors secreted by the retina trigger the proliferation of new blood vessels, which grow along the inside surface of the retina and into the fluid that fills the eye. The fragility of the new blood vessels makes them more likely to leak and bleed. Scar tissue can cause retinal detachment (pulling away of the retina from underlying tissue). Retinal detachment can lead to permanent vision loss.

Retinitis Pigmentosa (RP)

[0106] Retinitis pigmentosa (RP) is an inherited, degenerative eye disease that causes severe vision impairment due to the progressive degeneration of the rod photoreceptor cells in the retina. This form of retinal dystrophy manifests initial symptoms independent of age--diagnosis occurs from early infancy to late adulthood. Patients in the early stages of RP first notice compromised peripheral and dim light vision due to the decline of the rod photoreceptors. The progressive rod degeneration is later followed by abnormalities in the adjacent retinal pigment epithelium (RPE) and the deterioration of cone photoreceptor cells. As peripheral vision becomes increasingly compromised, patients experience progressive "tunnel vision" and eventual blindness. Afflicted individuals may additionally experience the accumulation of bone spicules in the fundus, defective light-dark adaptations, and nyctalopia (night blindness).

[0107] The initial retinal degenerative symptoms of Retinitis Pigmentosa are characterized by decreased night vision (nyctalopia) and the loss of the mid-peripheral visual field. The rod photoreceptor cells, which are responsible for low-light vision and are orientated in the retinal periphery, are the retinal processes affected first during non-syndromic forms of this disease. Visual decline progresses relatively quickly to the far peripheral field, eventually extending into the central visual field as tunnel vision increases. Visual acuity and color vision can become compromised due to accompanying abnormalities in the cone photoreceptor cells, which are responsible for color vision, visual acuity, and sight in the central visual field. The progression of disease symptoms occurs in a symmetrical manner, with both the left and right eyes experiencing symptoms at a similar rate.

[0108] A variety of indirect symptoms characterize Retinitis Pigmentosa along with the direct effects of the initial rod photoreceptor degeneration and later cone photoreceptor decline. Phenomena such as photophobia, which describes the event in which light is perceived as an intense glare, and photopsia, the presence of blinking or shimmering lights within the visual field, often manifest during the later stages of RP. Findings related to RP have often been characterized in the fundus of the eye as the Ophthalamic triad. This includes the development of a mottled appearance of the retinal pigment epithelium (RPE) caused by bone spicule formation, a waxy appearance of the optic nerve, and the attentuation of blood vessels in the retina.

[0109] A variety of retinal molecular pathway defects have been matched to multiple known RP gene mutations. Mutations in the rhodopsin gene, which is responsible for the majority of autosomal-dominantly inherited RP cases, disrupts the rod-opsin protein essential for translating light into decipherable electrical signals within the phototransduction cascade of the central nervous system. Defects in the activity of this G-protein-coupled receptor are classified into distinct classes that depend on the specific folding abnormality and the resulting molecular pathway defects. The Class I mutant protein's activity is compromised as specific point mutations in the protein-coding amino acid sequence affect the pigment protein's transportation into the outer segment of the eye, where the phototransduction cascade is localized. Additionally, the misfolding of Class II rhodopsin gene mutations disrupts the protein's conjunction with 11-cis-retinal to induce proper chromophore formation. Additional mutants in this pigment-encoding gene affect protein stability, disrupt mRNA integrity post-translationally, and affect the activation rates of transducin and opsin optical proteins. Additionally, animal models suggest that the retinal pigment epithelium fails to phagocytose the outer rod segment discs that have been shed, leading to an accumulation of outer rod segment debris. In mice that are homozygous recessive for retinal degeneration mutation, rod photoreceptors stop developing and undergo degeneration before cellular maturation completes. A defect in cGMP-phosphodiesterase has also been documented; this leads to toxic levels of cGMP.

Fibroblast Growth Factor 21 (FGF21)

[0110] Fibroblast growth factor 21 is a protein that in mammals is encoded by the FGF21 gene. The protein encoded by this gene is a member of the fibroblast growth factor (FGF) family and specifically a member of the "endocrine" subfamily which includes FGF23 and FGF15/19. FGF family members possess broad mitogenic and cell survival activities and are involved in a variety of biological processes including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. FGFs act through a family of four FGF receptors. Binding is complicated and requires both interaction of the FGF molecule with an FGF receptor and binding to heparin through an heparin binding domain. Endocrine FGFs lack a heparin binding domain and thus can be released into the circulation. FGF21 action through one of the FGF21 receptors thus requires interaction with a co-receptor, designated .beta.-klotho.

[0111] FGF21 is specifically induced by HMGCS2 activity. The oxidized form of ketone bodies (acetoacetate) in a cultured medium also induced FGF21, possibly via a SIRT1-dependent mechanism. HMGCS2 activity has also been shown to be increased by deacetylation of lysines 310, 447, and 473 via SIRT3 in the mitochondria. While FGF21 is expressed in numerous tissues, including liver, brown adipose tissue, white adipose tissue and pancreas, circulating levels of FGF21 are derived specifically from the liver in mice. In liver FGF21 expression is regulated by PPAR.alpha. and levels rise substantially with both fasting and consumption of ketogenic diets. LXR represses FGF21 in humans via an LXR response element located from -37 to -22 bp on the human FGF21 promoter.

[0112] FGF21 stimulates glucose uptake in adipocytes. This effect is additive to the activity of insulin. FGF21 treatment of adipocytes is associated with phosphorylation of FRS2, a protein linking FGF receptors to the Ras/MAP kinase pathway. FGF21 injection in ob/ob mice results in an increase in Glut1 in adipose tissue. FGF21 also protects animals from diet-induced obesity when overexpressed in transgenic mice and lowers blood glucose and triglyceride levels when administered to diabetic rodents. Treatment of animals with FGF21 results in increased energy expenditure, fat utilization and lipid excretion.

[0113] Serum FGF-21 levels were significantly increased in patients with type 2 diabetes mellitus (T2DM) which may indicate a role in the pathogenesis of T2DM. Elevated levels also correlate with liver fat content in non-alcoholic fatty liver disease and positively correlate with BMI in humans suggesting obesity as a FGF21-resistant state.

[0114] FGF21 stimulates phosphorylation of fibroblast growth factor receptor substrate 2 and ERK1/2 in the liver. Acute FGF21 treatment induced hepatic expression of key regulators of gluconeogenesis, lipid metabolism, and ketogenesis including glucose-6-phosphatase, phosphoenol pyruvate carboxykinase, 3-hydroxybutyrate dehydrogenase type 1, and carnitine palmitoyltransferase 1.alpha.. In addition, injection of FGF21 was associated with decreased circulating insulin and free fatty acid levels. FGF21 treatment induced mRNA and protein expression of PGC-1.alpha., but in mice PGC-1.alpha. expression was not necessary for the effect of FGF21 on glucose metabolism.

[0115] In mice FGF21 is strongly induced in liver by prolonged fasting via PPAR-alpha and in turn induces the transcriptional coactivator PGC-1.alpha. and stimulates hepatic gluconeogenesis, fatty acid oxidation, and ketogenesis. FGF21 also blocks somatic growth and sensitizes mice to a hibernation-like state of torpor, playing a key role in eliciting and coordinating the adaptive starvation response. FGF21 expression is also induced in white adipose tissue by PPAR-gamma, which may indicate it also regulates metabolism in the fed state.

[0116] Activation of AMPK and SIRT1 by FGF21 in adipocytes enhanced mitochondrial oxidative capacity as demonstrated by increases in oxygen consumption, citrate synthase activity, and induction of key metabolic genes. The effects of FGF21 on mitochondrial function require serine/threonine kinase 11 (STK11/LKB1), which activates AMPK. Inhibition of AMPK, SIRT1, and PGC-1.alpha. activities attenuated the effects of FGF21 on oxygen consumption and gene expression, indicating that FGF21 regulates mitochondrial activity and enhances oxidative capacity through an LKB1-AMPK-SIRT1-PGC-1.alpha.-dependent mechanism in adipocytes, resulting in increased phosphorylation of AMPK, increased cellular NAD+ levels and activation of SIRT1 and deacetylation of SIRT1 targets PGC-1.alpha. and histone 3.

Stabilized FGF21

[0117] Modifications to FGF21 stabilize the protein and increase the half-life compared to native FGF21 without any loss in efficacy or potency. A stabilized or long-acting FGF21, as described herein, comprises an FGF21 polypeptide conjugated to an antibody scaffold. In certain embodiments, the modified FGF21 is dHis/Ala129Cys, optionally the modified FGF21 is conjugated at Cys 129 to the antibody scaffold. Optionally, two or more FGF21 polypeptide molecules are conjugated to one antibody scaffold--the two or more FGF21 polypeptide molecules can be the same or different from each other. In some embodiments, the antibody scaffold is a humanized IgG1K monoclonal antibody. In other embodiments, the antibody scaffold is the Fab region of a humanized IgG1K monoclonal antibody. In some embodiments, the antibody scaffold is a CovX-2000 scaffold. In certain embodiments, the stabilized FGF21 agent is a long acting FGF21 analog, optionally PF-05231023.

[0118] In some embodiments, a stabilized FGF21 agent (i.e., long-acting FGF21) possesses a half-life of at least 1.5.times., at least 2.times., at least 3.times., at least 4.times., at least 5.times., at least 8.times., at least 10.times., at least 12.times., at least 15.times., at least 20.times., at least 30.times., at least 40.times., at least 50.times., at least 60.times., or at least 70.times. the half-life of a native FGF21 peptide, when assayed for stability under identical conditions. In other embodiments, a stabilized FGF21 agent (i.e., long-acting FGF21) possesses a half-life of at least 0.8 h, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 7 h, at least 10 h, at least 15 h, at least 20 h, at least 25 h, at least 28 h or at least 30 h in the circulation of a mammal, optionally wherein the mammal is human.

[0119] As described herein, the stability or half-life of a modified FGF21 (e.g., long-acting FGF21) composition may be determined by assessing the degree of resistance of such a composition to protease digestion, as compared to native FGF21 compositions, in vitro, in vivo, or both. Optionally, stability may be assessed by collecting blood and/or serum from subjects previously administered a candidate long-acting FGF21 agent and detecting the presence of the long-acting FGF21 agent with anti-FGF21 antibodies in such samples at different time points. Serum samples may be prepared and analyzed using an ELISA-based assay in which long-acting FGF21 compounds are captured in 96 well microtiter plates coated with anti-FGF21 mAb followed by detection with an anti-hIgG-horseradish peroxidase (HRP). Alternatively, the activity of a candidate long-acting FGF21 agent can also be assessed as a proxy for the continued presence of the long-acting FGF21 agent in the sample, at one or more time points post-administration.

[0120] Exemplary forms of long-acting FGF21 can be found in the art, for example as described in U.S. Pat. No. 9,163,277, issued Oct. 20, 2015; U.S. Pat. No. 8,722,622, issued May 13, 2014; U.S. Provisional Patent Application 61/644,831, filed May 9, 2012; Huang et al., (2013) Journal of Pharmacology and Experimental Therapeutics, 346:270-280; Weng et al., (2015) PLOS One 10(3) e0119104:1-18; Thompson et al., (2016) J. Pharmacokinet Pharmacodyn 43:411-425; and Talukdar et al., (2016) Cell Metabolism 23: 427-440. The disclosure of each of these publications is incorporated herein by reference in its entirety.

[0121] In certain embodiments, as depicted in FIG. 12, the long-acting FGF21 comprises the following polypeptide sequence (Huang et al., 2013):

TABLE-US-00001 (SEQ ID NO: 1) PIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLE DGYNVYQSEAHGLPLHLPGNKSPHRDPCPRGPARFLPLPGLPPALPEPPG ILAPQPPDVGSSDPLSMVGPSQGRSPSYAS..

[0122] For example, a provided long acting FGF21 may be a disclosed FGF21 peptide sequence such as the above, covalently attached to a combining site of an antibody or an antigen binding portion of a scaffold antibody (such as a humanized IgG1k mAb, or e.g., an aldolase catalytic antibody) via a chemical linker (e.g., 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(2-(3-oxo-3-((4-(3-oxo-3-(2- -oxoazetidin-1-yl)propyl)phenyl)amino)propoxy)ethoxy)ethyl]propanamide), wherein for example, the linker is covalently attached to a FGF21 peptide sequence through the thiol group of a cysteine in the FGF21 peptide sequence (e.g., A129C) and the linker is conjugated to the Fab region of the antibody. In some embodiments, two FGF21 peptide sequences (such as disclosed herein) are conjugated to a scaffold antibody (e.g., as depicted in FIG. 12, e.g., a FGF21 homologue (e.g., a modified FGF21)-linker-Ab-linker-FGF21 homologue. In other embodiments a FGF21 peptide sequence and an Exendin4 peptide sequence are conjugated to an antibody.

[0123] For example, provided herein is a long acting FGF21 comprising:

##STR00001##

[0124] In other embodiments, long acting FGF21 forms may include: pegylated FGF21 (e.g, that includes a FGFR21 with a R131AcF modification, coupled to PEG); modified FGF21 proteins with e.g., a FGF21 L118C, A1343C and/or a S167A modification, which has the effect, for example, to increase stability and/or limit or prevent glycosylation in yeast; and Fc-FGF21 fusion constructs ((e.g, with FGF21 modifications L98R and/or P171G, for example, a fusion protein having an IgG constant domain, a FGF21 protein or modified protein and linker sequence associated with the IgG constant domain FGF21 protein). Contemplated long acting FGF21 forms may include FGF21 mimetics, such as FGFR1b/1c agonist antibodies; mimAB1agonist antibodies, and FGF1c/.beta.-klothobispecific.

[0125] Exemplary wild type FGF21 human and mouse mRNA and protein sequences include:

TABLE-US-00002 Human FGF21 gene, complete cds (GenBank Accession No. DQ847413; SEQ ID NO: 2) CACAGCACAGCAGGATGACTGCGGGCAGGCCTAGATAATACCCAGCCTCCCACA AGAAGCTGGTGGAGCAGAGTGTTCCCTGACTCCTCCAAGAAAAGGAGATTCCCT TTCGTGGTCTGCTGAGTAACGGGTGCCTTCCCAGACACTGGCGTTCCCGCTTGAC CAAGGAGCCCTCAAGAGGCCCTTATGCCGGTGTGACAGAAGGCTCACCTCTTGC CTTCTAGGTCACTTCTCACAATGTCCCTTCAGTACCTGACCCTATACCCACCGGTT GTTTCCTGGTTATATTAGTTATACAACAAAGAATAAAAGTAATAGCTAATGATTA ATAATGTTTACACTAATGATTGATACTGTCCATGATCATCTCTATATCTAATTTGT ATGATAACTATTCTTATTCTAACTATTTTCTTTATTATACTGAAACAGTTTGTGCC TTCAGTCTCTTGCCTCGGCACCTGGGTAATCCTTCCCCACAGACTGACCCTCCCAT TCAAGATACATCAATGTCAAAGACTCAGGAGTTTGACTTGATTCCCAGAAGTTTA ACCATCATCTCCCCAGGCTCGGGACTCCCAGCACCCAGACCCTTCTGCTCACACC CAGCAGTCCAGGCCCCCAGACCCTCCTCCCTCAGACTTAGGAGTCCAGGCTCCCG GCCCCTCCTTCCTCAGACCCAGGAGTCCAAGCCCCCTGCCCCTCCTTCCTCAGAC CCAGGAGTCCAGGACCCCAGCCCCTCCTTCCTCACACCCACGAGTCCAGATCCCT AGCCCCTACTCCCTCAGACCCAGGAGTCCAGACCAAAGCTCCCTCCTCCCTCAGA CCCAGGAGCCCAAGTTCCCCAGCCCCTCCTCCCTCAGATCCAGGAGTACAGGCCC AGCCCCTCCTCCCTCAGACCCCTCCTCCCTCAGATCCAGGAGTACAGGCCCAGAC CCTCCTCCCTCAGACCCAGGAGTCCAGGCCCCCCACCCCTCCTCCCTCAGACCCA GGAGTCCAGAGCCCCAGCCCTCCTCCCTCAGACACAGAAGGCCTACCCTTGCACC CTTAGGGGCTCCAGGAAATTAGCCAACCTGTCTTCCCTCTGGGTGCCCACTCCAG GGCCTGGCTTGGCTGCCAACTCCAGTCAGGGACTTTCAGCCACCCCTCCCCCCAG GTTATTTCAGGAGCACCTGCCTGGGCCTGGGATGGCTTCTCTGGTGAAAGAAACA CCAGGATTGCATCAGGGAGGAGGAGGCTGGGATGTCCAGGGTCTGAGCATCTGA GCAGGGACAGATGAGGTTGAGGTTGGCCCACGGCCAGGTGAGAGGCTTCCAAGG CAGGATACTTGTGTCTCAGATGCGGTCGCTTCTTTCATACAGCAATTGCCGCCTT GCTGAGGATCAAGGAACCTCAGTGTCAGATCACGCCCTCCCCCCAAACTTAGAA ATTCAGATGGGGCGCAGAAATTTCTCTTGTTCTGCGTGATCTGCATAGATGGTCC AAGAGGTGGTTTTTCCAGGAGCCCAGCACCCCTCCTCCCTCCGACTCAGGTGCTT GAGACCCCAGATCCTTCTCTCTGAGACTCAGGAATGTGGGCCCCCAGCCCCTTTC ACCTGGGTCCCAGCTAACCCGATCCTCCCCTCCCTCATCCCCTAGACCCAGGAGT CTGGCCCTCCATTGAAAGGACCCCAGGTTACATCATCCATTCAGGCTGCCCTTGC CACGATGGAATTCTGTAGCTCCTGCCAAATGGGTCAAATATCATGGTTCAGGCGC AGGGAGGGTGATTGGGCGGGCCTGTCTGGGTATAAATTCTGGAGCTTCTGCATCT ATCCCAAAAAACAAGGGTGTTCTGTCAGCTGAGGATCCAGCCGAAAGAGGAGCC AGGCACTCAGGCCACCTGAGTCTACTCACCTGGACAACTGGAATCTGGCACCAA TTCTAAACCACTCAGCTTCTCCGAGCTCACACCCCGGAGATCACCTGAGGACCCG AGCCATTGATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGGCTGTGGGTTT CTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTC CAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGAT GATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGG GGGCGCTGCTGACCAGAGCCCCGAAAGTGAGTGTGGGCCAGAGCCTGGGTCTGA GGGAGGAGGGGCTGTGGGTCTGGATTCCTGGGTCTGAGGGAGGAGGGGCTGGGG GCCTTGGCCCCTGGGTCTGAGGGAGGAGGGGCTGGGGATCTGGACTCCTGGGTC TGAGGGAGGAGGGGCTGGGGATCTGGGCCCCTGGGTCTGAGGGAGGAGGGGCT GGGTCTGGACCCCTGGGTCTGAGGGAGGAGGGGCTGGGGGTCTGGACTCTTGGG TTTGAAGAAGGAAGGGCTGGGGTCCTGGACTCTTGGGTCTGAGTTGGGAGGGGG CTTTGGCTTGGGCTTCTCCTGGGTCTGAGGGAGGAGGTAGGCTGTGGGCTTGGAC TCCCAGGGCTGGGACAGAGCCGGATGGTGGGACAGAGTCGGGTGGTGGGACAGT CCCGGGTGGGAGAGGTCCTCGAACCACCTTATCGCTTTCACCCCTTAGGTCTCCT GCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATC CAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGGTGAGTTTCCAG GACCCTCCTCACCACCCACCATGCTCCTCCTATATGTCGCCCTCACAGCCTGGGG TGCCTTGTCTTGCTCATCCCCCCCGGAGCCAGACTTGATTCTATTTGCTCTGCACG CCCCCAGCTGCAACATTTGGAGGTTGAAGTTGTCATCAGTGTTTGCAAGATGAGG AAACTGAGGCCCAGGCCGGGGCGCCAGTGACCTCAATCATGTGATGTGTGGATG CTGGAGCGGCCTGAGGCTCAGGTTATTGGGAGTCTCGTGATTCAGTAACCCCTGC TCCTGCCCACACGGCCCCTGTGTGCACGGCTCATGCTGGGCACAGGGACACTCGG GGAAGCCATGGCCAGTAAAGTGACCAGGACCTTGAGTGCTAGGGAGACACCCCG CCTGGCC TGAGAGAGCACTGATGGCTCCGAGGGCTGGAATGTTCTCTGTGAAGTCTGAACT GGGAGGCAGGTCCCTGCAGGAGAGCCCTGGGGTAAAAAACAAAACCTGCCTTGC TGTTTTGTTTCCTAGAGGAGGGGCTGGGGGCCTGGACTCCTGGGTCTGAGGGAGG AGGGGCTGGGGGCCTGGACCCCTGGGTCTGAGGGAGGAGGGGCTGGGGGCCTG GAACCCCGGGTCTGAGGGAGGAGAGGCTGGGGCCTGGAACCCCGGGTCTGAGG GAGGAGAGGCTGGGGCCTGGAACCCCGGGTCTGAGGGAGGAGGCGCTGGGGGC CTGGACTCCTGGGTCGGATGGAGGAGAAACTAGGGTCTGGACCCCTGGGTCTGA GGGAGGAGGCGCTGGGGGCCTGGACCCCTGGGTCTGAGGGAGGCAGGGCTGGG GCCTGGATCCTGGGTCTTACATCAGGAAAACAGAGGAACCCTGTCTCTGATCCTG TTTTTGTCCCCTAGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTT CTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACC TGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCT TCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGC CCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCC CAGGGCCGAAGCCCCAGCTACGCTTCCTGAAGCCAGAGGCTGTTTACTATGACAT CTCCTCTTTATTTATTAGGTTATTTATCTTATTTATTTTTTTATTTTTCTTACTTGAG ATAATAAAGAGTTCCAGAGGAGGATAAGAATGAGCATGTGTGAGTGTCTGAGGG AAGACATGGCAGCTGTTTTGTCTCCCTTGGCCCGGACAATCCCCTCTACACCTCC CCTCACGTGGTCCGAGGGTCCTGGCTTCCCACTGGGCCTCACTTTTTTCTTTTCTT TTCTTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTCACCCAGGCTGGAGTGCAGT GGCGCGATCTTGGCTCACTCCAACCTCCGCCTCCCAGGTTCAAGCAATTCTCCTG CCTCAGCCACCCGAGTAGCTGTGATTACAGGCGTGCGCCACCACACCCAGCTAAT TTTGTAATTTTAGTAGAGACAGGGTTTCGCCATGTTGGCCAGGATGCTCTCCATCT CTTGACTTCATGACCTGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCTT GAGTCACTGTGCCCAGCCCAGCCTCACTTTTCTACTCTGCTAAAGTGTCCCCAGG GACTGTGGACTATCCCTGCTCTCTGAAAGGACAAGACTGGCCGGGAGTGGTGGC TTACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGTGGATCACGAGGTC AGGAGATTGAGACTATCCTGGCTAATACGATGAAACCCCGTCTCTACTAAAAAT ACAAAAACAAAATTAGCTGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTC CGGAGGCTGAGGCAGAATGGCGTGAATGCGGGAGGCGGAGCTTGCAGTGAGCC GAGATCGCGCCACTGCACTCCAGCCCAGGCCACAGAGCGAGATTCCATCTCAAA AAAATAAATAAATAAATAAATAAATAAATAAATATAAAAATAAAATGAAAGAG CAGGACTTCTTTCTACAACCCCTCAACTTGTGTGAGCGTTGTGTAACTATTTCATA GAGCTACCTCAATAACAGGGGAGCTTTTACGAGGTGACACAGCACACTCACATC CTCATGGGAGATGTAGTTTTCTGGCATCATTTAGCAGCAGGAATGAGATCTGTTG GGCCTCAAATCTGGGACAAGGACTCCTGGGTCCTGGAGTAGGTTTGGGGCTAGT GTAACACCCAAGTTCTGGGGAATCAGTGGGCTGGACATCTGGACACCTGGATCA CAGGAGAACTGGGGACTGCAGACTTAGGCATCCTGGTCTGAGAAAAAAGGGGCT GGAGGGTGGGAGTTTGGGTTCTCAGGAAAAGGAGCTGAAACCTGGAATTCTTCC ATCTGGGTCCTTATGAAC mRNA: Human FGF21 mRNA, complete cds (GenBank Accession NM_019113; SEQ ID NO: 3) GAGGCTTCCAAGGCAGGATACTTGTGTCTCAGATGCGGTCGCTTCTTTCATACAG CAATTGCCGCCTTGCTGAGGATCAAGGAACCTCAGTGTCAGATCACGCCCTCCCC CCAAACTTAGAAATTCAGATGGGGCGCAGAAATTTCTCTTGTTCTGCGTGATCTG CATAGATGGTCCAAGAGGTGGTTTTTCCAGGAGCCCAGCACCCCTCCTCCCTCCG ACTCAGACCCAGGAGTCTGGCCCTCCATTGAAAGGACCCCAGGTTACATCATCCA TTCAGGCTGCCCTTGCCACGATGGAATTCTGTAGCTCCTGCCAAATGGGTCAAAT ATCATGGTTCAGGCGCAGGGAGGGTGATTGGGCGGGCCTGTCTGGGTATAAATT CTGGAGCTTCTGCATCTATCCCAAAAAACAAGGGTGTTCTGTCAGCTGAGGATCC AGCCGAAAGAGGAGCCAGGCACTCAGGCCACCTGAGTCTACTCACCTGGACAAC TGGAATCTGGCACCAATTCTAAACCACTCAGCTTCTCCGAGCTCACACCCCGGAG ATCACCTGAGGACCCGAGCCATTGATGGACTCGGACGAGACCGGGTTCGAGCAC TCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCAC ACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCG GTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGA GGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCT GAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTT CCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAG GCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCG AAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACC CTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACT CCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGAC CCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCACTACGCTTCCTGAAG CCAGAGGCTGTTTACTATGACATCTCCTCTTTATTTATTAGGTTATTTATCTTATTT ATTTTTTTATTTTTCTTACTTGAGATAATAAAGAGTTCCAGAGGAGGATAAAAAA

AAAAAAAAAAAAAAAAA mRNA: Mus musculas FGF21, mRNA (GenBank Accession No. NM_020013; SEQ ID NO: 4) AGACAGCCTTAGTGTCTTCTCAGCTGGGGATTCAACACAGGAGAAACAGCCATT CACTTTGCCTGAGCCCCAGTCTGAACCTGACCCATCCCTGCTGGGCACCGGAGTC AGAACACAATTCCAGCTGCCTTGGCTCCTCAGCCGCTCGCTTGCCAGGGGCTCTC CCGAACGGAGCGCAGCCCTGATGGAATGGATGAGATCTAGAGTTGGGACCCTGG GACTGTGGGTCCGACTGCTGCTGGCTGTCTTCCTGCTGGGGGTCTACCAAGCATA CCCCATCCCTGACTCCAGCCCCCTCCTCCAGTTTGGGGGTCAAGTCCGGCAGAGG TACCTCTACACAGATGACGACCAAGACACTGAAGCCCACCTGGAGATCAGGGAG GATGGAACAGTGGTAGGCGCAGCACACCGCAGTCCAGAAAGTCTCCTGGAGCTC AAAGCCTTGAAGCCAGGGGTCATTCAAATCCTGGGTGTCAAAGCCTCTAGGTTTC TTTGCCAACAGCCAGATGG AGCTCTCTATGGATCGCCTCACTTTGATCCTGAGGCCTGCAGCTTCAGAGAACTG CTGCTGGAGGACGGTTACAATGTGTACCAGTCTGAAGCCCATGGCCTGCCCCTGC GTCTGCCTCAGAAGGACTCCCCAAACCAGGATGCAACATCCTGGGGACCTGTGC GCTTCCTGCCCATGCCAGGCCTGCTCCACGAGCCCCAAGACCAAGCAGGATTCCT GCCCCCAGAGCCCCCAGATGTGGGCTCCTCTGACCCCCTGAGCATGGTAGAGCCT TTACAGGGCCGAAGCCCCAGCTATGCGTCCTGACTCTTCCTGAATCTAGGGCTGT TTCTTTTTGGGTTTCCACTTATTTATTACGGGTATTTATCTTATTTATTTATTTTAG TTTTTTTTTCTTACTTGGAATAATAAAGAGTCTGAAAGAAAAATGTGTGTT Mus musculas FGF21 protein (AAH49592) (SEQ ID NO: 5) MEWMRSRVGTLGLWVRLLLAVFLLGVYQAYPIPDSSPLLQFGGQVRQRYLYTDDD QDTEAHLEIREDGTVVGAAHRSPESLLELKALKPGVIQILGVKASRFLCQQPDGALY GSPHFDPEACSFRELLLEDGYNVYQSEAHGLPLRLPQKDSPNQDATSWGPVRFLPMP GLLHEPQDQAGFLPPEPPDVGSSDPLSMVEPLQGRSPSYAS Human FGF21 protein (GenBank accession AAQ89444) (SEQ ID NO: 6) MDSDETGFEHSGLWVSVLAGLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQT EAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLH FDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPA LPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS

Beta Klotho

[0126] Beta klotho contributes to the transcriptional repression of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis. Beta klotho increases the ability of FGFR1 and FGFR4 to bind FGF21. Beta klotho acts as a co-receptor for FGF21. The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1-4). Klotho and 1 Klotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Like FGF21, FGF19 also requires .beta.Klotho. Both FGF19 and FGF21 can signal through FGFR1-3 bound by .beta.Klotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the .beta.Klotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. The expression of .beta.Klotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21.

[0127] Exemplary Beta Klotho human and mouse mRNA and protein sequences include:

TABLE-US-00003 Human Beta Klotho gene, Cyp7a1 gene, linear DNA (GenBank Accession No. DD362478) (SEQ ID NO: 7) ACCCTGGGCCTGGCCCAAGAAACTATACATTCCTCCTGGGAATCTGGGGCTGTGA TGGGAGGGGTTGCCATGAAGACTTCTGACATGCCCTGGAGACATTTTCCCCATGG TCTTGGGGATTAACATTCAGCTCCTTGTTACTTATGCAAATTTCTGCAGCTGGCTT GAATTTCTCCTCAGAAAATGAGATTTTCTTTTCTATCGCATTGTCAGGCTGCAAAT TTTCCAAA CTTTTGTGCTCTGCTTCCCTTATAAAACTGAAGGCCTGGCCAGGTGTGGTGGCTC ACGCCTGTAATCCCAGCACTTTGGGGAGCTGAGACGGGCGAATCACGAGGTCAG GAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCTGTCTCTACTAAAAATAC AAAAAGTTAGCTGGGCATAGTCATGGGTGCCTTTAATCCCAGCTACTTGGGAGAC TGAGGCAGGAGAATCGTTTGAATCCAGGAGGCGGAGGTTGCAGTGAGTCGAGAT CACACCACTGCACTCCAGCCCTGGTAACATAGTAAGACTCTGTCTCAAAAAAAA AAAAAAAAAAACAAAACTGAATGCCTTTAACAACACCCAAGTTGCTTCTTGAAT GCTTTGCTGCTTAGAAATTTCTTCTGCCAGATACCCTAAATCATCTCTCTTAGGTT CCAAGTTCCACAAATCTCTAGGGCAGGGACAAAACGCTGCCAGTCTCTTTACTAA AACATAACAAGAGTCACCCTTGCTCTAGTTCCCAAAAAGTTCCTCATCTCCATCT GAAACCACCCCCGCCTAGATTTCGTTGTCCATATCATTATCAGCATTTTGGTCAA AGTCATTCAACAGGCCTCTAGGGAGTTCCAAACTTGCTCACATTCTCCTGTCTTCT TCTGAGCCCTCCAAACTGTTCCAATCCCTACCTGTTACCCAGTTCCAAAGTCGCTT CCACATTTTTGGTTATCTTTTCAGCCG TGCCCCACTCTACTGGTACCAGTTTACTGTATTAGTCGATTTTCATGCTGCTGATA AAGACATACCTGAAACTGGACAATTTACAAAAGAAAGAGGTTTATTGGACTTAC AATTCTACATCACTTGGGAGGCCTCACAATCATGATGGAAGGAGAAAGGCACAT CTCACATGGCAGCAGACAAGAAAAGAGCTTGTGCAGGGAAACTCCTCTTTTTAA AACCATCAGATCTCATGAAATTTATTCATTATCATGACAATAGCACAGGAAAGA ACTGCACCCATAATTCAGTCACCTCCTACCAGGTTCCTCCCACAACACGTGAGAA TTCAAGATGAGATTTGGATGGGGACACAGCCAAACCATGTCACACTACCATGCC TGACTTCCTTTCCATTTTTGTATATTTGCTTGTTCTTCATTTGCCCGAGAAGTAACT CTAAAGGGCTGTATTATTTGGATATTAGATTGGCATTTTATCTGACTGGGATATCT TGCTGTGATTGTCCATGTATAAGATCAGCTTTTCTATAAACCATATTTTTAAAAAG ATATATTAATTTTTTAAAAA TCCACCTGTCTAAATAAATGCACAAAGCCCCCCAAAAACCTAGATTCTAAGAAA AATCTATGTACTGCCATACAATGATTGATATTAATATTTATGGTGATAAATTACA CACAAAAAATGTGTGATCTCTGTTTAAACAGGCAAAAACAAAAAACACATGAAA TAAATCTATGGCATCTATAGCCAAAACTGGAAACAACCCACATATCCATCAATA GGAAATCAGTTAAATAAATTATAGTACATTTATCCAATGGAAGATTAAGCACATA TTCAATATAATTATTTATACACACATATAGATACACACATGTATAAATATAGAGA ATACTGTGGGTGTATGTGTGTGTGTGTTTATATACATATATATACACACACAGTA CTGTTGCCTACCTTCTTTTGTCTTAATTCTGTGAACTCTCATTCACTCTGCTTCAGT AGGATACATCCTTCTTTTTGGTTCTTAGACTCACCAAGTTGATCCTTGACTCAAGA CATTGCATTTGCTGCTTCCTCTTCCTGGAATATCCTTCCTTCTGATATTCACATGA GTAGTCTCTTCTTGTCATTCAGATCTCAAATGTCACAATTTCAGAGAGCCCATCTC TGATCATCATATCTAAAGTTGTC CTCATTCCCCCATAGCTTTCTATACCATGTTTTATTTTTTTCATAACATGTATTTTA TTACTCCTTTCTCCATTGGAATAGAATCTCCATTAGATTAGGAAATCTGCCTATCT TATTAATGCCTGCAACTGGAATACTTTTGAAGAGTTCTTGGCACGTAATAAATAC TCAACTAATATTTTTGTGTACACAGAAATAAAGTTTGGAAGAACAGATGCCAAAT TGTTACTAGTGGTTACTTCTGAGTAAAGGAGTAGCATGGTAGGTAAATTATTAAT AGATGTTCACTTTCCACCAAGATATGTTTTAGTTAGTCTTAACTTACTTGAAATGA AATTTATTACTTTAATAATTAGAAACATTGATAAACATTTTAGTCACAAGAATGA TAGATAAAATTTTGATGCTTCCAATAAGTTATATTTATCTAGAGGATGCACTTAT GTAGAATACTCTCTTGAGGATGTTAGGTGAGTAACATGTTACTATATGTAGTAAA ATATCTATGATTTTATAAAAGCACTGAAACATGAAGCAGCAGAAACGTTTTTCCC AGTTCTCTTTCCTCTGAACTTGATCACCGTCTCTCTGGCAAAGCACCTAAATTAAT TCTTCTTTAAAAGTTAACAAGACCAAATTATAAGCTTGATGAATAACTCATTCTT ATCTTTCTTTAAATGATTATAGTTT ATGTATTTATTAGCTATGCCCATCTTAAACAGGTTTATTTGTTCTTTTTACACATA CCAAACTCTTAATATTAGCTGTTGTCCCCAGGTCCGAATGTTAAGTCAACATATA TTTGAGAGAACTTCAACTTATCAAGTATTGCAGGTCTCTGATTGCTTTGGAACCA CTTCTGATACCTGTGGACTTAGTTCAAGGCCAGTTACTACCACTTTTTTTTTTCTA ATAGAATG AACAAATGGCTAATTGTTTGCTTTGTCAACCAAGCTCAAGTTAATGGATCTGGAT ACTATGTATATAAAAAGCCTAGCTTGAGTCTCTTTTCAGTGGCATCCTTCCCTTTC TAATCAGAGATTTTCTTCCTCAGAGATTTTGGCCTAGATTTGCAAA Homo sapiens, klotho beta (KLB) mRNA (GenBank Accession No. NM_175737) (SEQ ID NO: 8) ATCCTCAGTCTCCCAGTTCAAGCTAATCATTGACAGAGCTTTACAATCACAAGCT TTTACTGAAGCTTTGATAAGACAGTCCAGCAGTTGGTGGCAAATGAAGCCAGGC TGTGCGGCAGGATCTCCAGGGAATGAATGGATTTTCTTCAGCACTGATGAAATAA CCACACGCTATAGGAATACAATGTCCAACGGGGGATTGCAAAGATCTGTCATCC TGTCAGCACTTATTCTGCTACGAGCTGTTACTGGATTCTCTGGAGATGGAAGAGC TATATGGTCTAAAAATCCTAATTTTACTCCGGTAAATGAAAGTCAGCTGTTTCTCT ATGACACTTTCCCTAAAAACTTTTTCTGGGGTATTGGGACTGGAGCATTGCAAGT GGAAGGGAGTTGGAAGAAGGATGGAAAAGGACCTTCTATATGGGATCATTTCAT CCACACACACCTTAAAAATGTCAGCAGCACGAATGGTTCCAGTGACAGTTATATT TTTCTGGAAAAAGACTTATCAGCCCTGGATTTTATAGGAGTTTCTTTTTATCAATT TTCAATTTCCTGGCCAAGGCTTTTCCCCGATGGAATAGTAACAGTTGCCAACGCA AAAGGTCTGCAGTACTACAGTACTCTTCTGGACGCTCTAGTGCTTAGAAACATTG AACCTATAGTTACTTTATACCACTGGGATTTGCCTTTGGCACTACAAGAAAAATA TGGGGGGTGGAAAAATGATACCATAA TAGATATCTTCAATGACTATGCCACATACTGTTTCCAGATGTTTGGGGACCGTGT CAAATATTGGATTACAATTCACAACCCATATCTAGTGGCTTGGCATGGGTATGGG ACAGGTATGCATGCCCCTGGAGAGAAGGGAAATTTAGCAGCTGTCTACACTGTG GGACAC AACTTGATCAAGGCTCACTCGAAAGTTTGGCATAACTACAACACACATTTCCGCC CACATCAGAAGGGTTGGTTATCGATCACGTTGGGATCTCATTGGATCGAGCCAAA CCGGTCGGAAAACACGATGGATATATTCAAATGTCAACAATCCATGGTTTCTGTG CTTGGATGGTTTGCCAACCCTATCCATGGGGATGGCGACTATCCAGAGGGGATG AGAAAGAAGTTGTTCTCCGTTCTACCCATTTTCTCTGAAGCAGAGAAGCATGAGA TGAGAGGCACAGCTGATTTCTTTGCCTTTTCTTTTGGACCCAACAACTTCAAGCCC CTAAACACCATGGCTAAAATGGGACAAAATGTTTCACTTAATTTAAGAGAAGCG CTGAACTGGATTAAACTGGAATACAACAACCCTCGAATCTTGATTGCTGAGAATG GCTGGTTCACAGACAGTCGTGTGAAAACAGAAGACACCACGGCCATCTACATGA TGAAGAATTTCCTCAGCCAGGTGCTTCAAGCAATAAGGTTAGATGAAATACGAG TGTTTGGTTATACTGCCTGGTCTCTCCTGGATGGCTTTGAATGGCAGGATGCTTAC ACCATCCGCCGAGGATTATTTTATGTGGATTTTAACAGTAAACAGAAAGAGCGG AAACCTAAGTCTTCAGCACACTACTACAAACAGATCATACGAGAAAATGGTTTTT CTTTAAAAGAGTCCACGCCAGATGTGCAGGGCCAGTTTCCCTGTGACTTCTCCTG GGGTGTCACTGAATCTGTTCTTAAGCCCGAGTCTGTGGCTTCGTCCCCACAGTTC AGCGATCCTCATCTGTACGTGTGGAACGCCACTGGCAACAGACTGTTGCACCGA GTGGAAGGGGTGAGGCTGAAAACACGACCCGCTCAATGCACAGATTTTGTAAAC ATCAAAAAACAACTTGAGATGTTGGCAAGAATGAAAGTCACCCACTACCGGTTT GCTCTGGATTGGGCCTCGGTCCTTCCCACTGGCAACCTGTCCGCGGTGAACCGAC AGGCCCTGAGGTACTACAGGTGCGTGGTCAGTGAGGGGCTGAAGCTTGGCATCT CCGCGATGGTCACCCTGTATTATCCGACCCACGCCCACCTAGGCCTCCCCGAGCC TCTGTTGCATGCCGACGGGTGGCTGAACCCATCGACGGCCGAGGCCTTCCAGGCC TACGCTGGGCTGTGCTTCCAGGAGCTGGGGGACCTGGTGAAGCTCTGGATCACC ATCAACGAGCCTAACCGGCTAAGTGACATCTACAACCGCTCTGGCAACGACACC TACGGGGCGGCGCACAACCTGCTGGTGGCCCACGCCCTGGCCTGGCGCC TCTACGACCGGCAGTTCAGGCCCTCACAGCGCGGGGCCGTGTCGCTGTCGCTGCA CGCGGACTGGGCGGAACCCGCCAACCCCTATGCTGACTCGCACTGGAGGGCGGC CGAGCGCTTCCTGCAGTTCGAGATCGCCTGGTTCGCCGAGCCGCTCTTCAAGACC GGGGAC TACCCCGCGGCCATGAGGGAATACATTGCCTCCAAGCACCGACGGGGGCTTTCC AGCTCGGCCCTGCCGCGCCTCACCGAGGCCGAAAGGAGGCTGCTCAAGGGCACG GTCGACTTCTGCGCGCTCAACCACTTCACCACTAGGTTCGTGATGCACGAGCAGC TGGCCGGCAGCCGCTACGACTCGGACAGGGACATCCAGTTTCTGCAGGACATCA CCCGCCTGAGCTCCCCCACGCGCCTGGCTGTGATTCCCTGGGGGGTGCGCAAGCT GCTGCGGTGGGTCCGGAGGAACTACGGCGACATGGACATTTACATCACCGCCAG TGGCATCGACGACCAGGCTCTGGAGGATGACCGGCTCCGGAAGTACTACCTAGG GAAGTACCTTCAGGAGGTGCTGAAAGCATACCTGATTGATAAAGTCAGAATCAA AGGCTATTATGCATTC AAACTGGCTGAAGAGAAATCTAAACCCAGATTTGGATTCTTCACATCTGATTTTA AAGCTAAATCCTCAATACAATTTTACAACAAAGTGATCAGCAGCAGGGGCTTCC CTTTTGAGAACAGTAGTTCTAGATGCAGTCAGACCCAAGAAAATACAGAGTGCA CTGTCTG

CTTATTCCTTGTGCAGAAGAAACCACTGATATTCCTGGGTTGTTGCTTCTTCTCCA CCCTGGTTCTACTCTTATCAATTGCCATTTTTCAAAGGCAGAAGAGAAGAAAGTT TTGGAAAGCAAAAAACTTACAACACATACCATTAAAGAAAGGCAAGAGAGTTGT TAGCT AAACTGATCTGTCTGCATGATAGACAGTTTAAAAATTCATCCCAGTTCCATATGC TGGTAACTTACAGGAGATATACCTGTATTATAGAAAGACAATCTGAGATACAGCT GTAACCAAGGTGATGACAATTGTCTCTGCTGTGTGGTTCAAAGAACATTCCCTTA GGTGT TGACATCAGTGAACTCAGTTCTTGGATGTAAACATAAAGGCTTCATCCTGACAGT AAGCTATGAGGATTACATGCTACATTGCTTCTTAAAGTTTCATCAACTGTATTCCA TCATTCTGCTTTAGCTTTCATCTCTACCAATAGCTACTTGTGGTACAATAAATTAT TTTTAAGAAGTAAAACTCTGGGGCTGGACGCTGTGGCTCACACCTGTAATCTCAG CACTTTGG GAGGCCGAGGCGGGGAGATCACCTGAGGTGAGGAGTTCGAGACCAGCCTGGCC AACATGGTGAAACCATGTCTCTACTAAAAATACAAAAAATTAGCCAGGCGTGGT GACAGTGGCACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGAAGTTTGAAC CCAGGAAACAGGTTACAGTAGGCCAAAATTGCGCCACTGCACTCCAGCCTAGGC GACAACAGCAAGACTGTGTCCAAAAAAAAAAAAAAAAGCAAAAGCAAAACTTT GTTTTGTTAGACTCTACAGCAGAGATTTAACACCCTTCTTTAAACTGGGTAGTCA GTGATAGATAATATATATTCTGTCACTTCTAATAAGGTGCCTTCTCCTTTAGGTCA GGGTGGTTCTAAAATGGAAAGAAAACACAATAGGGTAAGTAGTGCTTGTCTAAG CCAGTTACAACACAGAC TCTTAAAGAGGATCAAGCCCTTCATTTTTCTAACAACAAAAAATCACCTATAGAA TATCTAATTTGTGATCTTTTACTAGATCTGATTTTTTAAAATAATGTAATTTCCGG CCAGGCACGGTGGCACCGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGT GGATCACCTGAGGTTAGGAGTTCGAGACTAGCCTGGCCAACATGGCAAAACCCC ATCTCTACTAAAAATACAAAAGTTAGCCGGGCATGGTGGTGGGCACCTGTAATC CCAGCTACTCAGGAGGCCGAGGCAGGAGAATCGCTTGAACCCGAGAGGCAGAG GTTGCAATGAGCCAAGATCGTGCCATTGCACTCCAGCCTGGGGGACAGGGCAAG ACTGTCTCTCAAAATAAAAAAAAATAATAAAAATAAAAATAAAAGTAATTTCCA AAACCTCATCTCATGG AAAGATCACAGGATGAAGGAAAGCTAGACTCAACTCTGTGAATAGAAGTTGCTA TACTGTAAGTAAAGCAACAATTCAGAATACTGAATGAGTTTAAATTGTTTTATAT AGCACCCTTTTGGGCTAGGGTTAATTACTAGATCTGACTTGGATAATTTGACACT TTGGGA AATGAACTCTGTTCTTGAGACTTGTTCAGTGTATTTTAAACATCTGAGGAAGAAA ACTTAAATATGCACCTATTTATACCTATTCTTTCTTTAGGTCAACATTTAACACCC ACTGCATACATTAATTTGTCCTTGTCTGCTCACTCCAGCAATTTAGACCTTAACAG TCACAAGAGACGTTCTTCTGTTACAAAGCCTTAGTAAATTAAGGCAGTTTTGATT ATATTCTA GGTCCACCTATGTCTGAAGCTAAATTCAGTATCTAACTGCTAATGAACAAGTTTC CAAAATACTGTAAAAATACAATTAGTCAATTTGAGTAAATGCAAATATGATGAG AAATCAATTTGCTATTTGGCCTGGCAAATGGGAACAGTAAAATTCTGCTTTACTC TTCTCTAGTCTCCTTGCCCCAGCTGCACCCACTACCCCAAAGTTGGCAGTTTTGAG GTATGATTTTCAAGGAATTTTTTTAGTATTAACATCTCCCTCTGAGAACTATGTAC CTAAGGTCACGCATACAACTAGTCAATTCTGTTTTTATTACTCTAACTATGTAGAA ACAGTAAGTCACTTAAAACAATCACTTGGCTGGGTTTTTTCCCCTTTGTGCCACAT TGATTCACCCTGACCCAAGAACTCCAGGGAAAATTCTTTAATGTCAACTGGGCAA CTCATTAACCTCTCTTTAACATCAAGGGCTTGGGAAAAAAAAAAAAAAGGTTAG CCACAGGAATAACAAAAACCTGGAATTTATCTTTCAGGTTTTGCTTTCTCTTTCTC ACTTTGTTTAAAGTATCTCGTACTCACAGTTCACAAATTAACCTTCACTGTCTCTT TCACATTAAGAGCTTATGCTTAAAGCATGCCCCCCTTTTCTAACTTGCTGGTTTAC CATAAACTCCCCTAAGTAATAAAAT TCCTAACCCAGTACTGAGAGTCCTCCTTCTCTGCCACTTGGGCATTATTTTACTAG TTTTTAAGCCATCATCGCACAAGAATCCAAAAACCCTTAAATTTTTTAACCACTG GCAAATATGTACAGCAAATTAGGTTAAGCATTTAATCTGGCTCATGCTCTATCAT ACTAAATATTCAGGTTTATCATAAACTCCTTAAAAACCATCAAAGGTCAACCAGA AACTGATAACTCTTGAAAGGAGCAAACAGGTAAGATCTTTGGAGTTTAAGCTTTT CTGAGATGTGTTGTGAAAAATCTAACGTGTTTATCGTATATTCAATGTAACAACC TGGAGAATCACAACTATATTTAAAGAGCCTCTGGAAAATGAGGCCAGTACAGTG TGACTACATGTTTAA TTTTCAATGTAATTTATTCCAAATAAACTGGTTCATGCTGACCACTTGTATTCAAC TAA Human Beta Klotho (GenBank Accession No. NP_783864) (SEQ ID NO: 9) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGR AIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIH THLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQY YSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGD RVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYN THFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPE GMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLRE ALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFG YTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKES TPDVQGQFPCDFSWGVT ESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLE MLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLY YPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSD IYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYA DSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAER RLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWG VRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVR IKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENT ECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKR VVS Mus musculas beta klotho (KLB), mRNA (GenBank Accession No. NM_031180) (SEQ ID NO: 10) AATGAAGACAGGCTGTGCAGCAGGGTCTCCGGGGAATGAATGGATTTTCTTCAG CTCTGATGAAAGAAACACACGCTCTAGGAAAACAATGTCCAACAGGGCACTGCA AAGATCTGCCGTGCTGTCTGCGTTTGTTCTGCTGCGAGCTGTTACCGGCTTCTCCG GAGACGGGAAAGCAATATGGGATAAAAAACAGTACGTGAGTCCGGTAAACCCA AGTCAGCTGTTCCTCTATGACACTTTCCCTAAAAACTTTTCCTGGGGCGTTGGGA CCGGAGCATTTCAAGTGGAAGGGAGTTGGAAGACAGATGGAAGAGGACCCTCG ATCTGGGATCGGTACGTCTACTCACACCTGAGAGGTGTCAACGGCACAGACAGA TCCACTGACAGTTACATCTTTCTGGAAAAAGACTTGTTGGCTCTGGATTTTTTAGG AGTTTCTTTTTATCAGTTCTCAATCTCCTGGCCACGGTTGTTTCCCAATGGAACAG TAGCAGCAGTGAATGCGCAAGGTCTCCGGTACTACCGTGCACTTCTGGACTCGCT GGTACTTAGGAATATCGAGCCCATTGTTACCTTGTACCATTGGGATTTGCCTCTG ACGCTCCAGGAAGAATATGGGGGCTGGAAAAATGCAACTATGATAGATCTCTTC AACGACTATGCCACATACTGCTTCCAGACCTTTGGAGACCGTGTCAAATATTGGA TTACAATTCACAACCCTTACCTTGTTGCTTGGCATGGGTTTGGCACAGGTATGCA TGCACCAGGAGAGAAGGGAAATTTAACAGCTGTCTACACTGTGGGACACAACCT GATCAAGGCACATTCGAAAGTGTGGCATAACTACGACAAAAACTTCCGCCCTCA TCAGAAGGGTTGGCTCTCCATCACCTTGGGGTCCCAT TGGATAGAGCCAAACAGAACAGACAACATGGAGGACGTGATCAACTGCCAGCA CTCCATGTCCTCTGTGCTTGGATGGTTCGCCAACCCCATCCACGGGGACGGCGAC TACCCTGAGTTCATGAAGACGGGCGCCATGATCCCCGAGTTCTCTGAGGCAGAG AAGGAGGAGGTGAGGGGCACGGCTGATTTCTTTGCCTTTTCCTTCGGGCCCAACA ACTTCAGGCCCTCAAACACCGTGGTGAAAATGGGACAAAATGTATCACTCAACT TAAGGCAGGTGCTGAACTGGATTAAACTGGAATACGATGACCCTCAAATCTTGA TTTCGGAGAACGGCTGGTTCACAGATAGCTATATAAAGACAGAGGACACCACGG CCATCTACATGATGAAGAATTTCCTAAACCAGGTTCTTCAAGCAATAAAATTTGA TGAAATCCGCGTGTTTGGTTATACGGCCTGGACTCTCCTGGATGGCTTTGAGTGG CAGGATGCCTATACGACCCGACGAGGGCTGTTTTATGTGGACTTTAACAGTGAGC AGAAAGAGAGGAAACCCAAGTCCTCGGCTCATTACTACAAGCAGATCATACAAG ACAACGGCTTCCCTTTGAAAGAGTCCACGCCAGACATGAAGGGTCGGTTCCCCTG TGATTTCTCTTGGGGAGTCACTGAGTCTGTTCTTAAGCCCGAGTTTACGGTCTCCT CCCCGCAGTTTACCGATCCTCACCTGTATGTGTGGAATGTCACTGGCAACAGATT GCTCTACCGAGTGGAAGGGGTAAGGCTGAAAACAAGACCATCCCAGTGCACAGA TTATGTGAGCATCAAAAAACGAGTTGAAATGTTGGCAAAAATGAAAGTCACCCA CTACCAGTTTGCTCTGGACTGGACCTCTATCCTTCCCACTGGCAATCTGTCCAAA GTTAACAGACAAGTGTTAAGGTACTATAGGTGTGTGGTGAGCGAAGGACTGAAG CTGGGCGTCTTCCCCATGGTGACGTTGTACCACCCAACCCACTCCCATCTCGGCC TCCCCCTGCCACTTCTGAGCAGTGGGGGGTGGCTAAACATGAACACAGCCAAGG CCTTCCAGGACTACGCTGAGCTGTGCTTCCGGGAGTTGGGGGACTTGGTGAAGCT CTGGATCACCATCAATGAGCCTAACAGGCTGAGTGACATGTACAACCGCACGAG TAATGACACCTACCGTGCAGCCCACAACCTGATGATCGCCCATGCCCAGGTCTGG

CACCTCTATGATAGGCAGTATAGGCCGGTCCAGCATGGGGCTGTGTCGCTGTCCT TACATTGCGACTGGGCAGAACCTGCCAACCCCTTTGTGGATTCACACTGGAAGGC AGCCGAGCGCTTCCTCCAGTTTGAGATCGCCTGGTTTGCAGATCCGCTCTTCAAG ACTGGCGACTATCCATCGGTTATGAAGGAATACATCGCCTCCAAGAACCAGCGA GGGCTGTCTAGCTCAGTCCTGCCGCGCTTCACCGCGAAGGAGAGCAGGCTGGTG AAGGGTACCGTCGACTTCTACGCACTGAACCACTTCACTACGAGGTTCGTGATAC ACAAGCAGCTGAACACCAACCGCTCAGTTGCAGACAGGGACGTCCAGTTCCTGC AGGACATCACCCGCCTAAGCTCGCCCAGCCGCCTGGCTGTAACACCCTGGGGAG TGCGCAAGCTCCTTGCGTGGATCCGGAGGAACTACAGAGACAGGGATATCTACA TCACAGCCAATGGCATCGATGACCTGGCTCTAGAGGATGATCAGATCCGAAAGT ACTACTTGGAGAAGTATGTCCAGGAGGCTCTGAAAGCATATCTCATTGACAAGG TCAAAATCAAAGGCTACTATGCATTCAAACTGACTGAAGAGAAATCTAAGCCTA GATTTGGATTTTTCACCTCTGACTTCAGAGCTAAGTCCTCTGTCCAGTTTTACAGC AAGCTGATCAGCAGCAGTGGCCTCCCCGCTGAGAACAGAAGTCCTGCGTGTGGT CAGCCTGCGGAAGACACAGACTGCACCATTTGCTCATTTCTCGTGGAGAAGAAA CCACTCATCTTCTTCGGTTGCTGCTTCATCTCCACTCTGGCTGTACTGCTATCCAT CACCGTTTTTCATCATCAAAAGAGAAGAAAATTCCAGAAAGCAAGGAACTTACA AAATATACCATTGAAGAAAGGCCACAGCAGAGTTTTCAGCTAAACTGCCATTTCT GTCATAGTTTCAAGATTCACTCCGGCTCCATGTACTGGTAACTTACGATGTGAGA GACAGCTGTAACCAAGGTGAAGACAATCGATGCCTCTGAAGTGTGGTTCAAATA ATTCCTTCAGGTCCCGACAATCAGTGAGTCCGTTCTCCGAGCTGAAGACACCCTG ACAGTAACTCTGGGCGTGACCCTAAACATCGCTTCAGGAAGTGTGAATCACGAC TTCACATCCTTTTTCTCTAGCATTCTTCTGTAAATAACAATCACTATTCATGGTCA AGAAATTAATTTTAAAAAGT Mus musculas beta klotho protein (GenBank Accession No. NP_112457) (SEQ ID NO: 11) MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLAVTGSGDGKAW DKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLR GVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYR ALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRV KYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKN FRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFM KTGAMIP EFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDP QILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDA YTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGV TESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVE MLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYH PTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMY NRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSH WKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVK GTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLL AWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAF KLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFL VEKKPLIFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQNIPLKKGHSRVFS

Pharmaceutical Compositions

[0128] Another aspect of the disclosure pertains to pharmaceutical compositions of the compounds of the disclosure. The pharmaceutical compositions of the disclosure typically comprise a compound of the disclosure and a pharmaceutically acceptable carrier. 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 that are physiologically compatible. The type of carrier can be selected based upon the intended route of administration. In various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0129] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. 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. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the compounds can be administered in a time release formulation, for example in a composition which includes a slow release polymer, or in a fat pad described herein. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are generally known to those skilled in the art.

[0130] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a 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, certain methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0131] Depending on the route of administration, the compound may be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the agent. For example, the compound can be administered to a subject in an appropriate carrier or diluent co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluoro-phosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27). 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 may contain a preservative to prevent the growth of microorganisms.

[0132] The active agent in the composition (i.e., long-acting FGF21) optionally is formulated in the composition in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result to thereby influence the therapeutic course of a particular disease state. A therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. In another embodiment, the active agent is formulated in the composition in a prophylactically effective amount. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0133] The amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0134] Exemplary dosages of compounds (e.g., long-acting FGF21) of the disclosure include e.g., about 0.0001% to 5%, about 0.0001% to 1%, about 0.0001% to 0.1%, about 0.001% to 0.1%, about 0.005%-0.1%, about 0.01% to 0.1%, about 0.01% to 0.05% and about 0.05% to 0.1%.

[0135] Exemplary or long-acting or stable forms of FGF21, as described herein, may have a half-life greater than that of the native or wild type FGF21. The half-life of long-acting FGF21 may be greater than the half-life of native FGF21 by at least 1 hour, at least 2 hours, at least 3 hours, at 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, and at least a month, but at least some amount over that of the native FGF21.

[0136] Exemplary stabilized or long-acting forms of FGF21 (i.e., long-acting FGF21) as described herein, may have a half-life of at least 1.5.times., at least 2.times., at least 3.times., at least 4.times., at least 5.times., at least 8.times., at least 10.times., at least 12.times., at least 15.times., at least 20.times., at least 30.times., at least 40.times., at least 50.times., at least 60.times., or at least 70.times. the half-life of a native FGF21 peptide, when assayed for stability under identical conditions. Exemplary stabilized or long-acting forms of FGF21 (i.e., long-acting FGF21) as described herein, may possesses a half-life of at least 0.8 h, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 7 h, at least 10 h, at least 15 h, at least 20 h, at least 25 h, at least 28 h or at least 30 h in the circulation of a mammal, optionally wherein the mammal is human.

[0137] The compound(s) of the disclosure can be administered in a manner that prolongs the duration of the bioavailability of the compound(s), increases the duration of action of the compound(s) and the release time frame of the compound by an amount selected from the group consisting of at least 3 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, and at least a month, but at least some amount over that of the compound(s) in the absence of the fat pad delivery system. Optionally, the duration of any or all of the preceding effects is extended by at least 30 minutes, at least an hour, at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks or at least a month.

[0138] A compound of the disclosure can be formulated into a pharmaceutical composition wherein the compound is the only active agent therein. Alternatively, the pharmaceutical composition can contain additional active agents. For example, two or more compounds of the disclosure may be used in combination. Moreover, a compound of the disclosure can be combined with one or more other agents that have modulatory effects on cancer.

Kits

[0139] The disclosure also includes kits that include a composition of the disclosure, optionally also including a compound (e.g., a long-acting FGF21), and instructions for use.

[0140] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures, are incorporated herein by reference.

EXAMPLES

Example I: Materials and Methods

Mouse Models of Retinal/Choroidal Neovascularization

[0141] Oxygen-Induced Retinopathy:

[0142] mice were exposed to 75% oxygen from P7 to P12. The mice were then returned to room air until P17 (Connor et al., 2009; Smith et al., 1994). High oxygen led to vessel loss and the relative hypoxia induced neovascularization which reached to maximum at P17 (Smith et al., 1994). Both genders of mouse pups were used with body weight ranging from 5.5 to 7 grams (Connor et al., 2009). The mice were randomly assigned to either native FGF21 (5 mg/kg, twice per day) or long-acting FGF21 (1 mg/kg PF-05231023) or phosphate buffered saline (PBS). The mice were intraperitoneally injected daily from P12 to P16. For the intra-vitreal injection of PF-05231023 (0.5 .mu.l 10 .mu.g/.mu.l stock per eye) was delivered using 10 .mu.l syringe with 35G needle (World Precision Instruments, Inc.) at P12. The contralateral eye was injected with vehicle control. At P17, retinal neovascularization was quantified (Connor et al., 2009; Smith et al., 1994). Mice were sacrificed at P17 using a lethal intraperitoneal injection of ketamine/xylazine, and the eyes were enucleated, followed by fixation in 4% paraformaldehyde for 1 hour at room temperature. The retinas were dissected and stained overnight at room temperature with fluorescent Griffonia Bandeiraea Simplicifolia Isolectin B4 (Alexa Fluor 594, 121413, Molecular Probes, 10 .mu.g/ml) in 1 mM CaCl2 in PBS. The images of whole mounted retina were taken at 5.times. magnification on a Zeiss AxioObserver.Z1 microscope and merged to form one image with AxioVision 4.6.3.0 software. Retinal neovascularization was quantified as previously reported (Connor et al., 2009). Percentages of neovascularization were calculated by comparing the number of pixels in the neovascular areas with the total number of pixels in the retina. n is the number of eyes quantified.

[0143] Vldlr.sup.-/- Mice:

[0144] Vldlr.sup.-/- mice develop pathological neovascularization (retinal angiomatous proliferation or RAP as well as later onset choroidal neovascularization). RAP extends from the deep retinal vascular layer of the outer plexiform layer (OPL) towards the retinal pigment epithelium (RPE). The mouse pups were treated with a long-acting FGF21 analogue (0.5 mg/kg PF-05231023) or PBS from P8 to P15. At P16, neovascular lesions were quantified (Joyal et al., 2016). The eyes were enucleated and fixed in 4% paraformaldehyde for 1 hour at room temperature. The retinas were stained overnight with fluorescent Griffonia Bandeiraea Simplicifolia Isolectin B4 (Alexa Fluor 594, 121413, Molecular Probes, 10 .mu.g/ml) in 1 mM CaCl2 in PBS then washed with PBS and whole mounted with photoreceptors facing up. The images of whole mounted retina were taken at 5.times. magnification on a Zeiss AxioObserver.Z1 microscope and merged to form one image with AxioVision 4.6.3.0 software. Vascular lesions were analyzed using the SWIFT_MACTEL method, a plugin in Image J (Joyal et al., 2016).

[0145] Laser-Induced Choroidal Neovascularization:

[0146] Four laser burns were induced by a green Argon laser pulse, with duration of 70 ms and power of 240 mW in 6-8-week-old C57BL/6J mice. Both male and female mice were used (Lambert et al., 2013). A long-acting FGF21 analogue (10 mg/kg PF-05231023) or PBS were intraperitoneally injected every other day one week before and after the laser photocoagulation induction. The eyes were enucleated and fixed in 4% paraformaldehyde for 1 hour at room temperature. The choroid were penetrated with 1% Triton X-100 PBS for one hour at room temperature and stained overnight with fluorescent Griffonia Bandeiraea Simplicifolia Isolectin B4 (Alexa Fluor 594, 121413, Molecular Probes, 10 .mu.g/ml) in 1 mM CaCl2 in PBS. The choroids were washed with PBS and whole mounted. The images of whole mounted choroid were taken at 10.times. or 20.times. magnification on a Zeiss AxioObserver.Z1 microscope. Lesion area was quantified (Gong et al., 2015).

Real-Time PCR

[0147] RNA from retinas or choroid-retina complex was extracted and reverse-transcribed to cDNA. PCR was conducted for Fgfr1, Fgfr2, Fgfr3, Fgfr4, Klb, Apn, Vegfa, Dynamin2 and Tnf.alpha.. Cyclophilin A was used as internal control. Freshly isolated retinas were lysed with QIAzol.TM. lysis reagent and incubated on ice for 15 minutes. 20% chloroform was added and samples were incubated for 5 minutes at room temperature. RNA was extracted according to the manufacturer's instructions using a PureLink.RTM. RNA Mini Kit (#12183018A, Ambion). RNA was then reverse transcribed using iScript.TM. cDNA synthesis kit (#1708891, Bio-Rad). The sequences of primers were Fgfr1 ((F: 5'-ACT CTG CGC TGG TTG AAA AAT-3' (SEQ ID NO: 12), R: 5'-GGT GGC ATA GCG AAC CTT GTA-3' (SEQ ID NO: 13)); Fgfr2 ((F: 5'-GCT ATA AGG TAC GAA ACC AGC AC-3' (SEQ ID NO: 14), R: 5'-GGT TGA TGG ACC CGT ATT CAT TC-3' (SEQ ID NO: 15)); Fgfr3 ((F: 5'-GCC TGC GTG CTA GTG TTC T-3' (SEQ ID NO: 16), R: 5'-TAC CAT CCT TAG CCC AGA CCG-3'(SEQ ID NO: 17)); Fgfr4 ((F: 5'-TCC ATG ACC GTC GTA CAC AAT-3' (SEQ ID NO: 18), R: 5'-ATT TGA CAG TAT TCC CGG CAG-3' (SEQ ID NO: 19)); .beta.-klotho (Klb) ((F: 5'-TGT TCT GCT GCG AGC TGT TAC-3' (SEQ ID NO: 20), R: 5'-CCG GAC TCA CGT ACT GTT TTT-3' (SEQ ID NO: 21)); Adipoq: ((F: 5'-GAA GCC GCT TAT GTG TAT CGC-3' (SEQ ID NO: 22), R: 5'-GAA TGG GTA CAT TGG GAA CAG T-3' (SEQ ID NO: 23)); Vegfa ((F: 5'-GGA GAT CCT TCG AGG AGC ACT T-3' (SEQ ID NO: 24), R: 5'-GCG ATT TAG CAG CAG ATA TAA GAA-3' (SEQ ID NO: 25)); Tnf.alpha. ((F: 5'-AAG GAC CTG GTA CAT GAA CTG G-3' (SEQ ID NO: 26), R: 5'-GGT TCT GGG TGT CAA GTG TCG-3' (SEQ ID NO: 27)); Dynamin 2 ((F: 5'-TTT GGC GTT CGA GGC CAT T-3' (SEQ ID NO: 28); R: 5'-CAG GTC CAC GCA TTT CAG AC-3' (SEQ ID NO: 29)) Quantitative analysis of gene expression was performed using an Applied Biosystems 7300 Sequence Detection System with the SYBR Green Master mix kit, and gene expression was calculated relative to Cyclophilin A ((F: 5'-CAG ACG CCA CTG TCG CTT T-3' (SEQ ID NO: 30); R: 5'-TGT CTT TGG AAC TTT GTC TGC AA-3' (SEQ ID NO: 31)) using the .DELTA..DELTA.Ct method.

ELISA for Mouse Serum FGF21

[0148] Neonatal mouse serum levels of FGF21 were measured with ELISA following the manufacturer's protocol (Eagle Biosciences, #F2131-K01). Briefly, 50 .mu.l mouse serum or standards were first added on the designated microwell. 50 .mu.l tracer antibody was then added and the plate was incubated at room temperature for 2 hours. The wells were washed and incubated with 100 .mu.l ELISA HRP substrate at room temperature in dark for 20 minutes. Finally, 100 .mu.l ELISA stop solution were added and the signals were read at 450/650 nm.

Statistics

[0149] All data were used except for low quality images that were insufficient for analysis. Data represent mean.+-.SEM. 2-tailed unpaired t-test or ANOVA with Bonferroni's multiple comparison test was used for comparison of results as specified (Prism v5.0; GraphPad Software, Inc., San Diego, Calif.). Statistically significant difference was set at P.ltoreq.0.05. For phenotypic data, a dot represents each retina or choroid; for qPCR data, each dot represents a number of replicates from 4-6 pooled retinas.

Example 2: Long-Acting FGF21 Suppressed Hypoxia-Induced Retinal Neovascularization

[0150] Early vessel growth cessation or vessel loss in retinopathy of prematurity and diabetic retinopathy has been previously described to lead to hypoxia and nutrient deficits, which drives vessel overgrowth (Chen and Smith, 2007; Shin et al., 2014). To investigate the effects of FGF21 on pathologic neovessel growth under hypoxia, FGF21 was administered to mice having oxygen-induced retinopathy (Smith et al., 1994). After five-day exposure to 75% oxygen, mouse pups with their nursing dam were returned to room air. The oxygen exposure led to vaso-obliteration in the central retina, and the relatively avascular hypoxic retina induced neovascularization (Smith et al., 1994) extending from the retina into the vitreous at the boundary between vascular and non-vascularized areas (Connor et al., 2009). The mouse pups were intraperitoneally injected with native FGF21 or a long-acting FGF21 analog, PF-05231023 (Talukdar et al., 2016), or vehicle control for five days. Native FGF21, which exhibits a short half-life (0.4 hours; Huang et al., 2013), did not affect neovascularization (FIG. 1A) whereas administration of PF-05231023, which possesses a biological half-life of 28 hours (Huang et al., 2013) significantly decreased neovascularization (FIG. 1A). To confirm the role of FGF21 in retinal neovascularization, the impact of FGF21 deficiency on oxygen-induced retinopathy was examined by comparing wild-type (Fgf21.sup.+/+) and knockout (Fgf21.sup.-/-) mice. Fgf21.sup.-/- mice exhibited increased neovascularization (FIG. 1B). Noting the strong inhibition of pathologic neovessel formation observed when PF-05231023 was administered, the relatively smaller effects of native FGF21 and the effects of endogenous FGF21 deficiency observed upon retinal neovascularization, these latter effects were likely due to the short half-life of native FGF21 (0.4 hours) (Huang et al., 2013) and low endogenous FGF21 levels (about 658.3.+-.66.4 pg/ml in normal neonatal mouse serum detected by ELISA), respectively, while the former effects of long-acting FGF21 were also reflective of the short half-life of native FGF21. To determine if long-acting FGF21 directly inhibited neovascularization, intra-vitreal injection of PF-05231023 was performed, and indeed was found to reduce retinal neovascularization, as compared to the extent of retinal neovascularization observed in a vehicle-injected contralateral eye (FIG. 5). FGF21 also promoted retinal revascularization through APN, although APN might not be required for "basal" revascularization in OIR (FIG. 6A-6D). In some embodiments, improved revascularization has likely decreased the stimulus for proliferative neovascularization.

[0151] Without wishing to be bound by theory, in some embodiments, FGF21 apparently exerted its effects through interaction with its receptor, FGFR1, and co-receptor .beta.-klotho (KLB) (Ding et al., 2012; Foltz et al., 2012; Suzuki et al., 2008). As described herein, FGF21 receptor 1, 2, 3, 4 and Klb mRNA were all expressed in the mouse retina (FIG. 2A). Fgfr1 and fgfr3 were highly expressed in neovessels, and co-localized with APN (FIG. 7A-7B). PF-05231023 administration at P17 increased retinal Apn (FIG. 2B), an important mediator of FGF21 effects on metabolic function (Holland et al., 2013; Lin et al., 2013). APN receptor agonist AdipoRon inhibited endothelial cell function in vitro (FIG. 7C-7D). To determine if APN also mediated the protection conferred by long-acting FGF21 against retinal neovascularization, the retinal vasculature in APN knockout (Apn.sup.-/-) mice having oxygen-induced retinopathy was examined, in the presence or absence of PF-05231023 administration. APN deficiency worsened retinal neovascularization (FIG. 2C), and lack of APN completely abolished the beneficial effects of PF-05231023 otherwise exhibited in reducing neovascularization in hypoxic retinas (FIG. 2D). APN has been described as inhibiting retinal neovascularization by decreasing the levels of tumor necrosis factor (TNF).alpha. (Higuchi et al., 2009). Consistent with such observations, PF-05231023 suppressed Tnf.alpha. expression in neovascular WT retina, but the suppression was abolished with APN deficiency (FIG. 2E). In some embodiments, long-acting FGF21 inhibited pathological retinal neovessel growth by targeting APN and reducing TNF.alpha. (FIG. 2F), a key risk factor for oxygen-induced retinopathy (Kociok et al., 2006).

[0152] Each year, over 15 million babies are born preterm and possessing an incompletely vascularized retina, which--if normal vascularization does not occur postnatally--sets the stage for the progression to proliferative retinopathy (Hellstrom et al., 2013). Retinopathy of prematurity is a leading cause of blindness in children (Gilbert et al., 1997). As described herein, low serum APN levels positively correlated with proliferative retinopathy in premature infants (Fu et al., 2015). Increasing circulating APN levels were associated with less retinal neovascularization in mice (Fu et al., 2015; Higuchi et al., 2010). In mouse oxygen-induced retinopathy, FGF21 administration increased retinal Apn levels and reduced Tnf.alpha. levels, and also suppressed pathologic neovessel growth (FIG. 2).

[0153] Diabetic retinopathy currently afflicts approximately 93 million people worldwide, and of those, 28 million have vision-threatening proliferative retinopathy (Abcouwer and Gardner, 2014). The levels of FGF21 in type 1 diabetes have been observed as lower than those in healthy controls (Xiao et al., 2012; Zibar et al., 2014). In streptozotocin-induced type 1 diabetic mice, a FGF21 analog reduced blood glucose levels with improved glucose uptake in brown adipose tissue (Kim et al., 2015). FGF21 prevented renal lipid accumulation, and attenuated renal dysfunction in type 1 diabetic mice (Zhang et al., 2013). In type 2 diabetes, serum FGF21 levels have been observed as higher in patients with retinopathy versus no retinopathy (Esteghamati et al., 2016; Lin et al., 2014). FGF21 treatment decreased body weight and improved the lipid profile (decreases triglycerides and increases HDL cholesterol levels) in type 2 diabetes patients, non-human primates and in obese rodents (Bernardo et al., 2015; Gaich et al., 2013; Schlein et al., 2016; Talukdar et al., 2016). It was hypothesized that FGF21 could play a beneficial role in diabetes and diabetic complications, such as diabetic retinopathy. The mouse model of oxygen-induced retinopathy has been commonly used to model hypoxia-induced neovascularization in proliferative diabetic retinopathy (Lai and Lo, 2013). As described elsewhere herein, long-acting FGF21 is also contemplated as helping to prevent proliferative diabetic retinopathy, optionally via a direct effect upon early diabetic retinal neurovascular loss and/or late neovascularization, optionally also related to the hyperglycemic aspect of diabetic retinopathy.

Example 3: Long-Acting FGF21 Administration Protected Against Retinal Neovascularization Induced by Energy-Deficiency in Vldlr.sup.-/- Mice

[0154] In addition to lack of oxygen, an inadequate fuel supply can also drive retinal neovascularization (Joyal et al., 2016). Absence of the very low density lipoprotein receptor (VLDLR) has previously been described as associated with retinal angiomatous proliferation and choroidal neovascularization, similar to the neovascularization that has been seen with macular telangiectasia and late proliferative age-related macular degeneration (Engelbert and Yannuzzi, 2012; Lambert et al., 2016). In Vldlr.sup.-/- mice, abnormal blood vessels extended towards starved photoreceptors (Joyal et al., 2016) (FIG. 3A). To assess whether long-acting FGF21 protected against metabolism-induced pathologic neovessel growth, PF-05231023 was administered intraperitoneally at 0.5 mg/kg daily from P8 to P15 to Vldlr.sup.-/- mice. PF-05231023 administration attenuated the neovascular lesions (FIG. 3A, 3B, 3C) and increased retinal Apn, while decreasing Tnf.alpha. (FIG. 3D) in Vldlr.sup.-/- mice. Thus, long-acting FGF21 administration protected against retinal neovascularization induced by energy-deficiency in Vldlr.sup.-/- mice.

Example 4: Long-Acting FGF21 Inhibited Laser-Induced Choroidal Neovascularization in Mice

[0155] Choroidal neovascularization, a condition in which neovessels extend from the choriocapillaris into the subretinal space, is vision-threatening in age-related macular degeneration. In a laser-induced choroidal neovascularization mouse model, laser burns disrupt Bruch's membrane to induce choroidal neovessel growth (Ryan, 1979) (FIG. 4A). To test the effects of long-acting FGF21 in such a model, 10 mg/kg PF-05231023 was administered every other day one week before and after the laser injury in 6-8-week-old C57B/6J mice. As shown in FIG. 4B, PF-05231023 administration inhibited choroidal neovessel formation. In the mouse choroidal neovascularization model, PF-05231023 also induced Apn and reduced Tnf.alpha. in the choroid-retina complex (FIG. 4C).

[0156] Neovascularization more generally is a leading cause of vision loss in both age-related macular degeneration and macular telangiectasia in the population over age 50 years (Heeren et al., 2014; Yonekawa et al., 2015). Metabolic alterations in retinal pigment epithelial cells and photoreceptors contribute to disease progression (Barron et al., 2001; Joyal et al., 2016). In Vldlr.sup.-/- mice, which have an inadequate fuel supply driving retinal neovascularization, long acting FGF21 administration was observed to reduce neovascular lesion formation. As described herein, long-acting FGF21 administration likely attenuated the development of metabolically driven retinal neovascularization, modeling macular telangiectasia and some aspects of neovascular age-related macular degeneration. Furthermore, in laser-induced choroidal neovascularization modeling inflammatory aspects of neovascular age-related macular degeneration (Parmeggiani et al., 2012), FGF21 also suppressed pathological choroidal angiogenesis in adult mice.

[0157] In summary, there is an unmet need for effective treatment of vision-threatening eye neovessel growth. As described herein, long-acting FGF21 inhibited retinal and choroidal neovascularization mediated by APN in mice. APN suppresses TNF.alpha. transcription and mRNA stability in macrophages (Park et al., 2008; Wulster-Radcliffe et al., 2004). Inhibition of TNF.alpha. has been previously characterized as leading to decreased retinal and choroidal neovascularization (Kociok et al., 2006; Shi et al., 2006), possibly through increased endothelial cell sprouting (Hangai et al., 2006; Sainson et al., 2008). Long-acting FGF21 was observed herein to have promoted migration in HRMECs in vitro (FIG. 7C). Different target systems are likely affected by long-acting FGF21 with opposite angiogenic responses. As described herein, long-acting FGF21 inhibitory effects on retinal and choroidal neovascularization were identified as independent of VEGFA (FIG. 4D). It has previously been reported in clinical data that little to no effect of long-acting FGF21 has been observed upon glycemic endpoints, which has tempered enthusiasm for potential further development of long-acting FGF21 in the clinic for metabolic diseases; however, the instant disclosure offers a phenotypic basis and some mechanistic insights into a new indication where long-acting FGF21 is likely to provide an effective prophylactic and/or therapeutic against retinopathy, thereby providing a next-generation standard of care for patients with pathological vascular proliferation in retinopathy of prematurity, diabetic retinopathy, macular telangiectasia and age-related macular degeneration.

Example 5: FGF21 in Retinopathy of Prematurity

[0158] Hyperglycemia is a recently described risk factor for retinopathy of prematurity (ROP) in premature infants. In streptozotocin (STZ)-induced hyperglycemic ROP mouse model, retinal vascular development was confirmed as delayed. Specifically, FGF21 deficiency delayed normal retinal vascular network formation and worsened hyperglycemic ROP in such mice. Native (nFGF21) and long-acting FGF21 (PF-05231023) treatment were both observed to have promoted retinal vessel growth in hyperglycemic ROP. The protection of FGF21 observed was also confirmed as dependent upon adiponectin (APN).

[0159] As seen in FIGS. 8A and 8B, FGF21 deficiency delayed normal retinal vascular development (FIG. 12A) and worsened hyperglycemic retinopathy (FIG. 8B) in neonatal mice. As seen in FIGS. 9A and 9B, FGF21 promoted retinal vascular development in hyperglycemic mouse neonates. Both native FGF21 (nFGF21; FIG. 9A) and long-acting FGF21 (PF-05231023, FIG. 9B) administration improved retinal vascular growth. In some embodiments, FGF21 promoted and/or improved retinal vascular growth. In some embodiments, long-acting FGF21 promoted and/or improved retinal vascular growth.

[0160] As seen in FIGS. 10A and 10B, FGF21 protection against hyperglycemic retinopathy was dependent on adiponectin (APN). As shown in FIG. 10A, APN deficiency completely abolished FGF21 effects on STZ-induced hyperglycemic retinopathy. As demonstrated in FIG. 10B, FGF21 increased APN levels in serum. In some embodiments, FGF21 therefore prevented and/or reduced retinopathy of prematurity. In certain embodiments, long-acting FGF21 therefore prevented and/or reduced retinopathy of prematurity.

Example 6: Long-Acting FGF21 in Retinitis Pigmentosa (RP)

[0161] As seen in FIG. 11, long-acting FGF21 increased the photoreceptor layer thickness (ONL) in photoreceptor-degenerating mice rd10. To determine if FGF21 preserved photoreceptor function, rd10 mice are treated with long-acting FGF21 analog PF-05231023. Retinal histology, retinal function with electroretinogram (ERG), and cone survival with immunohistochemistry are used to assess preservation of photoreceptor function by long-acting FGF21. Retinal mitochondrial function and glycolysis with Seahorse Fx96 oxygen consumption rate (OCR) as well as an extracellular acidification rate (ECAR) analysis are also performed to assess preservation of photoreceptor function by long-acting FGF21. Furthermore, retinal key metabolic enzyme levels (Krebs cycle, glycolysis, fatty acid oxidation) are also performed with quantitative proteomic analysis to assess preservation of photoreceptor function by long-acting FGF21. Finally, mitochondrial function and glycolysis (Seahorse OCR, ECAR analysis) in cone-like photoreceptors (661W) in vitro are also assessed in concert with PF-05231023 treatment.

[0162] To determine if FGF21 administration improves retinal vascular development in rd10, as retinal vessels reflect photoreceptor metabolic needs, P30 deep vasculature in retinal whole mount in rd10 vs. control mice with PF-05231023 treatment is quantified. Neovascular growth in laser-captured microdissected retinal layers and vessels in WT and rd10 mice, as well as PF-05231023 versus vehicle-treated rd10 mice are also quantified.

[0163] In some embodiments, these analyses and quantifications thereby demonstrate that long-acting FGF21 preserves photoreceptor function. In some embodiments, these analyses and quantifications demonstrate that long-acting FGF21 improves retinal vascular development. Optionally, these analyses and quantifications demonstrate that long-acting FGF21 prevents and/or reduces retinitis pigmentosa in a subject having or at risk of developing RP.

Example 7: FGF21 Protects Against Early Diabetic Retinopathy (DR)

[0164] Retinal neuronal abnormalities occur before vascular changes in diabetic retinopathy. Accumulating experimental evidence suggests that neurons control vascular pathology in diabetic and other neovascular retinal disease. Whether fibroblast growth factor 21 (FGF21) prevented retinal neuronal dysfunction in insulin-deficient diabetic mice was investigated.

Materials and Methods

[0165] Animals: All animal studies adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee at Boston Children's Hospital.

[0166] Mouse Models of Type 1 Diabetes--Akita Mice:

[0167] Ins2.sup.Akita male mice have a spontaneous mutation in the insulin 2 gene which leads to incorrect folding of insulin protein. Heterozygous Akita mice develop diabetes within one month and retinal complications around 6-months of age (Lai A K, Lo A C. Journal of Diabetes Research 2013:106594). 7-to-8-month-old Akita mice were compared with littermate wild-type (WT) mice. Akita mice with retinal functional abnormalities were screened with electroretinography (ERG) and those with ERG changes were intraperitoneally injected with 10 mg/kg long-acting FGF21 analog, PF-05231023 (Pfizer) or vehicle control twice a week for four weeks. Retinal function was again examined with ERG after 4 weeks of treatment and body weight, blood glucose were recorded. Serum triglycerides levels were measured using the Wako L-Type TG M test.

[0168] Streptozotocin (STZ)-Induced Diabetic Mice:

[0169] 6-to-8-week-old male WT and adiponectin-deficient (Apn.sup.-/-) mice were starved six hours prior to an intraperitoneal injection of 55 mg/kg/day STZ on day 1 and 2 followed by an injection of 60 mg/kg/day STZ from day 3 to 5. Diabetes was induced within one week of injection (Lai A K et al. 2013). 7-to-8-month-old diabetic WT and Apn.sup.-/- mice were screened with ERG. Mice with retinal functional abnormalities were intraperitoneally injected with 10 mg/kg PF-05231023 or control twice a week for four weeks. Retinal function was then re-examined with ERG.

[0170] Electroretinography (ERG):

[0171] ERG was used to assess the function of retinal neurons. Flash ERGs were obtained using an Espion e.sup.2 (Diagnosys LLC, Lowell, Mass.) in dark-adapted, mydriatic (Cyclomydril; Alcon, Fort Worth, Tex.), anesthetized (ketamine/xylazine) subjects. Stimuli were "green" light emitting diode flashes of doubling intensity from .about.0.0064 to .about.2.05 cd.quadrature.s/m.sup.2 and then "white" xenon-arc flashes from .about.8.2 to .about.1,050 cd.quadrature.s/m.sup.2 presented in an integrating sphere (Colordome, Diagnosys LLC). As shown (FIG. 14A), the saturating amplitude (Rm.sub.P3) and sensitivity (S) of the rod photoreceptors was estimated from the a-waves elicited by the white flashes (Lamb T D, Pugh E N, Jr., J Physiol 1992; 449:719-758). The saturating amplitude (Rm.sub.P2) and sensitivity (1/K.sub.P2) of second-order neurons, principally bipolar cells (Wurziger K, et al., Vision Res 2001; 41:1091-1101), was measured by subtracting this model from the intact ERG waveform (Robson J G, et al., Vis Neurosci 1995; 12:837-850) and fitting the Naka-Rushton equation (Fulton A B, Rushton W A. Vision Research 1978; 18:793-800) to the response vs. intensity relationship of the resulting waveform, "P2." The oscillatory potentials (OPs), which characterize activity in inner retinal cells distinct from the generators of the a- and b-waves (Dong C J, et al., Vis Neurosci 2004; 21:533-543), were filtered from P2 (Lei B, et al., Invest Ophthalmol Vis Sci 2006; 47:2732-2738) and assessed in the frequency domain to determine their energy (Akula J D, et al., Invest Ophthalmol Vis Sci 2007; 48:5788-5797); the saturating energy (Em) and sensitivity (1/i.sub.1/2) of the OPs were then assessed similarly to those of P2. Finally, retinal sensitivity at threshold (Sm), was calculated by scaling the amplitude of each P2 by the intensity used to elicit it and fitting a generalized logistic growth curve, with the exponent set to negative unity, to the resulting sensitivities, and determining the limit of this function as intensity approached zero (Akula J D, et al., Mol Vis 2008; 14:2499-2508).

[0172] Optical Coherence Tomography (OCT):

[0173] Mice were anesthetized (ketamine/xylazine), and their pupils dilated (Cyclomydril; Alcon, Fort Worth, Tex.). Spectral domain OCT with guidance of bright-field live fundus image was performed with the image-guided OCT system (Micron IV, Phoenix Research Laboratories). Photoreceptor inner and outer segment thickness was measured using Insight (provided by Micron IV). The thickness of photoreceptor segments was plotted at six distances (50, 100, 150, 200, 250 and 300 .mu.m) from the optic nerve head both on the nasal and on the temporal side.

[0174] Immunohistochemistry:

[0175] For IHC on retinal cross-sections, eyes were fixed in 4% PFA, frozen in optimal cutting temperature compound (OCT, Tissue-Tek) and then were cut into 10-.mu.m sections and rinsed with PBS. The sections with optic nerve were treated with ice-cold methanol for 15 minutes and then 0.1% triton PBS for 45 minutes at room temperature. The sections were blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature and stained with primary antibody against cone arrestin (1:500, AB15282, Millipore), rhodopsin (1:500, MABN15, Millipore) overnight at 4.degree. C. The sections were stained with corresponding secondary antibody, covered in mounting medium with 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI, H-1200, Vector laboratories), and visualized with a Leica SP2 confocal microscope or a Zeiss AxioObserver.Z1 microscope at 200.times. magnification.

[0176] Laser-Captured Microdissection:

[0177] Fresh mouse eyes were embedded in OCT compound and sectioned at 12 .mu.m in a cryostat, mounted on RNase-free polyethylene naphthalate glass slides (Ser. No. 11/505,189, Leica). Frozen sections were fixed in 70% ethanol for 15 seconds, followed by 30 seconds in 80% ethanol and 30 seconds in absolute ethanol, and then washed with DEPC-treated water for 15 seconds. Laser-dissection of retinal neuronal layers was performed immediately thereafter with the Leica LMD 6000 system (Leica Microsystems) and samples were collected directly into lysis buffer from the RNeasy Micro kit (Qiagen, Chatsworth, Calif.).

[0178] Photoreceptors (661W) Cell Culture:

[0179] Photoreceptor 661W cells were cultured at 37.degree. Celsius, 5% CO.sub.2 in a humidified atmosphere in Dulbecco's modified Eagle's medium (DMEM, #1196502, Gibco) supplemented with 10% fetal bovine serum (#S12450, Atlanta Biologicals) and 1% antibiotic/antimycotic solution. An equal number of cells per well was plated on a 6-well dish. Oxidative stress was induced with 0.5 mM paraquat (PQ, Sigma, #856177) for 1 hour at 37.degree. Celsius. The culture media were changed and the cells were treated with vehicle (PBS) or 500 ng/ml PF-05231023 for 24 hours. Cells were collected for protein and RNA.

[0180] Real-Time PCR:

[0181] Retinas or 661W cells were lysed with QIAzol lysis reagent and incubated on ice for 15 minutes. 20% chloroform was added and incubated for 5 minutes at room temperature. RNA was extracted according to the manufacturer's instructions using a PureLink.RTM. RNA Mini Kit (#12183018A, Ambion). RNA was then reverse transcribed using iScript.TM. cDNA synthesis kit (#1708891, Bio-Rad). qPCR were performed for Arrestin4: 5'-GAG CAA GGG CTG CTA CTC AAG-3' (SEQ ID NO: 32), 5'-AAC CGC AGG TTC AAG TAT TCC-3' (SEQ ID NO: 33); Rhodopsin: 5'-TCA TGG TCT TCG GAG GAT TCA C-3' (SEQ ID NO: 34), 5'-TCA CCT CCA AGT GTG GCA AAG-3' (SEQ ID NO: 35); IL-1.beta.: 5'-TTC AGG CAG GCA GTA TCA CTC-3' (SEQ ID NO: 36), 5'-GAA GGT CCA CGG GAA AGA CAC-3' (SEQ ID NO: 37); IL-6: 5'-AAG AGC CGG AAA TCC ACG AAA-3' (SEQ ID NO: 38), 5'-GTC TCA AAA GGG TCA GGG TAC T-3' (SEQ ID NO: 39); Vegfa F: 5'-GGA GAT CCT TCG AGG AGC ACT T-3' (SEQ ID NO: 24), R: 5'-GCG ATT TAG CAG CAG ATA TAA GAA-3' (SEQ ID NO: 25); Tnf.alpha. 5'-AAG GAC CTG GTA CAT GAA CTG G-3' (SEQ ID NO: 26), 5'-GGT TCT GGG TGT CAA GTG TCG-3' (SEQ ID NO: 27); IL-10: 5'-CTT ACT GAC TGG CAT GAG GAT CA-3' (SEQ ID NO: 40), 5'-GCA GCT CTA GG AGC ATG TGG-3' (SEQ ID NO: 41); Apn: 5'-GAAGCCGCTTATGTGTATCGC-3' (SEQ ID NO: 42), 5'-GAATGGGTACATTGGGAACAGT-3' (SEQ ID NO: 43); Nrf2: 5'-TAG ATG ACC ATG AGT CGC TTG C-3' (SEQ ID NO: 44), 5'-GCC AAA CTT GCT CCA TGT CC-3' (SEQ ID NO: 45); Nf.kappa.b: F: 5'-GGA GAG TCT GAC TCT CCC TGA GAA-3' (SEQ ID NO: 46), R: 5'-CGA TGG GTT CCG TCT TGG T-3' (SEQ ID NO: 47). Quantitative analysis of gene expression was generated using an Applied Biosystems 7300 Sequence Detection System with the SYBR Green Master mix kit and gene expression was calculated relative to Cyclophilin A ((5'-CAG ACG CCA CTG TCG CTT T-3' (SEQ ID NO: 30); 5'-TGT CTT TGG AAC TTT GTC TGC AA-3'(SEQ ID NO: 31)) (retinas) or .beta.-actin ((5'-CGG TTC CGA TGC CCT GAG GCT CTT-3' (SEQ ID NO: 48), 5'-CGT CAC ACT TCA TGA TGG AAT TGA-3' (SEQ ID NO: 49)) (photoreceptor 661W cells) using the .DELTA..DELTA.Ct method. Each sample was repeated in triplicate.

[0182] Western Blot:

[0183] 5 .mu.l protein lysate from photoreceptor 661W cells were used to detect the levels of NRF2 (1:500, R&D, MAB3925), phospho-NF.kappa.B (1:200, Cell Signaling, #3037S), NF.kappa.B (1:1000, Cell Signaling, #3034), p-AKT (1:500, Cell Signaling, #9271), AKT (1:1000, Cell Signaling, #4691) in 5% bovine serum albumin (BSA) overnight at 4.degree. Celsius degree. Signals were detected using 1:5000 corresponding horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL, Pierce), then the digital images were visualized with Bio-Rad ChemiDoc Touch Imaging System. .beta.-ACTIN (1:5000, Sigma; A1978) was used as internal control.

[0184] Statistical Analysis:

[0185] All ERG data were presented as the log change from control (.DELTA. Log Normal); by expressing the data in log values, changes in observations of fixed proportion become linear, consistent with a constant fraction for physiologically meaningful changes in parameter values (Akula J D, et al., Mol Vis 2008; 14:2499-2508). .DELTA. Log Normal ERG data were plotted as mean.+-.SEM and evaluated for significant effects using mixed-effects linear models (MLMs) (Fitzmaurice G M, Laird N M, Ware J H: Applied longitudinal analysis. Hoboken, N.J., Wiley, 2011). In each analysis, two MLMs were employed. The first MLM, carried out on the saturating a-wave, P2 and OP parameters, had factors group (Akita vs. WT; or STZ vs. Control), treatment (before vs. after PF-05231023), parameter (Amplitude vs. Sensitivity), and retinal depth (photoreceptor vs. bipolar vs. inner retina). The second MLM, carried out on Sm, had factors group, and treatment. Data from both eyes was included in all analyses. Differences in ERG parameters were detected by ANOVA followed by Tukey's test. Two-tailed unpaired t-test, ANOVA with Bonferroni's multiple comparison test was used for comparison of results as specified (Prism v5.0; GraphPad Software, Inc., San Diego, Calif.). The threshold for statistical significance (a) was set at 0.05.

[0186] Results

[0187] PF-05231023 Administration Restored Retinal Function in Akita Mice:

[0188] Circulating FGF21 levels (ELISA) were reduced in >7 month old Akita mice versus littermate wild-type (WT) control mice (FIG. 13A). Retinal expression of Fgf21 (qPCR) was not changed significantly (FIG. 13B). The physiologic and pharmacologic actions of FGF21 are dependent on the receptor FGFR1 and co-receptor .beta.-klotho (Foltz I N, et al., Science Translational Medicine 2012; 4:162ra153; Ding X, et al., Cell Metabolism 2012; 16:387-393). Gene expression of FGF21 receptor Fgfr1 was comparable and .beta.-klotho was mildly increased in Akita versus WT mouse retinas (FIGS. 13C-13D) (Foltz I N, et al., Science Translational Medicine 2012; 4:162ra153; Ding X, et al., Cell Metabolism 2012; 16:387-393). Fgfr1 was expressed in retinal neurons isolated from retinal cross sections with laser-captured microdissection (FIG. 20).

[0189] To examine if FGF21 protects against retinal dysfunction in DR, PF-05231023 (10 mg/kg intraperitoneally was first administered, twice a week for a month) or vehicle control to those Akita mice which had ERG deficits at 7-8-month of age (50% of Akita mice (6/12) had ERG deficits versus their age-matched WT control mice). In Akita mice with ERG deficits, PF-05231023 administration did not change the body weight and serum triglyceride levels versus controls but mildly reduced blood glucose levels (FIGS. 14A-14C). Prior to PF-05231023 treatment, ERG responses were examined in Akita mice. Although the Akita mouse responses were slightly attenuated overall compared to WT controls (FIGS. 14A-14C), retinal sensitivity (Sm), as determined by mixed-linear modeling, was significantly attenuated (F=15.9; df=1,27.0; p=0.00046) relative to WT. PF-05231023 showed protective effects on Akita retinas (FIGS. 14B-14C). Notably, retinal sensitivity (Sm) in Akita mice was improved following treatment (FIG. 14D) (F=27.9, df=1,29.8, p=1.1.times.10.sup.-5) to levels that were supranormal, a result of better a-wave (5) and b-wave (1/K.sub.P2) sensitivities (FIG. 14E). There was no decline in baseline retinal function (ERG signals) in any individual mouse between 7 and 8 months of age (F=0.166, df=1,2, p=0.723, FIG. 22). In Akita mice, the change in post-receptor sensitivity (log 1/K.sub.P2) was positively correlated with the sum of changes in photoreceptor sensitivity and saturated amplitudes (log S+log Rm.sub.P3, a-wave) (FIG. 15A), providing evidence that the changes in post-receptor cells were reflecting the deficits in photoreceptor function (Hansen R M, et al., Progress in Retinal and Eye Research 2017; 56:32-57; Hood D C, et al., Visual Neuroscience 1992; 8:107-126).

[0190] PF-05231023 administration restored photoreceptor morphology in Akita mice: In addition to neuronal function, cone and rod photoreceptor structure was examined to determine if it was influenced by PF-05231023 administration. PF-05231023 administration increased cone-specific arrestin4 expression (Craft C M, et al., The Journal of Biological Chemistry 1994; 269:4613-4619) and did not change rhodopsin expression at the mRNA levels in Akita mice (FIG. 15B). Cone photoreceptor outer and inner segments were oriented in parallel in WT mice but disorganized in Akita mice. PF-05231023 administration normalized the photoreceptor segment arrangement (FIG. 15C). Rhodopsin staining was comparable between WT and PF-05231023-treated Akita mouse retinas, while there appeared to be a reduction in the thickness of rod photoreceptor segments in Akita mice (FIG. 15D). With OCT measurements, there was a significant reduction in the total thickness of photoreceptor inner and outer segment in Akita (blue line) versus WT (black line) mice, but the inner and outer segment thickness was restored with PF-05231023 administration (orange line), particularly the photoreceptor outer segments (FIG. 15E). These observations provide evidence that PF-05231023 protection against DR is through the restoration of photoreceptor function and structure. The possibility of the contribution from other retinal cells was not excluded as Fgfr1 is also expressed in INL and RGC (FIG. 20).

[0191] PF-05231023 Decreased Retinal Inflammation in Diabetic Mice:

[0192] Retinal inflammation induces retinal neurovascular abnormalities in diabetes (Du Y, et al., Proc. Nat'l. Acad. Sci. USA 2013; 110:16586-16591; Liu H, et al., Inv. Ophthal. & Vis. Sci. 2016; 57:4272-4281; Tonade D. et al., Invest. Ophthal. & Vis. Sci. 2016; 57:4264-4271; Joussen A M, et al., FASEB J. 2004; 18:1450-1452). Significantly increased retinal IL-1.beta. and decreased Vegfa mRNA expression was observed in Akita versus WT mice (FIG. 16A). PF-05231023 administration reduced retinal IL-1.beta. mRNA expression in Akita mice (FIG. 16B). IL-1.beta. inhibits energy production in retinal neurons and induces retinal microvascular changes in rats. PF-05231023 administration did not change the expression levels of Vegfa, Tnf.alpha., IL-6, IL-10 and Apn in Akita mouse retinas (FIG. 16B).

[0193] PF-05231023 Inhibited Oxidative-Stress-Induced Inflammation in Photoreceptors:

[0194] Hyperglycemia induces oxidative stress, a crucial contributor to diabetic retinopathy (Madsen-Bouterse S A, Kowluru R A. Reviews in Endocrine & Metabolic Disorders 2008; 9:315-327). Photoreceptors are the most metabolically active cell in the body and very susceptible to metabolic derangement and resulting oxidative stress (Kern T S, Berkowitz B A. J. Diabetes Invest. 2015; 6:371-380). Modulating photoreceptor oxidative stress protects against retinal neurodegeneration (Xiong W, et al., J. Clin. Invest. 2015; 125:1433-1445). In PF-05231023-versus vehicle-treated Akita mouse retinas, there was a significant increase in total AKT levels although there was no significant change in the ratio of p-AKT/AKT (FIG. 17A), evidencing that the absolute level of p-AKT was higher in PF-05231023-treated mouse retinas. Activation of the AKT pathway regulates NRF2 activity in retinal pigment epithelium in vitro (Wang L, et al., Invest. Ophthal. & Vis. Sci. 2008; 49:1671-1678). There was a large variation of NRF2 protein levels in Akita versus WT mice. PF-05231023 administration decreased the variability of retinal NRF2 levels in Akita (FIG. 17A). Taken together, the results provide evidence that PF-05231023 modulates retinal NRF2 levels by activating the AKT pathway. To test if PF-05231023 protects photoreceptors against oxidative stress, oxidative stress was induced with the use of paraquat (PQ), a nonselective herbicide to induce the production of reactive oxygen species in mitochondria (McCarthy S, et al., Toxicol. Applied Pharma. 2004; 201:21-31). In 661W cells (the only photoreceptor cell line available currently) in vitro, oxidative stress induced with PQ increased IL-1.beta. expression; PF-05231023 treatment prevented IL-1.beta. induction (FIG. 17B). Both the activation of antioxidant transcriptional factor NRF2 and the phosphorylation of NF.kappa.B modulate IL-1.beta. transcription, which can be modulated by FGF21 (Yu Y, et al., International Immunopharmacology 2016; 38:144-152; Kobayashi E H, et al., Nature Communications 2016; 7:11624; Cogswell J P, et al., J Immunol 1994; 153:712-723). PF-05231023 treatment increased gene expression of Nrf2 but not Nf.kappa.b in 661W (FIG. 17C). In PQ (to induce oxidative stress)-treated 661W cells, the induction of NRF2 expression by PF-05231023 was dose-dependently inhibited by perifosine, an AKT inhibitor (Zitzmann K, et al., Endocrine-Related Cancer 2012; 19:423-434) (FIG. 17D). PF-05231023 treatment also increased NRF2 production at the protein level but did not change NF.kappa.B phosphorylation in photoreceptors with PQ-induced oxidative stress (FIG. 17E), providing evidence that FGF21 inhibition of IL-1.beta. was through activation of the NRF2 pathway.

[0195] PF-05231023 Administration Protected STZ-Induced Diabetic Mice Against DR, Partly Independent of Adiponectin (APN):

[0196] APN is a key mediator of FGF21 modulation of glucose and lipid metabolism in mice (Lin Z, et al. Cell Metabolism 2013; 17:779-78947; Holland W L, et al., Cell Metabolism 2013; 17:790-797). Changes in the APN pathway may contribute to the development of neovascular eye diseases (Fu Z, et al., Biochimica et Biophysica Acta 2016; 1862:1392-1400). To test if APN mediated the protective effects of PF-05231023, diabetes was induced with injection of STZ in 6-8-week-old WT and in APN-deficient (Apn.sup.-/-) mice. Retinal function was then examined by ERG at 7-8-months of age. Again, PF-05231023 administration did not change body weight, blood glucose levels or serum triglyceride levels (FIGS. 23A-23C). Furthermore, neither the amplitude nor sensitivity of the a-wave, b-wave, or the OPs differed significantly between STZ-treated mice (FIGS. 18A-18C) but, retinal sensitivity at threshold, Sm, was significantly attenuated following STZ-treatment (F=12.2, df=1,6.0, p=0.013, FIG. 18D). PF-05231023, administered as above, again improved Sm (F=45.2, df=1,5.9, p=0.001) in the STZ treated mice to levels that were supranormal (FIGS. 18D-18E). The protective effects of PF-05231023 on retinal sensitivity (Sm) in STZ-induced WT diabetic mice were again found in Apn diabetic mice (F=23.8, df=1,2, p=0.040, FIGS. 18F-18H), providing evidence that the rescue was partly independent of APN. In the STZ-induced diabetic mice, PF-05231023 decreased IL-1.beta. expression in diabetic WT and Apn.sup.-/- retinas (FIG. 18I), suggesting that PF-05231023-induced reduction in IL-1.beta. was independent of APN, in line with the phenotypic observation above (FIGS. 18F-18H).

DISCUSSION

[0197] Dysfunction in photoreceptors and post receptor neurons are among the early retinal changes seen in diabetic patients, anteceding ophthalmoscopic signs of retinopathy (Pescosolido N. et al., J. Diabetes Research 2015; 2015: 319692). In diabetes, hyperglycemia induces oxidative stress, a crucial contributor leading to diabetic retinopathy (Madsen-Bouterse S A, Kowluru R A.: Oxidative stress and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Reviews in Endocrine & Metabolic Disorders 2008; 9:315-327). Activation of the anti-oxidant protein NRF2 protects against retinal neuronal degeneration (Xiong W., et al. The Journal of Clin. Invest. 2015; 125:1433-1445), particularly in photoreceptors, as photoreceptors are the most metabolically active cells in the body (Wong-Riley M T. Eye and Brain 2010; 2:99-116; Okawa H, et al., Current Biology: CB 2008; 18:1917-1921) and more vulnerable to oxidative stress damage. It was demonstrated herein that, in insulin-deficient diabetic mice, administration of the long-acting FGF21 analog PF-05231023 reversed diabetes-induced retinal neuronal deficits with improved photoreceptor function and morphology, and decreased photoreceptor-derived inflammation (FIG. 19). PF-05231023 administration regulated retinal NRF2 levels through activation of the AKT pathway, and suppression of pro-inflammatory IL-1.beta. expression. IL-1.beta. causes neurovascular damages in the retina (Abcouwer S F, et al., Invest. Ophthal. & Vis. Sci. 2008; 49:5581-5592; Kowluru R A, Odenbach S. Invest. Ophthal. & Vis. Sci. 2004; 45:4161-4166; Liu Y. et al., PloS one 2012; 7:e36949). Therefore, it was proposed that FGF21 (PF-05231023) regulates retinal NRF2 levels to reduce IL-1.beta. production and photoreceptor dysfunction in DR.

[0198] Photoreceptor high energy consumption makes it susceptible to neurovascular disease. Blood vessels supply nutrients and oxygen to neurons, and evacuate waste. Disturbances in neuronal activity trigger vascular remodeling (Fulton A B, et al., Documenta Ophthalmologica Advances in Ophthalmology 2009; 118:55-61). In diabetic animal models, photoreceptor responses to hyperglycemia induce retinal blood vessel loss (Du Y. et al., Proc. Nat'l. Acad. Sci. USA 2013; 110:16586-165916; Liu H., et al., Invest. Ophthal. &Vis. Sci. 2016; 57:4272-4281; Tonade D. et al., Invest. Ophthal. & Vis. Sci. 2016; 57:4264-4271). In addition, low rod sensitivity is associated with abnormal retinal vasculature. Rod photoreceptor demands contribute to the vascular recovery in hypoxia-induced retinal neovascularization (Akula J D et al., Invest. Ophthal. & Vis. Sci. 2007; 48:4351-4359). Photoreceptor metabolic dysfunction dictates pathological retinal angiogenesis (Joyal J S et al., Nature Medicine 2016; 22:439-445). Therefore, maintaining photoreceptor function may prevent vascular abnormalities in DR. In insulin-deficient Akita mice, reduced sensitivity was found in the post-receptor retina, in line with clinical observations (Pescosolido N., et al., J. Diabetes Res. 2015; 2015:319692). Meanwhile, the changes in post-receptor cells were actually reflecting the deficits in photoreceptor function (Hansen R M, et al., Prog. Retinal and Eye Res. 2017; 56:32-57; Hood D C, et al. Vis. Neurosci. 1992; 8:107-126). Administration of a FGF21 analogue, PF-05231023, reversed the diabetes-induced morphological changes in photoreceptors and restored retinal sensitivity; it also reduced disorganization of the photoreceptor segments. This provides evidence that PF-05231023''s effect on retinal function ERG may be due to improved photoreceptor structure and function.

[0199] There is a strong correlation between hyperglycemia and the development of DR. Hyperglycemia leads to many cellular metabolic alterations that could serve as therapeutic targets. However, while pharmacologic interventions that disrupt putative biochemical signaling pathways between hyperglycemia and DR, an effective and safe drug is not yet available. Type 1 diabetic patients have low circulating FGF21 levels (Zibar K, et al., Endocrine 2014; Xiao Y, et al., J. Clin. Endocrinol. Metabol. 2012; 97:E54-58) and FGF21 administration reduces hyperglycemia and lessens renal dysfunction in type 1 diabetic mice (Jiang X, et al., Toxicol. Letters 2013; 219:65-76). FGF21 also improves the lipid profile (decreased triglycerides) of obese monkeys and type 2 diabetic patients (Talukdar S, et al., Cell Metabolism 2016; 23:427-440), indicating that FGF21 may have positive effects on diabetes and diabetic complications.

[0200] No significant impact of PF-05231023 administration on serum triglyceride levels in either Akita or STZ-induced diabetic mice. Although PF-05231023 slightly reduced hyperglycemia in Akita mice, this finding was not replicated in STZ-induced mice. The protective effects of PF-05231023 on retinal neurons is, therefore, likely to be independent of circulating glucose and lipid modulation. It was also discovered that PF-05231023 protection against retinal neuronal deficits was preserved with APN deficiency. In the oxygen-induced retinopathy mouse model of late vaso-proliferative retinopathy, FGF21 inhibits pathologic neovessel growth mediated by APN (Fu Z, et al., Cell Reports 2017; 18:1606-1613). The current findings indicate that FGF21 regulates retinal neuron and neovessel growth through other mechanisms.

[0201] Oxidative stress resulting from highly metabolic photoreceptors induces inflammation, which induces DR (Du Y, et al., Proc. Nat'l. Acad. Sci. USA 2013; 110:16586-16591; Liu H, et al., Inv. Ophthal. & Vis. Sci. 2016; 57:4272-4281; Tonade D. et al., Invest. Ophthal. & Vis. Sci. 2016; 57:4264-4271; Joussen A M et al., FASEB J. 2004; 18:1450-1452; Kern T S, Berkowitz B A: J. Diabetes Invest. 2015; 6:371-380). Modulating oxidative stress prevents the progression of DR (Williams M, et al., Current Diabetes Reports 2013; 13:481-487). PF-05231023 administration attenuated the diabetes-induced IL-1.beta. expression in Akita mice. FGF21 reduces oxidative stress and inhibits the NF.kappa.B pathway in mice (Yu Y, et al., International Immunopharmacology 2016; 38:144-152). In photoreceptors in vitro with paraquat-induced oxidative stress, it was observed that PF-05231023 treatment decreased IL-1.beta. expression through the activation of the NRF2 pathway, which is known for its antioxidant capability (Xiong W, et al., J. Clin. Invest. 2015; 125:1433-1445) and regulation of IL-1.beta. transcription (Yu Y, et al., 2016; Kobayashi E H, et al., Nature Communications 2016; 7:1162443; 44). Additionally, PF-05231023-induced effect on IL-1.beta. was independent of APN in diabetic retinas, in line with the neuronal observation. APN inhibits retinal neovessel growth via TNF.alpha. (Higuchi A, et al., Circulation Research 2009; 104:1058-1065) and FGF21 also reduces TNF.alpha. in neovascular mouse retinas (Fu Z, et al., 2017). However, in Akita mice, there was no significant change in retinal Tnf.alpha. expression between PF-05231023- and vehicle-treated groups. Taken together, it was concluded that in diabetic retinas, PF-05231023 protected neuronal activity through the NRF2-IL-1.beta. pathway, which was at least to some degree independent of the APN-TNF.alpha. pathway that the inventors showed to be involved in retinal neovascularization in OIR (Fu Z, et al., Am. J. Clin. Nutrition 2015; 101:879-888). IL-10 causes a decline in mitochondrial membrane potential and ATP production in retinal neurons (Abcouwer S F, et al., Invest. Ophthal. & Vis. Sci. 2008; 49:5581-5592). Reduction of retinal IL-10 may prevent the induction of early vessel loss in DR as IL-1.beta. induces retinal microvascular abnormalities in rats (Kowluru R A, Odenbach S. Invest. Ophthal. & Vis. Sci. 2004; 45:4161-4166; Liu Y, et al., PloS one 2012; 7:e36949).

[0202] In this study, PF-05231023 was administrated intraperitoneally and circulating FGF21 levels were measured. PF-05231023 administration did not alter retinal Fgf21, Fgfr1 and Klb expression in Akita mice (FIGS. 24A-24C). These data implicate circulating/peripheral FGF21 as a primary driver of retinal protection rather than autocrine/paracrine effects of FGF21 in the retina. Although FGF21 is expressed in liver, white adipose tissue and brown adipose tissue, it is a hepatokine and liver is the primary source of circulating FGF21 under fasting and refeeding conditions (Markan K R, et al., Diabetes 2014; 63:4057-4063). In humans, liver is also the primary source of circulating FGF21 in a pattern consistent with a hormonal response (Yang C, et al., BMC Gastroenterology 2013; 13:67). While liver-derived FGF21 is critical for the adaptive response to fasting or starvation in rodents, in humans, FGF21 plays an important role in fructose metabolism (Dushay J R, et al. Molecular Metabolism 2015; 4:51-57). Circulating FGF21 has been shown to cross the blood brain barrier in humans in a non-saturable, unidirectional manner (Hsuchou H, et al., Peptides 2007; 28:2382-2386). FGF21 regulates metabolism and circadian behavior, sweet and alcohol preferences by directly acting on the nervous system (Bookout A L, et al. Nature Medicine 2013; 19:1147-1152; Talukdar S, et al. Cell Metabolism 2016; 23:344-349). The blood retinal barrier (BRB), which is essential for normal visual function (Cunha-Vaz J: The Blood-Retinal Barrier in the Management of Retinal Disease: EURETINA Award Lecture. Ophthalmologica Journal international d'ophtalmologie International journal of ophthalmology Zeitschrift fur Augenheilkunde 2017; 237:1-10), is broken down in DR (Klaassen I, et al. Progress in Retinal and Eye Research 2013; 34:19-48). The leaky BRB potentiates the transport of FGF21 from blood into retina. As local expression of FGF21 receptors has been detected in total retina and in retinal neurons, circulating FGF21 could directly act on retinal neurons to exert protective effects in DR.

[0203] In summary, there is an unmet need for the prevention and treatment of DR. Maintaining retinal structure and function particularly photoreceptor activity improves retinal vascular stability, which can be achieved by two ways: i) modulating photoreceptor metabolism to match the energy supply; ii) slowing down the visual cycle to reduce the energy demand.

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EQUIVALENTS

[0259] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence CWU 1

1

491180PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 1Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg1 5 10 15Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu 20 25 30Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro 35 40 45Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile 50 55 60Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala65 70 75 80Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu 85 90 95Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly 100 105 110Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Cys 115 120 125Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala 130 135 140Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly145 150 155 160Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro 165 170 175Ser Tyr Ala Ser 18025324DNAHomo sapiens 2cacagcacag caggatgact gcgggcaggc ctagataata cccagcctcc cacaagaagc 60tggtggagca gagtgttccc tgactcctcc aagaaaagga gattcccttt cgtggtctgc 120tgagtaacgg gtgccttccc agacactggc gttcccgctt gaccaaggag ccctcaagag 180gcccttatgc cggtgtgaca gaaggctcac ctcttgcctt ctaggtcact tctcacaatg 240tcccttcagt acctgaccct atacccaccg gttgtttcct ggttatatta gttatacaac 300aaagaataaa agtaatagct aatgattaat aatgtttaca ctaatgattg atactgtcca 360tgatcatctc tatatctaat ttgtatgata actattctta ttctaactat tttctttatt 420atactgaaac agtttgtgcc ttcagtctct tgcctcggca cctgggtaat ccttccccac 480agactgaccc tcccattcaa gatacatcaa tgtcaaagac tcaggagttt gacttgattc 540ccagaagttt aaccatcatc tccccaggct cgggactccc agcacccaga cccttctgct 600cacacccagc agtccaggcc cccagaccct cctccctcag acttaggagt ccaggctccc 660ggcccctcct tcctcagacc caggagtcca agccccctgc ccctccttcc tcagacccag 720gagtccagga ccccagcccc tccttcctca cacccacgag tccagatccc tagcccctac 780tccctcagac ccaggagtcc agaccaaagc tccctcctcc ctcagaccca ggagcccaag 840ttccccagcc cctcctccct cagatccagg agtacaggcc cagcccctcc tccctcagac 900ccctcctccc tcagatccag gagtacaggc ccagaccctc ctccctcaga cccaggagtc 960caggcccccc acccctcctc cctcagaccc aggagtccag agccccagcc ctcctccctc 1020agacacagaa ggcctaccct tgcaccctta ggggctccag gaaattagcc aacctgtctt 1080ccctctgggt gcccactcca gggcctggct tggctgccaa ctccagtcag ggactttcag 1140ccacccctcc ccccaggtta tttcaggagc acctgcctgg gcctgggatg gcttctctgg 1200tgaaagaaac accaggattg catcagggag gaggaggctg ggatgtccag ggtctgagca 1260tctgagcagg gacagatgag gttgaggttg gcccacggcc aggtgagagg cttccaaggc 1320aggatacttg tgtctcagat gcggtcgctt ctttcataca gcaattgccg ccttgctgag 1380gatcaaggaa cctcagtgtc agatcacgcc ctccccccaa acttagaaat tcagatgggg 1440cgcagaaatt tctcttgttc tgcgtgatct gcatagatgg tccaagaggt ggtttttcca 1500ggagcccagc acccctcctc cctccgactc aggtgcttga gaccccagat ccttctctct 1560gagactcagg aatgtgggcc cccagcccct ttcacctggg tcccagctaa cccgatcctc 1620ccctccctca tcccctagac ccaggagtct ggccctccat tgaaaggacc ccaggttaca 1680tcatccattc aggctgccct tgccacgatg gaattctgta gctcctgcca aatgggtcaa 1740atatcatggt tcaggcgcag ggagggtgat tgggcgggcc tgtctgggta taaattctgg 1800agcttctgca tctatcccaa aaaacaaggg tgttctgtca gctgaggatc cagccgaaag 1860aggagccagg cactcaggcc acctgagtct actcacctgg acaactggaa tctggcacca 1920attctaaacc actcagcttc tccgagctca caccccggag atcacctgag gacccgagcc 1980attgatggac tcggacgaga ccgggttcga gcactcaggg ctgtgggttt ctgtgctggc 2040tggtcttctg ctgggagcct gccaggcaca ccccatccct gactccagtc ctctcctgca 2100attcgggggc caagtccggc agcggtacct ctacacagat gatgcccagc agacagaagc 2160ccacctggag atcagggagg atgggacggt ggggggcgct gctgaccaga gccccgaaag 2220tgagtgtggg ccagagcctg ggtctgaggg aggaggggct gtgggtctgg attcctgggt 2280ctgagggagg aggggctggg ggccttggcc cctgggtctg agggaggagg ggctggggat 2340ctggactcct gggtctgagg gaggaggggc tggggatctg ggcccctggg tctgagggag 2400gaggggctgg gtctggaccc ctgggtctga gggaggaggg gctgggggtc tggactcttg 2460ggtttgaaga aggaagggct ggggtcctgg actcttgggt ctgagttggg agggggcttt 2520ggcttgggct tctcctgggt ctgagggagg aggtaggctg tgggcttgga ctcccagggc 2580tgggacagag ccggatggtg ggacagagtc gggtggtggg acagtcccgg gtgggagagg 2640tcctcgaacc accttatcgc tttcacccct taggtctcct gcagctgaaa gccttgaagc 2700cgggagttat tcaaatcttg ggagtcaaga catccaggtt cctgtgccag cggccagatg 2760gggccctgta tggatcggtg agtttccagg accctcctca ccacccacca tgctcctcct 2820atatgtcgcc ctcacagcct ggggtgcctt gtcttgctca tcccccccgg agccagactt 2880gattctattt gctctgcacg cccccagctg caacatttgg aggttgaagt tgtcatcagt 2940gtttgcaaga tgaggaaact gaggcccagg ccggggcgcc agtgacctca atcatgtgat 3000gtgtggatgc tggagcggcc tgaggctcag gttattggga gtctcgtgat tcagtaaccc 3060ctgctcctgc ccacacggcc cctgtgtgca cggctcatgc tgggcacagg gacactcggg 3120gaagccatgg ccagtaaagt gaccaggacc ttgagtgcta gggagacacc ccgcctggcc 3180tgagagagca ctgatggctc cgagggctgg aatgttctct gtgaagtctg aactgggagg 3240caggtccctg caggagagcc ctggggtaaa aaacaaaacc tgccttgctg ttttgtttcc 3300tagaggaggg gctgggggcc tggactcctg ggtctgaggg aggaggggct gggggcctgg 3360acccctgggt ctgagggagg aggggctggg ggcctggaac cccgggtctg agggaggaga 3420ggctggggcc tggaaccccg ggtctgaggg aggagaggct ggggcctgga accccgggtc 3480tgagggagga ggcgctgggg gcctggactc ctgggtcgga tggaggagaa actagggtct 3540ggacccctgg gtctgaggga ggaggcgctg ggggcctgga cccctgggtc tgagggaggc 3600agggctgggg cctggatcct gggtcttaca tcaggaaaac agaggaaccc tgtctctgat 3660cctgtttttg tcccctagct ccactttgac cctgaggcct gcagcttccg ggagctgctt 3720cttgaggacg gatacaatgt ttaccagtcc gaagcccacg gcctcccgct gcacctgcca 3780gggaacaagt ccccacaccg ggaccctgca ccccgaggac cagctcgctt cctgccacta 3840ccaggcctgc cccccgcacc cccggagcca cccggaatcc tggcccccca gccccccgat 3900gtgggctcct cggaccctct gagcatggtg ggaccttccc agggccgaag ccccagctac 3960gcttcctgaa gccagaggct gtttactatg acatctcctc tttatttatt aggttattta 4020tcttatttat ttttttattt ttcttacttg agataataaa gagttccaga ggaggataag 4080aatgagcatg tgtgagtgtc tgagggaaga catggcagct gttttgtctc ccttggcccg 4140gacaatcccc tctacacctc ccctcacgtg gtccgagggt cctggcttcc cactgggcct 4200cacttttttc ttttcttttc tttttttttt tttgagacgg agtctcgctc tgtcacccag 4260gctggagtgc agtggcgcga tcttggctca ctccaacctc cgcctcccag gttcaagcaa 4320ttctcctgcc tcagccaccc gagtagctgt gattacaggc gtgcgccacc acacccagct 4380aattttgtaa ttttagtaga gacagggttt cgccatgttg gccaggatgc tctccatctc 4440ttgacttcat gacctgcctg ccttggcctc ccaaagtgct gggattacag gcttgagtca 4500ctgtgcccag cccagcctca cttttctact ctgctaaagt gtccccaggg actgtggact 4560atccctgctc tctgaaagga caagactggc cgggagtggt ggcttacgcc tgtaatccca 4620gcactttggg aggccgaggc aggtggatca cgaggtcagg agattgagac tatcctggct 4680aatacgatga aaccccgtct ctactaaaaa tacaaaaaca aaattagctg ggcgtggtgg 4740cgggcgcctg tagtcccagc tactccggag gctgaggcag aatggcgtga atgcgggagg 4800cggagcttgc agtgagccga gatcgcgcca ctgcactcca gcccaggcca cagagcgaga 4860ttccatctca aaaaaataaa taaataaata aataaataaa taaatataaa aataaaatga 4920aagagcagga cttctttcta caacccctca acttgtgtga gcgttgtgta actatttcat 4980agagctacct caataacagg ggagctttta cgaggtgaca cagcacactc acatcctcat 5040gggagatgta gttttctggc atcatttagc agcaggaatg agatctgttg ggcctcaaat 5100ctgggacaag gactcctggg tcctggagta ggtttggggc tagtgtaaca cccaagttct 5160ggggaatcag tgggctggac atctggacac ctggatcaca ggagaactgg ggactgcaga 5220cttaggcatc ctggtctgag aaaaaagggg ctggagggtg ggagtttggg ttctcaggaa 5280aaggagctga aacctggaat tcttccatct gggtccttat gaac 532431332DNAHomo sapiens 3gaggcttcca aggcaggata cttgtgtctc agatgcggtc gcttctttca tacagcaatt 60gccgccttgc tgaggatcaa ggaacctcag tgtcagatca cgccctcccc ccaaacttag 120aaattcagat ggggcgcaga aatttctctt gttctgcgtg atctgcatag atggtccaag 180aggtggtttt tccaggagcc cagcacccct cctccctccg actcagaccc aggagtctgg 240ccctccattg aaaggacccc aggttacatc atccattcag gctgcccttg ccacgatgga 300attctgtagc tcctgccaaa tgggtcaaat atcatggttc aggcgcaggg agggtgattg 360ggcgggcctg tctgggtata aattctggag cttctgcatc tatcccaaaa aacaagggtg 420ttctgtcagc tgaggatcca gccgaaagag gagccaggca ctcaggccac ctgagtctac 480tcacctggac aactggaatc tggcaccaat tctaaaccac tcagcttctc cgagctcaca 540ccccggagat cacctgagga cccgagccat tgatggactc ggacgagacc gggttcgagc 600actcaggact gtgggtttct gtgctggctg gtcttctgct gggagcctgc caggcacacc 660ccatccctga ctccagtcct ctcctgcaat tcgggggcca agtccggcag cggtacctct 720acacagatga tgcccagcag acagaagccc acctggagat cagggaggat gggacggtgg 780ggggcgctgc tgaccagagc cccgaaagtc tcctgcagct gaaagccttg aagccgggag 840ttattcaaat cttgggagtc aagacatcca ggttcctgtg ccagcggcca gatggggccc 900tgtatggatc gctccacttt gaccctgagg cctgcagctt ccgggagctg cttcttgagg 960acggatacaa tgtttaccag tccgaagccc acggcctccc gctgcacctg ccagggaaca 1020agtccccaca ccgggaccct gcaccccgag gaccagctcg cttcctgcca ctaccaggcc 1080tgccccccgc actcccggag ccacccggaa tcctggcccc ccagcccccc gatgtgggct 1140cctcggaccc tctgagcatg gtgggacctt cccagggccg aagccccact acgcttcctg 1200aagccagagg ctgtttacta tgacatctcc tctttattta ttaggttatt tatcttattt 1260atttttttat ttttcttact tgagataata aagagttcca gaggaggata aaaaaaaaaa 1320aaaaaaaaaa aa 13324947DNAMus musculus 4agacagcctt agtgtcttct cagctgggga ttcaacacag gagaaacagc cattcacttt 60gcctgagccc cagtctgaac ctgacccatc cctgctgggc accggagtca gaacacaatt 120ccagctgcct tggctcctca gccgctcgct tgccaggggc tctcccgaac ggagcgcagc 180cctgatggaa tggatgagat ctagagttgg gaccctggga ctgtgggtcc gactgctgct 240ggctgtcttc ctgctggggg tctaccaagc ataccccatc cctgactcca gccccctcct 300ccagtttggg ggtcaagtcc ggcagaggta cctctacaca gatgacgacc aagacactga 360agcccacctg gagatcaggg aggatggaac agtggtaggc gcagcacacc gcagtccaga 420aagtctcctg gagctcaaag ccttgaagcc aggggtcatt caaatcctgg gtgtcaaagc 480ctctaggttt ctttgccaac agccagatgg agctctctat ggatcgcctc actttgatcc 540tgaggcctgc agcttcagag aactgctgct ggaggacggt tacaatgtgt accagtctga 600agcccatggc ctgcccctgc gtctgcctca gaaggactcc ccaaaccagg atgcaacatc 660ctggggacct gtgcgcttcc tgcccatgcc aggcctgctc cacgagcccc aagaccaagc 720aggattcctg cccccagagc ccccagatgt gggctcctct gaccccctga gcatggtaga 780gcctttacag ggccgaagcc ccagctatgc gtcctgactc ttcctgaatc tagggctgtt 840tctttttggg tttccactta tttattacgg gtatttatct tatttattta ttttagtttt 900tttttcttac ttggaataat aaagagtctg aaagaaaaat gtgtgtt 9475210PRTMus musculus 5Met Glu Trp Met Arg Ser Arg Val Gly Thr Leu Gly Leu Trp Val Arg1 5 10 15Leu Leu Leu Ala Val Phe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile 20 25 30Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg 35 40 45Tyr Leu Tyr Thr Asp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile 50 55 60Arg Glu Asp Gly Thr Val Val Gly Ala Ala His Arg Ser Pro Glu Ser65 70 75 80Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly 85 90 95Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr 100 105 110Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu 115 120 125Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro 130 135 140Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp145 150 155 160Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln 165 170 175Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser 180 185 190Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr 195 200 205Ala Ser 2106208PRTHomo sapiens 6Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser1 5 10 15Val Leu Ala Gly Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro Asp 20 25 30Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu 35 40 45Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu 50 55 60Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu65 70 75 80Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys 85 90 95Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser 100 105 110Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu 115 120 125Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His 130 135 140Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro145 150 155 160Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro 165 170 175Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro 180 185 190Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser 195 200 20573247DNAHomo sapiens 7accctgggcc tggcccaaga aactatacat tcctcctggg aatctggggc tgtgatggga 60ggggttgcca tgaagacttc tgacatgccc tggagacatt ttccccatgg tcttggggat 120taacattcag ctccttgtta cttatgcaaa tttctgcagc tggcttgaat ttctcctcag 180aaaatgagat tttcttttct atcgcattgt caggctgcaa attttccaaa cttttgtgct 240ctgcttccct tataaaactg aaggcctggc caggtgtggt ggctcacgcc tgtaatccca 300gcactttggg gagctgagac gggcgaatca cgaggtcagg agttcgagac cagcctggcc 360aacatggtga aaccctgtct ctactaaaaa tacaaaaagt tagctgggca tagtcatggg 420tgcctttaat cccagctact tgggagactg aggcaggaga atcgtttgaa tccaggaggc 480ggaggttgca gtgagtcgag atcacaccac tgcactccag ccctggtaac atagtaagac 540tctgtctcaa aaaaaaaaaa aaaaaaacaa aactgaatgc ctttaacaac acccaagttg 600cttcttgaat gctttgctgc ttagaaattt cttctgccag ataccctaaa tcatctctct 660taggttccaa gttccacaaa tctctagggc agggacaaaa cgctgccagt ctctttacta 720aaacataaca agagtcaccc ttgctctagt tcccaaaaag ttcctcatct ccatctgaaa 780ccacccccgc ctagatttcg ttgtccatat cattatcagc attttggtca aagtcattca 840acaggcctct agggagttcc aaacttgctc acattctcct gtcttcttct gagccctcca 900aactgttcca atccctacct gttacccagt tccaaagtcg cttccacatt tttggttatc 960ttttcagccg tgccccactc tactggtacc agtttactgt attagtcgat tttcatgctg 1020ctgataaaga catacctgaa actggacaat ttacaaaaga aagaggttta ttggacttac 1080aattctacat cacttgggag gcctcacaat catgatggaa ggagaaaggc acatctcaca 1140tggcagcaga caagaaaaga gcttgtgcag ggaaactcct ctttttaaaa ccatcagatc 1200tcatgaaatt tattcattat catgacaata gcacaggaaa gaactgcacc cataattcag 1260tcacctccta ccaggttcct cccacaacac gtgagaattc aagatgagat ttggatgggg 1320acacagccaa accatgtcac actaccatgc ctgacttcct ttccattttt gtatatttgc 1380ttgttcttca tttgcccgag aagtaactct aaagggctgt attatttgga tattagattg 1440gcattttatc tgactgggat atcttgctgt gattgtccat gtataagatc agcttttcta 1500taaaccatat ttttaaaaag atatattaat tttttaaaaa tccacctgtc taaataaatg 1560cacaaagccc cccaaaaacc tagattctaa gaaaaatcta tgtactgcca tacaatgatt 1620gatattaata tttatggtga taaattacac acaaaaaatg tgtgatctct gtttaaacag 1680gcaaaaacaa aaaacacatg aaataaatct atggcatcta tagccaaaac tggaaacaac 1740ccacatatcc atcaatagga aatcagttaa ataaattata gtacatttat ccaatggaag 1800attaagcaca tattcaatat aattatttat acacacatat agatacacac atgtataaat 1860atagagaata ctgtgggtgt atgtgtgtgt gtgtttatat acatatatat acacacacag 1920tactgttgcc taccttcttt tgtcttaatt ctgtgaactc tcattcactc tgcttcagta 1980ggatacatcc ttctttttgg ttcttagact caccaagttg atccttgact caagacattg 2040catttgctgc ttcctcttcc tggaatatcc ttccttctga tattcacatg agtagtctct 2100tcttgtcatt cagatctcaa atgtcacaat ttcagagagc ccatctctga tcatcatatc 2160taaagttgtc ctcattcccc catagctttc tataccatgt tttatttttt tcataacatg 2220tattttatta ctcctttctc cattggaata gaatctccat tagattagga aatctgccta 2280tcttattaat gcctgcaact ggaatacttt tgaagagttc ttggcacgta ataaatactc 2340aactaatatt tttgtgtaca cagaaataaa gtttggaaga acagatgcca aattgttact 2400agtggttact tctgagtaaa ggagtagcat ggtaggtaaa ttattaatag atgttcactt 2460tccaccaaga tatgttttag ttagtcttaa cttacttgaa atgaaattta ttactttaat 2520aattagaaac attgataaac attttagtca caagaatgat agataaaatt ttgatgcttc 2580caataagtta tatttatcta gaggatgcac ttatgtagaa tactctcttg aggatgttag 2640gtgagtaaca tgttactata tgtagtaaaa tatctatgat tttataaaag cactgaaaca 2700tgaagcagca gaaacgtttt tcccagttct ctttcctctg aacttgatca ccgtctctct 2760ggcaaagcac ctaaattaat tcttctttaa aagttaacaa gaccaaatta taagcttgat 2820gaataactca ttcttatctt tctttaaatg attatagttt atgtatttat tagctatgcc 2880catcttaaac aggtttattt gttcttttta cacataccaa actcttaata ttagctgttg 2940tccccaggtc cgaatgttaa gtcaacatat atttgagaga acttcaactt atcaagtatt 3000gcaggtctct gattgctttg gaaccacttc tgatacctgt ggacttagtt caaggccagt 3060tactaccact tttttttttc taatagaatg aacaaatggc taattgtttg ctttgtcaac 3120caagctcaag ttaatggatc tggatactat gtatataaaa agcctagctt gagtctcttt 3180tcagtggcat ccttcccttt ctaatcagag attttcttcc tcagagattt tggcctagat 3240ttgcaaa 324786079DNAHomo sapiens 8atcctcagtc tcccagttca agctaatcat tgacagagct ttacaatcac aagcttttac 60tgaagctttg ataagacagt ccagcagttg gtggcaaatg aagccaggct gtgcggcagg 120atctccaggg aatgaatgga ttttcttcag cactgatgaa ataaccacac gctataggaa 180tacaatgtcc aacgggggat tgcaaagatc tgtcatcctg tcagcactta ttctgctacg

240agctgttact ggattctctg gagatggaag agctatatgg tctaaaaatc ctaattttac 300tccggtaaat gaaagtcagc tgtttctcta tgacactttc cctaaaaact ttttctgggg 360tattgggact ggagcattgc aagtggaagg gagttggaag aaggatggaa aaggaccttc 420tatatgggat catttcatcc acacacacct taaaaatgtc agcagcacga atggttccag 480tgacagttat atttttctgg aaaaagactt atcagccctg gattttatag gagtttcttt 540ttatcaattt tcaatttcct ggccaaggct tttccccgat ggaatagtaa cagttgccaa 600cgcaaaaggt ctgcagtact acagtactct tctggacgct ctagtgctta gaaacattga 660acctatagtt actttatacc actgggattt gcctttggca ctacaagaaa aatatggggg 720gtggaaaaat gataccataa tagatatctt caatgactat gccacatact gtttccagat 780gtttggggac cgtgtcaaat attggattac aattcacaac ccatatctag tggcttggca 840tgggtatggg acaggtatgc atgcccctgg agagaaggga aatttagcag ctgtctacac 900tgtgggacac aacttgatca aggctcactc gaaagtttgg cataactaca acacacattt 960ccgcccacat cagaagggtt ggttatcgat cacgttggga tctcattgga tcgagccaaa 1020ccggtcggaa aacacgatgg atatattcaa atgtcaacaa tccatggttt ctgtgcttgg 1080atggtttgcc aaccctatcc atggggatgg cgactatcca gaggggatga gaaagaagtt 1140gttctccgtt ctacccattt tctctgaagc agagaagcat gagatgagag gcacagctga 1200tttctttgcc ttttcttttg gacccaacaa cttcaagccc ctaaacacca tggctaaaat 1260gggacaaaat gtttcactta atttaagaga agcgctgaac tggattaaac tggaatacaa 1320caaccctcga atcttgattg ctgagaatgg ctggttcaca gacagtcgtg tgaaaacaga 1380agacaccacg gccatctaca tgatgaagaa tttcctcagc caggtgcttc aagcaataag 1440gttagatgaa atacgagtgt ttggttatac tgcctggtct ctcctggatg gctttgaatg 1500gcaggatgct tacaccatcc gccgaggatt attttatgtg gattttaaca gtaaacagaa 1560agagcggaaa cctaagtctt cagcacacta ctacaaacag atcatacgag aaaatggttt 1620ttctttaaaa gagtccacgc cagatgtgca gggccagttt ccctgtgact tctcctgggg 1680tgtcactgaa tctgttctta agcccgagtc tgtggcttcg tccccacagt tcagcgatcc 1740tcatctgtac gtgtggaacg ccactggcaa cagactgttg caccgagtgg aaggggtgag 1800gctgaaaaca cgacccgctc aatgcacaga ttttgtaaac atcaaaaaac aacttgagat 1860gttggcaaga atgaaagtca cccactaccg gtttgctctg gattgggcct cggtccttcc 1920cactggcaac ctgtccgcgg tgaaccgaca ggccctgagg tactacaggt gcgtggtcag 1980tgaggggctg aagcttggca tctccgcgat ggtcaccctg tattatccga cccacgccca 2040cctaggcctc cccgagcctc tgttgcatgc cgacgggtgg ctgaacccat cgacggccga 2100ggccttccag gcctacgctg ggctgtgctt ccaggagctg ggggacctgg tgaagctctg 2160gatcaccatc aacgagccta accggctaag tgacatctac aaccgctctg gcaacgacac 2220ctacggggcg gcgcacaacc tgctggtggc ccacgccctg gcctggcgcc tctacgaccg 2280gcagttcagg ccctcacagc gcggggccgt gtcgctgtcg ctgcacgcgg actgggcgga 2340acccgccaac ccctatgctg actcgcactg gagggcggcc gagcgcttcc tgcagttcga 2400gatcgcctgg ttcgccgagc cgctcttcaa gaccggggac taccccgcgg ccatgaggga 2460atacattgcc tccaagcacc gacgggggct ttccagctcg gccctgccgc gcctcaccga 2520ggccgaaagg aggctgctca agggcacggt cgacttctgc gcgctcaacc acttcaccac 2580taggttcgtg atgcacgagc agctggccgg cagccgctac gactcggaca gggacatcca 2640gtttctgcag gacatcaccc gcctgagctc ccccacgcgc ctggctgtga ttccctgggg 2700ggtgcgcaag ctgctgcggt gggtccggag gaactacggc gacatggaca tttacatcac 2760cgccagtggc atcgacgacc aggctctgga ggatgaccgg ctccggaagt actacctagg 2820gaagtacctt caggaggtgc tgaaagcata cctgattgat aaagtcagaa tcaaaggcta 2880ttatgcattc aaactggctg aagagaaatc taaacccaga tttggattct tcacatctga 2940ttttaaagct aaatcctcaa tacaatttta caacaaagtg atcagcagca ggggcttccc 3000ttttgagaac agtagttcta gatgcagtca gacccaagaa aatacagagt gcactgtctg 3060cttattcctt gtgcagaaga aaccactgat attcctgggt tgttgcttct tctccaccct 3120ggttctactc ttatcaattg ccatttttca aaggcagaag agaagaaagt tttggaaagc 3180aaaaaactta caacacatac cattaaagaa aggcaagaga gttgttagct aaactgatct 3240gtctgcatga tagacagttt aaaaattcat cccagttcca tatgctggta acttacagga 3300gatatacctg tattatagaa agacaatctg agatacagct gtaaccaagg tgatgacaat 3360tgtctctgct gtgtggttca aagaacattc ccttaggtgt tgacatcagt gaactcagtt 3420cttggatgta aacataaagg cttcatcctg acagtaagct atgaggatta catgctacat 3480tgcttcttaa agtttcatca actgtattcc atcattctgc tttagctttc atctctacca 3540atagctactt gtggtacaat aaattatttt taagaagtaa aactctgggg ctggacgctg 3600tggctcacac ctgtaatctc agcactttgg gaggccgagg cggggagatc acctgaggtg 3660aggagttcga gaccagcctg gccaacatgg tgaaaccatg tctctactaa aaatacaaaa 3720aattagccag gcgtggtgac agtggcacct gtaatcccag ctacttggga ggctgaggca 3780gaagtttgaa cccaggaaac aggttacagt aggccaaaat tgcgccactg cactccagcc 3840taggcgacaa cagcaagact gtgtccaaaa aaaaaaaaaa aagcaaaagc aaaactttgt 3900tttgttagac tctacagcag agatttaaca cccttcttta aactgggtag tcagtgatag 3960ataatatata ttctgtcact tctaataagg tgccttctcc tttaggtcag ggtggttcta 4020aaatggaaag aaaacacaat agggtaagta gtgcttgtct aagccagtta caacacagac 4080tcttaaagag gatcaagccc ttcatttttc taacaacaaa aaatcaccta tagaatatct 4140aatttgtgat cttttactag atctgatttt ttaaaataat gtaatttccg gccaggcacg 4200gtggcaccgc ctgtaatccc agcactttgg gaggccaagg caggtggatc acctgaggtt 4260aggagttcga gactagcctg gccaacatgg caaaacccca tctctactaa aaatacaaaa 4320gttagccggg catggtggtg ggcacctgta atcccagcta ctcaggaggc cgaggcagga 4380gaatcgcttg aacccgagag gcagaggttg caatgagcca agatcgtgcc attgcactcc 4440agcctggggg acagggcaag actgtctctc aaaataaaaa aaaataataa aaataaaaat 4500aaaagtaatt tccaaaacct catctcatgg aaagatcaca ggatgaagga aagctagact 4560caactctgtg aatagaagtt gctatactgt aagtaaagca acaattcaga atactgaatg 4620agtttaaatt gttttatata gcaccctttt gggctagggt taattactag atctgacttg 4680gataatttga cactttggga aatgaactct gttcttgaga cttgttcagt gtattttaaa 4740catctgagga agaaaactta aatatgcacc tatttatacc tattctttct ttaggtcaac 4800atttaacacc cactgcatac attaatttgt ccttgtctgc tcactccagc aatttagacc 4860ttaacagtca caagagacgt tcttctgtta caaagcctta gtaaattaag gcagttttga 4920ttatattcta ggtccaccta tgtctgaagc taaattcagt atctaactgc taatgaacaa 4980gtttccaaaa tactgtaaaa atacaattag tcaatttgag taaatgcaaa tatgatgaga 5040aatcaatttg ctatttggcc tggcaaatgg gaacagtaaa attctgcttt actcttctct 5100agtctccttg ccccagctgc acccactacc ccaaagttgg cagttttgag gtatgatttt 5160caaggaattt ttttagtatt aacatctccc tctgagaact atgtacctaa ggtcacgcat 5220acaactagtc aattctgttt ttattactct aactatgtag aaacagtaag tcacttaaaa 5280caatcacttg gctgggtttt ttcccctttg tgccacattg attcaccctg acccaagaac 5340tccagggaaa attctttaat gtcaactggg caactcatta acctctcttt aacatcaagg 5400gcttgggaaa aaaaaaaaaa aggttagcca caggaataac aaaaacctgg aatttatctt 5460tcaggttttg ctttctcttt ctcactttgt ttaaagtatc tcgtactcac agttcacaaa 5520ttaaccttca ctgtctcttt cacattaaga gcttatgctt aaagcatgcc ccccttttct 5580aacttgctgg tttaccataa actcccctaa gtaataaaat tcctaaccca gtactgagag 5640tcctccttct ctgccacttg ggcattattt tactagtttt taagccatca tcgcacaaga 5700atccaaaaac ccttaaattt tttaaccact ggcaaatatg tacagcaaat taggttaagc 5760atttaatctg gctcatgctc tatcatacta aatattcagg tttatcataa actccttaaa 5820aaccatcaaa ggtcaaccag aaactgataa ctcttgaaag gagcaaacag gtaagatctt 5880tggagtttaa gcttttctga gatgtgttgt gaaaaatcta acgtgtttat cgtatattca 5940atgtaacaac ctggagaatc acaactatat ttaaagagcc tctggaaaat gaggccagta 6000cagtgtgact acatgtttaa ttttcaatgt aatttattcc aaataaactg gttcatgctg 6060accacttgta ttcaactaa 607991044PRTHomo sapiens 9Met Lys Pro Gly Cys Ala Ala Gly Ser Pro Gly Asn Glu Trp Ile Phe1 5 10 15Phe Ser Thr Asp Glu Ile Thr Thr Arg Tyr Arg Asn Thr Met Ser Asn 20 25 30Gly Gly Leu Gln Arg Ser Val Ile Leu Ser Ala Leu Ile Leu Leu Arg 35 40 45Ala Val Thr Gly Phe Ser Gly Asp Gly Arg Ala Ile Trp Ser Lys Asn 50 55 60Pro Asn Phe Thr Pro Val Asn Glu Ser Gln Leu Phe Leu Tyr Asp Thr65 70 75 80Phe Pro Lys Asn Phe Phe Trp Gly Ile Gly Thr Gly Ala Leu Gln Val 85 90 95Glu Gly Ser Trp Lys Lys Asp Gly Lys Gly Pro Ser Ile Trp Asp His 100 105 110Phe Ile His Thr His Leu Lys Asn Val Ser Ser Thr Asn Gly Ser Ser 115 120 125Asp Ser Tyr Ile Phe Leu Glu Lys Asp Leu Ser Ala Leu Asp Phe Ile 130 135 140Gly Val Ser Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg Leu Phe Pro145 150 155 160Asp Gly Ile Val Thr Val Ala Asn Ala Lys Gly Leu Gln Tyr Tyr Ser 165 170 175Thr Leu Leu Asp Ala Leu Val Leu Arg Asn Ile Glu Pro Ile Val Thr 180 185 190Leu Tyr His Trp Asp Leu Pro Leu Ala Leu Gln Glu Lys Tyr Gly Gly 195 200 205Trp Lys Asn Asp Thr Ile Ile Asp Ile Phe Asn Asp Tyr Ala Thr Tyr 210 215 220Cys Phe Gln Met Phe Gly Asp Arg Val Lys Tyr Trp Ile Thr Ile His225 230 235 240Asn Pro Tyr Leu Val Ala Trp His Gly Tyr Gly Thr Gly Met His Ala 245 250 255Pro Gly Glu Lys Gly Asn Leu Ala Ala Val Tyr Thr Val Gly His Asn 260 265 270Leu Ile Lys Ala His Ser Lys Val Trp His Asn Tyr Asn Thr His Phe 275 280 285Arg Pro His Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly Ser His Trp 290 295 300Ile Glu Pro Asn Arg Ser Glu Asn Thr Met Asp Ile Phe Lys Cys Gln305 310 315 320Gln Ser Met Val Ser Val Leu Gly Trp Phe Ala Asn Pro Ile His Gly 325 330 335Asp Gly Asp Tyr Pro Glu Gly Met Arg Lys Lys Leu Phe Ser Val Leu 340 345 350Pro Ile Phe Ser Glu Ala Glu Lys His Glu Met Arg Gly Thr Ala Asp 355 360 365Phe Phe Ala Phe Ser Phe Gly Pro Asn Asn Phe Lys Pro Leu Asn Thr 370 375 380Met Ala Lys Met Gly Gln Asn Val Ser Leu Asn Leu Arg Glu Ala Leu385 390 395 400Asn Trp Ile Lys Leu Glu Tyr Asn Asn Pro Arg Ile Leu Ile Ala Glu 405 410 415Asn Gly Trp Phe Thr Asp Ser Arg Val Lys Thr Glu Asp Thr Thr Ala 420 425 430Ile Tyr Met Met Lys Asn Phe Leu Ser Gln Val Leu Gln Ala Ile Arg 435 440 445Leu Asp Glu Ile Arg Val Phe Gly Tyr Thr Ala Trp Ser Leu Leu Asp 450 455 460Gly Phe Glu Trp Gln Asp Ala Tyr Thr Ile Arg Arg Gly Leu Phe Tyr465 470 475 480Val Asp Phe Asn Ser Lys Gln Lys Glu Arg Lys Pro Lys Ser Ser Ala 485 490 495His Tyr Tyr Lys Gln Ile Ile Arg Glu Asn Gly Phe Ser Leu Lys Glu 500 505 510Ser Thr Pro Asp Val Gln Gly Gln Phe Pro Cys Asp Phe Ser Trp Gly 515 520 525Val Thr Glu Ser Val Leu Lys Pro Glu Ser Val Ala Ser Ser Pro Gln 530 535 540Phe Ser Asp Pro His Leu Tyr Val Trp Asn Ala Thr Gly Asn Arg Leu545 550 555 560Leu His Arg Val Glu Gly Val Arg Leu Lys Thr Arg Pro Ala Gln Cys 565 570 575Thr Asp Phe Val Asn Ile Lys Lys Gln Leu Glu Met Leu Ala Arg Met 580 585 590Lys Val Thr His Tyr Arg Phe Ala Leu Asp Trp Ala Ser Val Leu Pro 595 600 605Thr Gly Asn Leu Ser Ala Val Asn Arg Gln Ala Leu Arg Tyr Tyr Arg 610 615 620Cys Val Val Ser Glu Gly Leu Lys Leu Gly Ile Ser Ala Met Val Thr625 630 635 640Leu Tyr Tyr Pro Thr His Ala His Leu Gly Leu Pro Glu Pro Leu Leu 645 650 655His Ala Asp Gly Trp Leu Asn Pro Ser Thr Ala Glu Ala Phe Gln Ala 660 665 670Tyr Ala Gly Leu Cys Phe Gln Glu Leu Gly Asp Leu Val Lys Leu Trp 675 680 685Ile Thr Ile Asn Glu Pro Asn Arg Leu Ser Asp Ile Tyr Asn Arg Ser 690 695 700Gly Asn Asp Thr Tyr Gly Ala Ala His Asn Leu Leu Val Ala His Ala705 710 715 720Leu Ala Trp Arg Leu Tyr Asp Arg Gln Phe Arg Pro Ser Gln Arg Gly 725 730 735Ala Val Ser Leu Ser Leu His Ala Asp Trp Ala Glu Pro Ala Asn Pro 740 745 750Tyr Ala Asp Ser His Trp Arg Ala Ala Glu Arg Phe Leu Gln Phe Glu 755 760 765Ile Ala Trp Phe Ala Glu Pro Leu Phe Lys Thr Gly Asp Tyr Pro Ala 770 775 780Ala Met Arg Glu Tyr Ile Ala Ser Lys His Arg Arg Gly Leu Ser Ser785 790 795 800Ser Ala Leu Pro Arg Leu Thr Glu Ala Glu Arg Arg Leu Leu Lys Gly 805 810 815Thr Val Asp Phe Cys Ala Leu Asn His Phe Thr Thr Arg Phe Val Met 820 825 830His Glu Gln Leu Ala Gly Ser Arg Tyr Asp Ser Asp Arg Asp Ile Gln 835 840 845Phe Leu Gln Asp Ile Thr Arg Leu Ser Ser Pro Thr Arg Leu Ala Val 850 855 860Ile Pro Trp Gly Val Arg Lys Leu Leu Arg Trp Val Arg Arg Asn Tyr865 870 875 880Gly Asp Met Asp Ile Tyr Ile Thr Ala Ser Gly Ile Asp Asp Gln Ala 885 890 895Leu Glu Asp Asp Arg Leu Arg Lys Tyr Tyr Leu Gly Lys Tyr Leu Gln 900 905 910Glu Val Leu Lys Ala Tyr Leu Ile Asp Lys Val Arg Ile Lys Gly Tyr 915 920 925Tyr Ala Phe Lys Leu Ala Glu Glu Lys Ser Lys Pro Arg Phe Gly Phe 930 935 940Phe Thr Ser Asp Phe Lys Ala Lys Ser Ser Ile Gln Phe Tyr Asn Lys945 950 955 960Val Ile Ser Ser Arg Gly Phe Pro Phe Glu Asn Ser Ser Ser Arg Cys 965 970 975Ser Gln Thr Gln Glu Asn Thr Glu Cys Thr Val Cys Leu Phe Leu Val 980 985 990Gln Lys Lys Pro Leu Ile Phe Leu Gly Cys Cys Phe Phe Ser Thr Leu 995 1000 1005Val Leu Leu Leu Ser Ile Ala Ile Phe Gln Arg Gln Lys Arg Arg 1010 1015 1020Lys Phe Trp Lys Ala Lys Asn Leu Gln His Ile Pro Leu Lys Lys 1025 1030 1035Gly Lys Arg Val Val Ser 1040103439DNAMus musculus 10aatgaagaca ggctgtgcag cagggtctcc ggggaatgaa tggattttct tcagctctga 60tgaaagaaac acacgctcta ggaaaacaat gtccaacagg gcactgcaaa gatctgccgt 120gctgtctgcg tttgttctgc tgcgagctgt taccggcttc tccggagacg ggaaagcaat 180atgggataaa aaacagtacg tgagtccggt aaacccaagt cagctgttcc tctatgacac 240tttccctaaa aacttttcct ggggcgttgg gaccggagca tttcaagtgg aagggagttg 300gaagacagat ggaagaggac cctcgatctg ggatcggtac gtctactcac acctgagagg 360tgtcaacggc acagacagat ccactgacag ttacatcttt ctggaaaaag acttgttggc 420tctggatttt ttaggagttt ctttttatca gttctcaatc tcctggccac ggttgtttcc 480caatggaaca gtagcagcag tgaatgcgca aggtctccgg tactaccgtg cacttctgga 540ctcgctggta cttaggaata tcgagcccat tgttaccttg taccattggg atttgcctct 600gacgctccag gaagaatatg ggggctggaa aaatgcaact atgatagatc tcttcaacga 660ctatgccaca tactgcttcc agacctttgg agaccgtgtc aaatattgga ttacaattca 720caacccttac cttgttgctt ggcatgggtt tggcacaggt atgcatgcac caggagagaa 780gggaaattta acagctgtct acactgtggg acacaacctg atcaaggcac attcgaaagt 840gtggcataac tacgacaaaa acttccgccc tcatcagaag ggttggctct ccatcacctt 900ggggtcccat tggatagagc caaacagaac agacaacatg gaggacgtga tcaactgcca 960gcactccatg tcctctgtgc ttggatggtt cgccaacccc atccacgggg acggcgacta 1020ccctgagttc atgaagacgg gcgccatgat ccccgagttc tctgaggcag agaaggagga 1080ggtgaggggc acggctgatt tctttgcctt ttccttcggg cccaacaact tcaggccctc 1140aaacaccgtg gtgaaaatgg gacaaaatgt atcactcaac ttaaggcagg tgctgaactg 1200gattaaactg gaatacgatg accctcaaat cttgatttcg gagaacggct ggttcacaga 1260tagctatata aagacagagg acaccacggc catctacatg atgaagaatt tcctaaacca 1320ggttcttcaa gcaataaaat ttgatgaaat ccgcgtgttt ggttatacgg cctggactct 1380cctggatggc tttgagtggc aggatgccta tacgacccga cgagggctgt tttatgtgga 1440ctttaacagt gagcagaaag agaggaaacc caagtcctcg gctcattact acaagcagat 1500catacaagac aacggcttcc ctttgaaaga gtccacgcca gacatgaagg gtcggttccc 1560ctgtgatttc tcttggggag tcactgagtc tgttcttaag cccgagttta cggtctcctc 1620cccgcagttt accgatcctc acctgtatgt gtggaatgtc actggcaaca gattgctcta 1680ccgagtggaa ggggtaaggc tgaaaacaag accatcccag tgcacagatt atgtgagcat 1740caaaaaacga gttgaaatgt tggcaaaaat gaaagtcacc cactaccagt ttgctctgga 1800ctggacctct atccttccca ctggcaatct gtccaaagtt aacagacaag tgttaaggta 1860ctataggtgt gtggtgagcg aaggactgaa gctgggcgtc ttccccatgg tgacgttgta 1920ccacccaacc cactcccatc tcggcctccc cctgccactt ctgagcagtg gggggtggct 1980aaacatgaac acagccaagg ccttccagga ctacgctgag ctgtgcttcc gggagttggg 2040ggacttggtg aagctctgga tcaccatcaa tgagcctaac aggctgagtg acatgtacaa 2100ccgcacgagt aatgacacct accgtgcagc ccacaacctg atgatcgccc atgcccaggt 2160ctggcacctc tatgataggc agtataggcc ggtccagcat ggggctgtgt cgctgtcctt 2220acattgcgac tgggcagaac ctgccaaccc ctttgtggat tcacactgga aggcagccga 2280gcgcttcctc cagtttgaga tcgcctggtt tgcagatccg ctcttcaaga ctggcgacta 2340tccatcggtt atgaaggaat acatcgcctc caagaaccag cgagggctgt ctagctcagt 2400cctgccgcgc ttcaccgcga aggagagcag gctggtgaag ggtaccgtcg acttctacgc 2460actgaaccac ttcactacga ggttcgtgat acacaagcag ctgaacacca accgctcagt 2520tgcagacagg gacgtccagt tcctgcagga catcacccgc ctaagctcgc ccagccgcct 2580ggctgtaaca ccctggggag tgcgcaagct ccttgcgtgg atccggagga actacagaga 2640cagggatatc tacatcacag ccaatggcat cgatgacctg gctctagagg atgatcagat 2700ccgaaagtac tacttggaga agtatgtcca ggaggctctg aaagcatatc tcattgacaa 2760ggtcaaaatc aaaggctact atgcattcaa

actgactgaa gagaaatcta agcctagatt 2820tggatttttc acctctgact tcagagctaa gtcctctgtc cagttttaca gcaagctgat 2880cagcagcagt ggcctccccg ctgagaacag aagtcctgcg tgtggtcagc ctgcggaaga 2940cacagactgc accatttgct catttctcgt ggagaagaaa ccactcatct tcttcggttg 3000ctgcttcatc tccactctgg ctgtactgct atccatcacc gtttttcatc atcaaaagag 3060aagaaaattc cagaaagcaa ggaacttaca aaatatacca ttgaagaaag gccacagcag 3120agttttcagc taaactgcca tttctgtcat agtttcaaga ttcactccgg ctccatgtac 3180tggtaactta cgatgtgaga gacagctgta accaaggtga agacaatcga tgcctctgaa 3240gtgtggttca aataattcct tcaggtcccg acaatcagtg agtccgttct ccgagctgaa 3300gacaccctga cagtaactct gggcgtgacc ctaaacatcg cttcaggaag tgtgaatcac 3360gacttcacat cctttttctc tagcattctt ctgtaaataa caatcactat tcatggtcaa 3420gaaattaatt ttaaaaagt 3439111040PRTMus musculus 11Met Lys Thr Gly Cys Ala Ala Gly Ser Pro Gly Asn Glu Trp Ile Phe1 5 10 15Phe Ser Ser Asp Glu Arg Asn Thr Arg Ser Arg Lys Thr Met Ser Asn 20 25 30Arg Ala Leu Gln Arg Ser Ala Val Leu Ser Ala Phe Val Leu Leu Ala 35 40 45Val Thr Gly Ser Gly Asp Gly Lys Ala Trp Asp Lys Lys Gln Tyr Val 50 55 60Ser Pro Val Asn Pro Ser Gln Leu Phe Leu Tyr Asp Thr Phe Pro Lys65 70 75 80Asn Phe Ser Trp Gly Val Gly Thr Gly Ala Phe Gln Val Glu Gly Ser 85 90 95Trp Lys Thr Asp Gly Arg Gly Pro Ser Ile Trp Asp Arg Tyr Val Tyr 100 105 110Ser His Leu Arg Gly Val Asn Gly Thr Asp Arg Ser Thr Asp Ser Tyr 115 120 125Ile Phe Leu Glu Lys Asp Leu Leu Ala Leu Asp Phe Leu Gly Val Ser 130 135 140Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg Leu Phe Pro Asn Gly Thr145 150 155 160Val Ala Ala Val Asn Ala Gln Gly Leu Arg Tyr Tyr Arg Ala Leu Leu 165 170 175Asp Ser Leu Val Leu Arg Asn Ile Glu Pro Ile Val Thr Leu Tyr His 180 185 190Trp Asp Leu Pro Leu Thr Leu Gln Glu Glu Tyr Gly Gly Trp Lys Asn 195 200 205Ala Thr Met Ile Asp Leu Phe Asn Asp Tyr Ala Thr Tyr Cys Phe Gln 210 215 220Thr Phe Gly Asp Arg Val Lys Tyr Trp Ile Thr Ile His Asn Pro Tyr225 230 235 240Leu Val Ala Trp His Gly Phe Gly Thr Gly Met His Ala Pro Gly Glu 245 250 255Lys Gly Asn Leu Thr Ala Val Tyr Thr Val Gly His Asn Leu Ile Lys 260 265 270Ala His Ser Lys Val Trp His Asn Tyr Asp Lys Asn Phe Arg Pro His 275 280 285Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly Ser His Trp Ile Glu Pro 290 295 300Asn Arg Thr Asp Asn Met Glu Asp Val Ile Asn Cys Gln His Ser Met305 310 315 320Ser Ser Val Leu Gly Trp Phe Ala Asn Pro Ile His Gly Asp Gly Asp 325 330 335Tyr Pro Glu Phe Met Lys Thr Gly Ala Met Ile Pro Glu Phe Ser Glu 340 345 350Ala Glu Lys Glu Glu Val Arg Gly Thr Ala Asp Phe Phe Ala Phe Ser 355 360 365Phe Gly Pro Asn Asn Phe Arg Pro Ser Asn Thr Val Val Lys Met Gly 370 375 380Gln Asn Val Ser Leu Asn Leu Arg Gln Val Leu Asn Trp Ile Lys Leu385 390 395 400Glu Tyr Asp Asp Pro Gln Ile Leu Ile Ser Glu Asn Gly Trp Phe Thr 405 410 415Asp Ser Tyr Ile Lys Thr Glu Asp Thr Thr Ala Ile Tyr Met Met Lys 420 425 430Asn Phe Leu Asn Gln Val Leu Gln Ala Ile Lys Phe Asp Glu Ile Arg 435 440 445Val Phe Gly Tyr Thr Ala Trp Thr Leu Leu Asp Gly Phe Glu Trp Gln 450 455 460Asp Ala Tyr Thr Thr Arg Arg Gly Leu Phe Tyr Val Asp Phe Asn Ser465 470 475 480Glu Gln Lys Glu Arg Lys Pro Lys Ser Ser Ala His Tyr Tyr Lys Gln 485 490 495Ile Ile Gln Asp Asn Gly Phe Pro Leu Lys Glu Ser Thr Pro Asp Met 500 505 510Lys Gly Arg Phe Pro Cys Asp Phe Ser Trp Gly Val Thr Glu Ser Val 515 520 525Leu Lys Pro Glu Phe Thr Val Ser Ser Pro Gln Phe Thr Asp Pro His 530 535 540Leu Tyr Val Trp Asn Val Thr Gly Asn Arg Leu Leu Tyr Arg Val Glu545 550 555 560Gly Val Arg Leu Lys Thr Arg Pro Ser Gln Cys Thr Asp Tyr Val Ser 565 570 575Ile Lys Lys Arg Val Glu Met Leu Ala Lys Met Lys Val Thr His Tyr 580 585 590Gln Phe Ala Leu Asp Trp Thr Ser Ile Leu Pro Thr Gly Asn Leu Ser 595 600 605Lys Val Asn Arg Gln Val Leu Arg Tyr Tyr Arg Cys Val Val Ser Glu 610 615 620Gly Leu Lys Leu Gly Val Phe Pro Met Val Thr Leu Tyr His Pro Thr625 630 635 640His Ser His Leu Gly Leu Pro Leu Pro Leu Leu Ser Ser Gly Gly Trp 645 650 655Leu Asn Met Asn Thr Ala Lys Ala Phe Gln Asp Tyr Ala Glu Leu Cys 660 665 670Phe Arg Glu Leu Gly Asp Leu Val Lys Leu Trp Ile Thr Ile Asn Glu 675 680 685Pro Asn Arg Leu Ser Asp Met Tyr Asn Arg Thr Ser Asn Asp Thr Tyr 690 695 700Arg Ala Ala His Asn Leu Met Ile Ala His Ala Gln Val Trp His Leu705 710 715 720Tyr Asp Arg Gln Tyr Arg Pro Val Gln His Gly Ala Val Ser Leu Ser 725 730 735Leu His Cys Asp Trp Ala Glu Pro Ala Asn Pro Phe Val Asp Ser His 740 745 750Trp Lys Ala Ala Glu Arg Phe Leu Gln Phe Glu Ile Ala Trp Phe Ala 755 760 765Asp Pro Leu Phe Lys Thr Gly Asp Tyr Pro Ser Val Met Lys Glu Tyr 770 775 780Ile Ala Ser Lys Asn Gln Arg Gly Leu Ser Ser Ser Val Leu Pro Arg785 790 795 800Phe Thr Ala Lys Glu Ser Arg Leu Val Lys Gly Thr Val Asp Phe Tyr 805 810 815Ala Leu Asn His Phe Thr Thr Arg Phe Val Ile His Lys Gln Leu Asn 820 825 830Thr Asn Arg Ser Val Ala Asp Arg Asp Val Gln Phe Leu Gln Asp Ile 835 840 845Thr Arg Leu Ser Ser Pro Ser Arg Leu Ala Val Thr Pro Trp Gly Val 850 855 860Arg Lys Leu Leu Ala Trp Ile Arg Arg Asn Tyr Arg Asp Arg Asp Ile865 870 875 880Tyr Ile Thr Ala Asn Gly Ile Asp Asp Leu Ala Leu Glu Asp Asp Gln 885 890 895Ile Arg Lys Tyr Tyr Leu Glu Lys Tyr Val Gln Glu Ala Leu Lys Ala 900 905 910Tyr Leu Ile Asp Lys Val Lys Ile Lys Gly Tyr Tyr Ala Phe Lys Leu 915 920 925Thr Glu Glu Lys Ser Lys Pro Arg Phe Gly Phe Phe Thr Ser Asp Phe 930 935 940Arg Ala Lys Ser Ser Val Gln Phe Tyr Ser Lys Leu Ile Ser Ser Ser945 950 955 960Gly Leu Pro Ala Glu Asn Arg Ser Pro Ala Cys Gly Gln Pro Ala Glu 965 970 975Asp Thr Asp Cys Thr Ile Cys Ser Phe Leu Val Glu Lys Lys Pro Leu 980 985 990Ile Phe Phe Gly Cys Cys Phe Ile Ser Thr Leu Ala Val Leu Leu Ser 995 1000 1005Ile Thr Val Phe His His Gln Lys Arg Arg Lys Phe Gln Lys Ala 1010 1015 1020Arg Asn Leu Gln Asn Ile Pro Leu Lys Lys Gly His Ser Arg Val 1025 1030 1035Phe Ser 10401221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 12actctgcgct ggttgaaaaa t 211321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 13ggtggcatag cgaaccttgt a 211423DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 14gctataaggt acgaaaccag cac 231523DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 15ggttgatgga cccgtattca ttc 231619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 16gcctgcgtgc tagtgttct 191721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 17taccatcctt agcccagacc g 211821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 18tccatgaccg tcgtacacaa t 211921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 19atttgacagt attcccggca g 212021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 20tgttctgctg cgagctgtta c 212121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 21ccggactcac gtactgtttt t 212221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 22gaagccgctt atgtgtatcg c 212322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23gaatgggtac attgggaaca gt 222422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24ggagatcctt cgaggagcac tt 222524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25gcgatttagc agcagatata agaa 242622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26aaggacctgg tacatgaact gg 222721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 27ggttctgggt gtcaagtgtc g 212819DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28tttggcgttc gaggccatt 192920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29caggtccacg catttcagac 203019DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30cagacgccac tgtcgcttt 193123DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31tgtctttgga actttgtctg caa 233221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 32gagcaagggc tgctactcaa g 213321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33aaccgcaggt tcaagtattc c 213422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 34tcatggtctt cggaggattc ac 223521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 35tcacctccaa gtgtggcaaa g 213621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36ttcaggcagg cagtatcact c 213721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37gaaggtccac gggaaagaca c 213821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38aagagccgga aatccacgaa a 213922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39gtctcaaaag ggtcagggta ct 224023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 40cttactgact ggcatgagga tca 234120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 41gcagctctag gagcatgtgg 204221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 42gaagccgctt atgtgtatcg c 214322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 43gaatgggtac attgggaaca gt 224422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 44tagatgacca tgagtcgctt gc 224520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 45gccaaacttg ctccatgtcc 204624DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 46ggagagtctg actctccctg agaa 244719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 47cgatgggttc cgtcttggt 194824DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 48cggttccgat gccctgaggc tctt 244924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 49cgtcacactt catgatggaa ttga 24

Claims

1. A method for treating or preventing neovascularization and/or angiogenesis in the eye of a subject, the method comprising: (a) identifying a subject having or at risk of neovascularization and/or angiogenesis in the eye; and (b) administering a pharmaceutical composition comprising a stabilized fibroblast growth factor 21 (FGF21) agent to the subject in the eye of a subject

2. (canceled)

3. The method of claim 1, wherein the subject has or is at risk of developing a condition selected from the group consisting of neovascular retinopathy, diabetic retinopathy in type I diabetes, retinopathy of prematurity (ROP), retinitis pigmentosa (RP) and macular telangiectasia (MacTel).

4. The method of claim 1, wherein neovascularization and/or angiogenesis in the choroid or retinal cells of the eye is treated or prevented.

5. (canceled)

6. The method of claim 1, wherein the stabilized FGF21 agent comprises an FGF21 polypeptide or a modified FGF21 polypeptide conjugated to an antibody scaffold.

7. (canceled)

8. The method of claim 6, wherein the modified FGF21 is dHis/Ala129Cys, or wherein the modified FGF21 is conjugated at Cys 129 to the antibody scaffold.

9. The method of claim 6, wherein two or more FGF21 polypeptide molecules are conjugated to one antibody scaffold.

10. The method of claim 6, wherein the antibody scaffold is a CovX-2000 scaffold.

11. The method of claim 1, wherein the stabilized FGF21 agent is a long acting FGF21 analog, comprising PF-05231023.

12. The method of claim 1, wherein the stabilized FGF21 agent possesses a half-life of at least 1.5.times. the half-life of a native FGF21 peptide when assayed for stability under identical conditions.

13. The method of claim 1, wherein the stabilized FGF21 agent possesses a half-life of at least 0.8 h in the circulation of a mammal, wherein the mammal is human.

14. The method of claim 1, wherein the pharmaceutical composition is administered to the eye of the subject.

15. (canceled)

16. A pharmaceutical composition for use in treating or preventing neovascularization, and/or angiogenesis in the eye of a subject comprising fibroblast growth factor 21 (FGF21), a stabilized FGF21 agent, a modified FGF21 molecule, or combinations thereof, and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, wherein the stabilized FGF21 agent comprises an FGF21 polypeptide or modified FGF21 polypeptide conjugated to an antibody scaffold.

18. (canceled)

19. The pharmaceutical composition of claim 17, wherein the modified FGF21 is dHis/Ala129Cys, or wherein the modified FGF21 is conjugated at Cys 129 to the antibody scaffold.

20. The pharmaceutical composition of claim 17, wherein two or more FGF21 polypeptide molecules are conjugated to one antibody scaffold.

21. (canceled)

22. The pharmaceutical composition of claim 17, wherein the stabilized FGF21 agent is a long acting FGF21 analog, comprising PF-05231023.

23. (canceled)

24. A method for treating or preventing hyperglycemic retinopathy of prematurity (ROP) in a subject, the method comprising: (a) identifying a subject having or at risk of hyperglycemic ROP; and (b) administering a pharmaceutical composition comprising a fibroblast growth factor 21 (FGF21) agent to a subject, thereby treating or preventing hyperglycemic ROP in the subject.

25. (canceled)

26-30. (canceled)

31. A method of treating or preventing against photoreceptor dysfunction, inflammation and/or morphology in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of fibroblast growth factor 21 (FGF21) or a long-acting FGF21 analog.

32-40. (canceled)

41. A pharmaceutical composition comprising one or more fibroblast growth factor 21 (FGF21) molecules, comprising: pegylated FGF21, modified FGF21 proteins, Fc-FGF21 fusion constructs, long acting FGF21 or combinations thereof.

42. The pharmaceutical composition of claim 41, wherein a pegylated FGF21 comprises an FGFR21 with a R131AcF modification, coupled to PEG.

43-46. (canceled)