Method for treating angiogenesis, and immunogenic composition for use in said method

Abstract

The present disclosure provides a method for treating angiogenesis in a target tissue in a subject. According to the present disclosure, there is provided a method which includes administering an immunogenic composition containing messenger RNA (mRNA) that encodes LRG1 or a fragment thereof to the subject.

Description

Methods of treating angiogenesis and immunogenic compositions for use therein

[0001] The present disclosure relates to methods of treating angiogenesis, and immunogenic compositions for use in such methods.

[0002] Abnormal angiogenesis is involved in the pathology of various diseases, including cancer and retinopathies such as age-related macular degeneration, and inhibiting angiogenesis is expected to have therapeutic value in these diseases. Age-related macular degeneration is considered a disease that significantly reduces quality of life. The first-line treatment for age-related macular degeneration is a VEGF inhibitor, which is treated by intravitreal administration of anti-VEGF antibodies. In Non-Patent Document 1, a peptide derived from VEGFR2 was administered to A2 / Kb transgenic mice to treat choroidal neovascularization (CNV). In Non-Patent Document 2, it was reported that leucine-rich α2 glycoprotein 1 (LRG1) induces vascular endothelial cell division in the presence of TGFβ, promoting angiogenesis, and that intraocular administration of anti-LRG1 antibodies attenuates angiogenesis. In Non-Patent Document 3, LRG1 is shown to be involved in various diseases.

[0003] Therapeutic vaccines targeting lifestyle-related diseases have been developed. Non-Patent Document 3 shows that an immune tolerance vaccine targeting GAD65 was effective in experimental animals, but in a large-scale test, it was shown that it did not significantly change insulin secretion levels, and that antibody production in the body due to administration of amyloid beta induced a reduction in amyloid plaques in the brain.

[0004] Y. Yanagi et al., Invest. Ophthalmol. Vis. Sci., 49, 2143-2147, 2008X. Wang et al., Nature, 499, 7458, 2013C. Camilli et al., J. Biomed. Sci., 29:6, 2022 Keitoku Nakagami, Japanese Pharmacological Journal, 154, 270-274, 2019

[0005] The present disclosure provides a method for treating angiogenesis, an immunogenic composition for use in the method, etc. The present disclosure provides, for example, an immunogenic composition that can be administered to a non-target tissue (e.g., a tissue other than the eyeball in the case of an ophthalmic disease) to thereby suppress angiogenesis in the target tissue.

[0006] The present disclosure provides the following inventions: (1) An immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or a fragment thereof as an immunogen, for use in a method for treating a disease or condition (preferably angiogenesis) associated with increased LRG1 expression in a target tissue of a subject, the method comprising administering to the subject an immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or an immunogenic fragment thereof, thereby expressing LRG1 or the fragment in cells of a tissue other than the target tissue (a non-target tissue), and inducing specific antibodies against LRG1 or the fragment in the subject, thereby inhibiting LRG1 with the antibodies. (2) The composition according to (1) above, wherein the subject has retinopathy and the target tissue is the retina. (3) The composition according to (1) or (2) above, wherein the retinopathy is age-related macular degeneration. (4) The composition according to any of (1) to (3) above, wherein the administration is intramuscular. (5) The composition according to any one of (1) to (4) above, wherein the mRNA comprises one or more modified nucleic acids. (6) The composition according to any one of (1) to (5) above, wherein the mRNA has a Cap 2 structure. (7) The composition according to any one of (1) to (6) above, wherein the purity of mRNA having the Cap 2 structure relative to the total mRNA is more than 90%. (8) The composition according to any one of (1) to (7) above, wherein the mRNA is encapsulated in lipid nanoparticles. (9) The composition according to (8) above, wherein the lipid nanoparticles comprise an ionizable lipid, a phospholipid, cholesterol, and a PEG-lipid.

[0007] The present disclosure provides the following inventions: (1A) An immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or a fragment thereof as an immunogen, for use in a method for treating angiogenesis in a target tissue of a subject, the method comprising administering to the subject an immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or an immunogenic fragment thereof, causing LRG1 or the fragment to be expressed in cells of a tissue other than the target tissue, and inducing specific antibodies against LRG1 or the fragment in the subject, and inhibiting LRG1 with the antibodies. (2A) The composition according to (1A) above, wherein the subject has retinopathy and the target tissue is the retina. (3A) The composition according to (1A) or (2A) above, wherein the retinopathy is age-related macular degeneration. (4A) The composition according to any of (1A) to (3A) above, wherein the administration is intramuscular. (5A) The composition according to any one of (1A) to (4A) above, wherein the mRNA comprises one or more modified nucleic acids. (6A) The composition according to any one of (1A) to (5A) above, wherein the mRNA has a Cap 2 structure. (7A) The composition according to any one of (1A) to (6A) above, wherein the purity of the mRNA having the Cap 2 structure relative to the total mRNA is more than 90%. (8A) The composition according to any one of (1A) to (7A) above, wherein the mRNA is encapsulated in lipid nanoparticles. (9A) The composition according to (8A) above, wherein the lipid nanoparticles comprise an ionizable lipid, a phospholipid, cholesterol, and a PEG-lipid.

[0008] The present disclosure provides the following inventions: (1B) An immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or an immunogenic fragment thereof as an immunogen, for use in a method for treating retinopathy (particularly age-related macular degeneration) in a subject, the method comprising administering to the subject an immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or an immunogenic fragment thereof, thereby expressing LRG1 or the fragment in cells of a tissue other than the target tissue, and inducing specific antibodies against LRG1 or the fragment in the subject, and inhibiting LRG1 with the antibodies. (3B) The composition according to (1B) above, wherein the retinopathy is age-related macular degeneration. (4B) The composition according to any of (1B) to (3B) above, wherein the administration is intramuscular administration. (5B) The composition according to any of (1B) to (4B) above, wherein the mRNA comprises one or more modified nucleic acids. (6B) The composition according to any one of (1B) to (5B) above, wherein the mRNA has a Cap 2 structure. (7B) The composition according to any one of (1B) to (6B) above, wherein the purity of the mRNA having the Cap 2 structure relative to the total mRNA is more than 90%. (8B) The composition according to any one of (1B) to (7B) above, wherein the mRNA is encapsulated in a lipid nanoparticle. (9B) The composition according to (8B) above, wherein the lipid nanoparticle comprises an ionizable lipid, a phospholipid, cholesterol, and a PEG-lipid.

[0009] This is an electrophoretic image of mRNA synthesized using an in vitro transcription system and purified by HPLC. Electrophoresis was performed on a 5% denaturing PAGE in 1x TBE buffer containing 7.5 M urea. Staining was performed with SYBR Green II. Approximately 5.5-6.0 μg of mRNA was cleaved with a DNAzyme (5'-TCTGTGGGGAGGCTAGCTACAACGACAGAAGAATACTAGTTT-3': SEQ ID NO: 4) and subjected to electrophoresis. Cleavage was performed in a buffer containing 50 mM Tris-HCl (pH 8.0) and 50 mM MgCl2 in the presence of 1 μM DNAzyme. RNA was precipitated with isopropyl alcohol and recovered, followed by electrophoresis on a 15% dPAGE in 1x TBE buffer containing 7.5 M urea. Staining was performed with SYBR Gold. This is a dot blot showing the presence of double-stranded RNA (dsRNA) in purified LRG1 mRNA with a cap structure, confirmed using the J2 antibody. 500 ng of synthetic mRNA was compared with Poly I:C, a double-stranded RNA, as a standard sample. While detectable levels of dsRNA were present in the crude product (Crude) before purification, no dsRNA contamination was observed in the purified mRNA (Purified). The treatment schedules for two types of angiogenesis induction models are shown. Specifically, the schedules for the untreated negative control group, the LRG1 mRNA vaccine group, and the anti-VEGFR2 antibody group are shown. The LRG1 mRNA vaccine group received a primary immunization on day 0 and a booster immunization on day 14, while the anti-VEGFR2 antibody group received the antibody intravitreally on day 18. Fluorescein angiography and enucleation were performed on day 21. Fluorescence area (including leakage of fluorescence) in fluorescein angiography of Vldlr knockout mice (2-3 months old) was compared between the LRG1 mRNA vaccine-administered group (n=6) and the negative control group (n=6). Representative fundus images obtained by fluorescein angiography are shown. The expression of each mRNA was measured by quantitative real-time PCR in tissues including the retina, RPE, choroid, and sclera isolated from the eyes of Vldlr knockout mice (2-3 months old) administered the LRG1 mRNA vaccine (n=6) and the negative control group (n=6).Flat-mount images of eyes from a laser-induced CNV model are shown. Isolectin B4 and Iba1 were fluorescently stained by immunohistochemistry, and the resulting fluorescent images (Panel A) and fluorescence intensity (Panel B) are shown. The above evaluations were performed in the untreated group, the LRG1 mRNA vaccine-administered group, and the anti-VEGFR2 antibody-administered group (n = 5 each). The scale bar is 200 μm. Hematoxylin and eosin-stained images (H&E) and CD31 expression are shown in fundus tissue sections from the untreated group and the LRG1 mRNA vaccine-administered group (n = 5 each) in the laser-induced CNV model. Arrowheads indicate the areas of CNV. The expression of isolectin B4 and Iba1 in fundus tissue sections from the untreated group and the LRG1 mRNA vaccine-administered group (n = 5 each) in the laser-induced CNV model are shown. The above evaluations were performed in the untreated group, the LRG1 mRNA vaccine-administered group, and the anti-VEGFR2 antibody-administered group (n = 5 each). The scale bar is 50 μm. The figure shows the fluorescence leakage area in fluorescein angiography for the untreated group, the LRG1 mRNA vaccine-administered group, and the VEGFR2 mRNA-administered group in the laser-induced CNV model. Each circle represents the fluorescence leakage area for each individual. The figure shows the time course of antibody levels before administration and 7, 14, 21, and 28 days after administration of LRG1 mRNA (ARCR mRNA) in C57BL / 6 mice. Representative fundus images obtained by fluorescein angiography for the unvaccinated group (A) and the LRG1 mRNA (PureCap mRNA)-encapsulated LNP-vaccinated group (B) are shown. Also, C shows the quantification of CNV size. The vertical axis represents the fold change in pixel area, with the LRG1 vaccine set as 1. The figure shows the results of H&E staining 1 day after administration of LRG1 mRNA (PureCap mRNA). The figure shows the results of H&E staining 21 days after administration of LRG1 mRNA (PureCap mRNA). In addition, the values ​​of AST, ALT, LDH, BUN, and CRE 21 days after administration are compared with those of the untreated control group and are shown in the table.

[0010] As used herein, a "subject" can be a vertebrate, a mammal (human or non-human mammal), a primate (such as a human, chimpanzee, gorilla, orangutan, monkey, marmoset, and bonobo), a non-primate mammal such as a mouse, rat, pig, cow, sheep, goat, llama, camel, horse, cat, and dog, a fish, an amphibian, a reptile, a crustacean, a bird, an insect, and a eukaryote, such as a plant.

[0011] As used herein, "angiogenesis" refers to the physiological phenomenon in which new blood vessels branch off from existing blood vessels to form a vascular network. Angiogenesis occurs when vascular endothelial cells are stimulated by vascular endothelial growth factor (VEGF), causing them to proliferate and migrate to form new lumens, and pericytes surround the new blood vessels to form new blood vessels. Angiogenesis includes pathological angiogenesis (e.g., angiogenesis in disease), such as angiogenesis in cancer tissue, angiogenesis in retinopathy, angiogenesis in glaucoma, and angiogenesis in age-related macular degeneration, as well as angiogenesis in psoriasis, angiogenesis in inflammatory tissues such as arthritis, and angiogenesis in diabetic nephropathy. Angiogenesis can be inhibited, for example, by administering an anti-VEGF antibody. Angiogenesis in ophthalmic diseases has been inhibited by intravitreal administration of an anti-VEGF antibody. Known anti-VEGF antibody products, such as Lucentis (trademark) and Eylea (trademark), have received market approval from pharmaceutical authorities. Inhibition of angiogenesis has also been used in the treatment of tumors, for example, Avastin (trademark) is known and has been approved for the market by pharmaceutical authorities.

[0012] As used herein, "age-related macular degeneration" (AMD) refers to a disease that results in impaired vision due to damage to the macula, the central part of the retina. AMD is a progressive disease that occurs with aging. Vision is impaired due to bleeding and swelling in the macula. The macula is a central area of ​​the retina, approximately 1.5 to 2 mm in diameter, that functions similarly to the film in a camera. Its yellow color is due to the abundance of a pigment called xanthophyll. The macula plays a key role in ensuring vision, and damage to the macula can significantly reduce vision even if the rest of the retina is intact. Proper function of the retina requires the proper functioning of the retinal pigment epithelium cells underlying the retina and the choroid, a tissue rich in blood vessels beneath them. AMD is broadly divided into dry AMD and wet AMD. In dry AMD, the retinal pigment epithelium gradually atrophies, damaging the retina and resulting in impaired vision. In wet AMD, abnormal choroidal neovascularization (ADV) occurs when abnormal choroidal neovascularization invades the retina from the choroid beneath the retinal pigment epithelium or between the retina and the retinal pigment epithelium, damaging the retina. These abnormal vessels, unlike normal blood vessels, leak blood components or are prone to rupture. Leakage of blood components can lead to retinal edema and subretinal fluid, which can impair retinal function and reduce vision. Rupture of blood vessels can also result in bleeding and retinal damage. Symptoms of AMD include metamorphopsia, decreased visual acuity, central scotoma, and / or color vision disorders. Exudative AMD includes polypoidal choroidal vasculopathy and retinal angiomatous proliferation. AMD can be diagnosed by visual acuity testing, Amsler testing, fundus examination (which can reveal the presence of bleeding and neovascularization), angiography (e.g., fluorescein angiography, indocyanine green angiography), and / or optical coherence tomography (OCT). Anti-VEGF drugs have been developed to treat wet AMD, and three types are currently on the market: Lucentis (trademark), Eylea (trademark), and Beovu (trademark). All of these drugs are administered by intravitreal injection three times every four weeks. Other treatments for wet AMD include photodynamic therapy (PDT), laser coagulation (only when choroidal neovascularization is away from the center), and surgical removal of choroidal neovascularization.

[0013] As used herein, "treatment" means intervening in a subject with the expectation of a therapeutic or preventive effect in the subject. Treatment that can be expected to have a therapeutic effect is called therapeutic treatment, and treatment that can be expected to have a preventive effect is called prophylactic treatment. As used herein, "treatment" means treating, curing, preventing, or improving the remission of a disease or disorder, or reducing the rate of progression of a disease or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or condition, or delaying the onset of a disease or condition.

[0014] As used herein, "disease" means a pathological condition for which treatment may be beneficial.

[0015] As used herein, "effective amount" refers to an amount of a drug effective to treat (prevent or cure) a disease or condition. A therapeutically effective amount of a drug is capable of slowing the rate of progression of, halting the progression of, ameliorating, curing, or inhibiting the onset or development of symptoms of a disease or condition. Treatment is accomplished by administering a therapeutically effective amount of the active ingredient.

[0016] As used herein, a "target tissue" refers to a tissue in a living organism, preferably one that is hypervascularized and that is targeted by the methods of the present disclosure. Target tissues can be, for example, hypervascularized tissues such as tumors, the eye (e.g., the retina), skin, and joints.

[0017] As used herein, "immunogenicity" refers to the property of a substance to induce the production of specific antibodies in an organism. An immunogenic substance can induce the production of antibodies specific to that substance in an organism. To enhance immunogenicity, a carrier protein can be linked to the substance. Examples of carrier proteins include hemocyanins such as keyhole limpet hemocyanin (KLH) and albumins such as bovine serum albumin (BSA) and ovalbumin (OVA). Alternatively, aluminum salts (e.g., aluminum hydroxide (alum)) can be mixed with the immunogenic substance and administered to an organism to enhance immunogenicity. An immunogenic composition is an immunogenic composition containing an immunogenic substance. Antibodies generated in an immunized individual by immunization with an immunogenic substance include antibodies specific to the immunogenic substance (antibodies that specifically bind to the immunogenic substance). A vaccine or vaccine composition is an immunogenic composition, particularly an immunogenic composition that generates antibodies in the subject's body that inhibit a specific pathological phenomenon or prevent or treat a pathological condition.

[0018] As used herein, "LRG1" refers to leucine-rich α2-glycoprotein 1. LRG1 promotes transforming growth factor-β (TGFβ) signaling in endothelial cells, thereby promoting the formation of new blood vessels. LRG1 has been implicated as a key player in pathogenic cancer angiogenesis. The lack of obvious phenotypic abnormalities in Lrg1 - / - mice suggests that LRG1 is essentially dispensable for development and homeostasis. LRG1 functions as an angiogenic factor, inhibiting cell-cell interactions normally required for blood vessel formation and maintenance. Significant increases in LRG1 expression have been observed during the ischemic proliferative phase of laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), recapitulating aspects of neovascular age-related macular degeneration (nvAMD) and proliferative diabetic retinopathy (PDR), major microvascular complications of diabetes. High levels of LRG1 have been detected in choroidal neovascular membranes and aqueous humor samples, which are explained by a model in which LRG1 is produced at the lesion site and subsequently released into the intraocular fluid. LRG1 has been proposed as a therapeutic target for nvAMD, diabetic retinopathy (e.g., preproliferative and proliferative retinopathy), and diabetic macular edema. A severe microvascular complication of diabetes is diabetic kidney disease (DKD). The early stage of DKD, called diabetic nephropathy, involves the proliferation of immature, leaky blood vessels. In DKD patients, increased LRG1 expression in glomerular endothelial cells precedes elevated VEGF levels, promoting angiogenesis in the kidney. LRG1 inhibition has also been discussed as a potential therapeutic option for these kidney diseases. In addition to DKD, IgA nephropathy and childhood idiopathic nephrotic syndrome are known kidney diseases characterized by abnormal LRG1 secretion. LRG1 is also involved in pulmonary disease, cardiovascular disease, and diabetes. LRG1 is also involved in inflammation, functioning as a pro-inflammatory mediator, and is highly expressed in adult-onset Still's disease, psoriasis, lupus nephritis, arthritis, rheumatoid arthritis, vasculitis, and gastrointestinal inflammation. LRG1 is also highly expressed in human cancers (e.g., biliary tract cancer, bladder cancer, lung cancer, head and neck cancer, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, glioblastoma, stem cell cancer, leukemia, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, retinal cancer, etc.). Thus, LRG1 expression is elevated in various diseases, potentially exacerbating symptoms.LRG1 may be a therapeutic target for these diseases (e.g., angiogenesis). For the role of LRG1 and its relationship to diseases, see C. Camilli et al., J. Biomed. Sci., 29:6, 2022, the entire contents of which are incorporated herein by reference. The amino acid sequence of human LRG1 may be, for example, the amino acid sequence registered in GenBank under accession number KAI4039707.1, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence. As used herein, the term "LRG1" refers to the full-length LRG1.

[0019] As used herein, "messenger RNA" (mRNA) refers to a nucleic acid that encodes a protein and has a structure suitable as a template for protein translation. mRNA typically has a cap structure and an untranslated region at the 5' end, a coding region encoding the protein, and an untranslated region and poly(A) at the 3' end. The cap structure is formed by a 7-methylguanosine (m7G) linked to the mRNA via a special bond known as a 5'-5' triphosphate linkage. The 2'-OH groups of one or two nucleotides following the 7-methylguanosine (m7G) cap may be methylated, resulting in structures called Cap 1 and Cap 2, respectively. Cap structures lacking such OH methylation are called Cap 0. mRNA with a Cap 2 structure is suitable for pharmaceutical use and can be purified to high purity (typically 99% or higher) using the PureCap method (see K. Inagaki et al., Nature Communications, 14, Article Number: 2657, 2023). mRNA may contain modified nucleic acids. For example, the mRNA can have pseudouridines in place of some or all of the uracils.

[0020] As used herein, a "drug delivery carrier" refers to a complex of a drug and a carrier encapsulating a drug. Drug delivery carriers improve the stability of drugs in the blood or promote cellular uptake, thereby enabling more effective intracellular drug delivery in vivo. Examples of drug delivery carriers include liposomes, micelles, polycationic molecules, lipid nanoparticles, viral vectors, and polyion complexes. Liposomes have a lipid bilayer membrane surrounding an internal space, and drugs are typically encapsulated within the membrane or internal space. Micelles are single-layer structures made of amphiphilic substances, with relatively hydrophobic interior regions and relatively hydrophilic exterior regions. In micelles, hydrophobic drugs are typically encapsulated in the internal hydrophobic region, while hydrophilic drugs are encapsulated in the external hydrophilic region. Polycationic molecules can be used to deliver negatively charged nucleic acids into cells. Viral vectors possess a virus-derived outer shell and nucleic acid (e.g., genome) and can deliver nucleic acids into cells. Viral vectors can deliver DNA and synthesize mRNA within cells, or they can deliver mRNA. Polyion complexes are vesicles (polymersomes or micelles) that spontaneously form when polycations and polyanions are mixed in water. In polyion complexes, uncharged hydrophilic polymers (e.g., polyalkylene glycols, particularly polyethylene glycols, or polyoxazolines) are attached to either or both of the polycation and polyanion, and can have either or both of the polycation and polyanion as block copolymers (see, for example, WO 2015 / 075942A, incorporated herein by reference in its entirety). In the present disclosure, drug delivery carriers can be used to deliver mRNA to cells in vivo. The polycation can be a polymer with cationic units as monomer units.

[0021] As used herein, "lipid nanoparticles" (LNPs) refer to nanovesicles composed of lipid molecules. Examples of lipid nanoparticles include the nucleic acid-lipid particles described in U.S. Pat. No. 8,058,069B, the entire contents of which are incorporated herein by reference. Amphiphilic lipids can form lipid nanoparticles in aqueous solution. mRNA-encapsulated LNPs have been put to practical use in COVID-19 vaccines and have become widely used as a delivery technology for mRNA. Those skilled in the art can appropriately select lipids to form lipid nanoparticles from the lipids. Lipid nanoparticles typically contain one or more, or preferably all, selected from the group consisting of cationic lipids, non-cationic lipids (or neutral lipids), sterols, and PEG-lipids. In some embodiments, the cationic lipid is an ionizable cationic lipid. Ionizable cationic lipids are electrically neutral at physiological pH and cationic in acidic environments (e.g., within endosomes). In some embodiments, the non-cationic lipid is a neutral lipid. The ionizable lipid becomes protonated and positively charged in the acidic environment of the endosome, disrupting the endosomal membrane and facilitating endosomal escape and delivery of the mRNA to the cytoplasm. In one embodiment, the sterol comprises cholesterol.

[0022] <Treatment Method> The present disclosure provides a method for treating angiogenesis in a target tissue of a subject. Angiogenesis accompanied by increased LRG1 expression occurs in the body in various situations, including pathological conditions. The method of the present disclosure can be beneficial in such situations, for example.

[0023] In some embodiments, the target tissue may be the retina. In some embodiments, the retina does not have neovascularization. Treatment at a stage where there is no obvious neovascularization may be beneficial in inhibiting age-related macular degeneration and neovascularization. In some embodiments, the retina is accompanied by macular neovascularization. In some embodiments, neovascularization may be neovascularization in age-related macular degeneration. Age-related macular degeneration is, for example, the exudative type. In some embodiments, the methods of the present disclosure may reduce exudate. In some embodiments, the methods of the present disclosure may inhibit an increase in choroidal neovascularization. In some embodiments, the methods of the present disclosure may reduce choroidal neovascularization. In some embodiments, the subject has precursor lesions of age-related macular degeneration. The precursor lesions include soft drusen and retinal pigment epithelial abnormalities. A precursor lesion can be determined when a subject has soft drusen with a diameter of 63 μm or greater in a 6 mm diameter area centered on the fovea at the center of the macula, and one or more abnormalities selected from the group consisting of depigmentation of the retinal pigment epithelium, pigmentation, uneven pigmentation, and small serous retinal pigment epithelial detachment (less than one papillary diameter in diameter). In one embodiment, the subject has early age-related macular degeneration associated with macular neovascularization but not exudative changes. In one embodiment, the subject has dry age-related macular degeneration. In one embodiment, the subject has neovascular (exudative) age-related macular degeneration, such as polypoidal choroidal vasculopathy and retinal angiomatous proliferation. In one embodiment, the subject has mild age-related macular degeneration (e.g., visual acuity of about 0.4 to 0.6). In one embodiment, the subject has moderate age-related macular degeneration (e.g., visual acuity of about 0.2 to 0.3). In some embodiments, the subject has severe age-related macular degeneration (eg, visual acuity ≦0.15).

[0024] The method of the present disclosure includes administering to a subject an immunogenic composition containing a nucleic acid (preferably messenger RNA (mRNA)) encoding LRG1 or an immunogenic fragment thereof. By expressing LRG1 or an immunogenic fragment thereof in non-target tissues of the subject, LRG1 or an immunogenic fragment thereof can be expressed in cells in the subject's body, and specific antibodies against LRG1 or an immunogenic fragment thereof can be induced. LRG1 and its immunogenic fragment can be secreted extracellularly. For extracellular secretion, LRG1 and its immunogenic fragment can be linked to a signal sequence.

[0025] Even short peptides are sufficient to induce antibody production in the body. In the present disclosure, fragments of LRG1 do not necessarily have to be short peptides, but may be, for example, fragments (preferably continuous fragments) comprising 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the entire length of LRG1. For example, they may be a region of the protein, such as a loop portion or a portion between loops, or may be short peptides, typically 10 to 20 amino acids in length, 15 to 25 amino acids in length, 20 to 25 amino acids in length, 25 to 30 amino acids in length, 20 to 30 amino acids in length, 30 to 40 amino acids in length, 12 to 18 amino acids in length, or 10 to 40 amino acids in length. Those skilled in the art would be able to design immunogenic fragments of LRG1 to induce antibody production. The immunogenic fragment may be MHC class I or class II restricted, but may not be MHC class I or class II restricted in order to prevent T cell activation. In one embodiment, it may be MHC class II restricted. In one embodiment, the immunogenic composition may comprise LRG1 or a fragment thereof linked to a peptide that is not MHC class I or class II restricted and a carrier protein. In order to promote the production of IgG2 or IgG4 without effector function, the immunogenic composition may contain an aluminum salt such as alum.

[0026] A protein or a fragment thereof can be conferred or improved in immunogenicity by linking it to an immune carrier protein (simply referred to as a "carrier protein"). Examples of carrier proteins include hemocyanins such as keyhole limpet hemocyanin (KLH), and albumins such as bovine serum albumin (BSA) and ovalbumin (OVA). In particular, fragments (especially short peptides) are preferably fused with a carrier protein.

[0027] The nucleic acid can be DNA or mRNA.

[0028] DNA encoding LRG1 or an immunogenic fragment thereof can be incorporated into a gene expression cassette equipped with the components necessary for intracellular transcription and translation. A gene expression cassette typically includes a promoter sequence, a coding region, a 3' untranslated region, and a poly(A) addition signal. Within the gene expression cassette, the promoter sequence can drive gene expression from the coding region. The promoter can be either a constitutive promoter or a promoter induced by a drug (e.g., tetracycline or doxycycline). Constitutive promoters include promoters derived from viruses such as CMV (cytomegalovirus), RSV (respiratory syncytial virus), and SV40 (simian virus 40), actin promoters, and RNA polymerase II promoters such as the EF (elongation factor) 1α promoter. Examples of inducible promoters include tetracycline response elements (TRE3G promoters), Cumate operator sequences, λ operator sequences (12×λOp), and heat shock promoters. The DNA may be incorporated into circular DNA such as a plasmid, or into a viral genome. Alternatively, the DNA may be incorporated into linear DNA.

[0029] The mRNA encoding LRG1 or an immunogenic fragment thereof comprises the components necessary for protein translation in cells. Specifically, the mRNA may contain a cap structure, a coding region, a 3' untranslated region, and a poly(A). The cap structure of the mRNA may be a Cap 0 structure, a Cap 1 structure, or a Cap 2 structure, preferably a Cap 2 structure. It is preferable that the amount of contaminating mRNA without a cap structure is low. The proportion of mRNA with a Cap 2 structure in the mRNA in the immunogenic composition is preferably 95 mol% or more, more preferably 96 mol% or more, even more preferably 97 mol%, even more preferably 98 mol% or more, and particularly preferably 99 mol% or more. The amount of contaminating double-stranded RNA in the immunogenic composition is preferably less than 0.1 wt%, more preferably less than 0.01 wt%. The amount of contaminating double-stranded RNA in the immunogenic composition is preferably below the detection limit in a dot blot using the J2 antibody. The J2 antibody is a double-stranded RNA-recognizing antibody commercially available from various companies, and is included in, for example, Abcam's product designated product number ab288755. The mRNA may also contain a 3' untranslated region and poly(A) on the 3' side of the coding region. Thus, the mRNA is suitable for protein translation in cells.

[0030] The mRNA encoding LRG1 or an immunogenic fragment thereof may itself be low immunogenic. The mRNA encoding LRG1 or an immunogenic fragment thereof may contain modified nucleosides. Examples of mRNA containing modified nucleosides include the modified nucleosides described in US Pat. No. 8,278,036B, the entirety of which is incorporated herein by reference. Modified nucleosides include, for example, pseudouridine, which is known as a modified nucleoside for in vivo expression of mRNA. Modified nucleosides can replace unmodified nucleosides. Examples of pseudouridine include 1-methyl-3-(amino-5-carboxypropyl)pseudouridine (m 1 acp 3Examples of pseudouridines that can replace uridine include pseudouridine (m1Ψ), 1-methylpseudouridine (m1Ψ), 2'-O-methylpseudouridine (Ψm), 5-methyldihydriuridine (m5D), and 3-methylpseudouridine (m3Ψ). The modified mRNA preferably contains pseudouridine, and may further contain preferably 5-methylcytidine, and more preferably 1-methylpseudouridine. In one embodiment, one or more or all of the uridines in the mRNA are pseudouridine.

[0031] The nucleic acid (preferably messenger RNA (mRNA)) encoding LRG1 or an immunogenic fragment thereof may be encapsulated in a drug delivery carrier, as this is expected to improve stability in the blood and / or improve the efficiency of uptake into cells.

[0032] In the present disclosure, the immunogenic composition may further comprise an adjuvant, such as an aluminum salt. Examples of aluminum salts include alum (i.e., aluminum hydroxide). In the present disclosure, the immunogenic composition may not comprise an adjuvant. In the immunogenic composition, the adjuvant may be contained within a drug delivery carrier.

[0033] In some embodiments, mRNA can be administered transdermally via a needleless syringe, in which case the mRNA does not need to be contained in a drug delivery carrier (see WO 2023 / 145755 A, which is incorporated herein by reference in its entirety).

[0034] In some embodiments, the mRNA is encapsulated in a drug delivery carrier. Drug delivery carriers include, but are not limited to, lipid nanoparticles. In some preferred embodiments, the lipid nanoparticles may contain an ionizable lipid (or cationic lipid), a non-cationic lipid (preferably a neutral lipid), cholesterol, and a PEG lipid.

[0035] In some embodiments, the cationic lipid includes 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530). In some embodiments, the cationic lipid can include or is L608. In some aspects, the cationic lipid can include or is L530.

[0036] In some embodiments, the cationic lipid is selected from the group consisting of 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 of US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[0037] In some embodiments, the ionizable cationic lipid (also referred to as an ionizable lipid) can be 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0038] In some embodiments, non-cationic lipids (particularly neutral lipids) include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), PO phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and sphingomyelin (SM). Neutral lipids can be neutral as a whole molecule under neutral pH conditions.

[0039] Examples of sterols include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as zymosterol and ergosterol. The lipid nanoparticles preferably contain a sterol, and more preferably contain cholesterol.

[0040] The lipid nanoparticles may contain a lipid having a polyalkylene glycol modification. Examples of polyalkylene glycols that can be used include polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and polyhexamethylene glycol. The weight-average molecular weight of the polyalkylene glycol (preferably polyethylene glycol) is, for example, about 300 to 10,000, preferably about 500 to 10,000, more preferably about 1,000 to 5,000, for example, 1,000 to 3,000, and particularly about 2,000. Lipids having a polyethylene glycol modification are called PEG lipids.

[0041] Modification of lipids with polyethylene glycol can be achieved by stearylated polyethylene glycol (e.g., PEG 45 stearate (STR-PEG45)). PEG lipids include N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and N- Polyethylene glycol derivatives such as [carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) can also be used.

[0042] The lipid nanoparticles preferably have an average particle diameter of 400 nm or less, and more preferably an average particle diameter of 300 nm or less. The average particle diameter of lipid nanoparticles refers to the number-average particle diameter measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be performed by conventional methods using a commercially available DLS device or the like. The polydispersity index (PDI) of the lipid nanoparticles can be approximately 0.05 to 0.5, approximately 0.05 to 0.4, approximately 0.05 to 0.3, preferably approximately 0.05 to 0.2, more preferably approximately 0.05 to 0.15, or approximately 0.1. Lipid nanoparticles can be prepared using a microfluidic device according to conventional methods. Furthermore, it is easy to control the PDI of LNPs to approximately 0.1 using conventional methods.

[0043] In some embodiments, the lipid nanoparticles may comprise about 20 mol% to about 60 mol% cationic or ionizable lipids, about 0.5 mol% to about 15% PEG-modified lipids, about 25 mol% to about 55% sterol, and about 20 mol% to about 60 mol% non-cationic lipids. In some preferred embodiments, the lipid nanoparticles comprise about 45 mol% to about 50 mol% cationic or ionizable lipids, about 9-10 mol% non-cationic lipids (preferably neutral lipids), about 35 mol% to about 45 mol% sterol (preferably cholesterol), and about 1.5 mol% to about 2 mol% PEG-lipids.

[0044] The lipid nanoparticles may have a positive charge in aqueous solution. Alternatively, the lipid nanoparticles may have a negative charge in aqueous solution. Alternatively, the lipid nanoparticles may be uncharged in aqueous solution. In some preferred embodiments, the lipid nanoparticles have an approximately neutral charge in aqueous solution.

[0045] The method of the present disclosure is a method for stimulating immunity, and therefore its effects can be expected to last for a long time. In the method of the present disclosure, the frequency of administration of mRNA is not particularly limited, but can be, for example, once every 3 to 6 months. The dosage and frequency of administration of mRNA can be changed depending on the degree of symptom recovery.

[0046] <Compositions and Kits for Use in the Treatment Methods of the Present Disclosure> The present disclosure provides immunogenic compositions containing messenger RNA (mRNA) encoding LRG1 or immunogenic fragments thereof for use in the above-described methods, or kits containing the immunogenic compositions. According to the present disclosure, LRG1 or immunogenic fragments thereof may be in the form of a fusion protein with an immune carrier protein. According to the present disclosure, the immunogenic composition may further contain an adjuvant such as alum. The mRNA may be modified as described above. The purity of the mRNA having the Cap2 structure may also be as described above. The mRNA may preferably be encapsulated in a drug delivery carrier.

[0047] The immunogenic composition contains an effective amount of the mRNA. In one embodiment, in the immunogenic composition, some or preferably all of the uridines contained in the mRNA are pseudouridines (preferably 1-methylpseudouridine) and have a Cap 2 structure, and the mRNA having the Cap 2 structure accounts for more than 90% (preferably 98%, more preferably more than 99%) of all mRNAs encoding LRG1 or immunogenic fragments thereof. In one embodiment, in such an immunogenic composition, the mRNA may be naked (i.e., not encapsulated in a drug delivery carrier). Such compositions may be administered by intramuscular injection, subcutaneous injection, intradermal injection, or other topical administration, or may be administered transdermally by spraying using the needleless injection device described above. In another embodiment, in such an immunogenic composition, the mRNA may be encapsulated in a drug delivery carrier (e.g., LNP). Such compositions may be administered by intramuscular injection, subcutaneous injection, intradermal injection, or other topical administration, or may be administered transdermally by spraying using the needleless injection device described above. In one embodiment, the LNP comprises an ionizable lipid (or cationic lipid), a non-cationic lipid (preferably a neutral lipid), cholesterol, and a PEG lipid, preferably in proportions of about 20 mol% to about 60 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 55%, and about 0.5 mol% to about 15%, more preferably about 45 mol% to about 50 mol% cationic lipid or ionizable lipid, about 9-10 mol% non-cationic lipid (preferably a neutral lipid), about 35 mol% to about 45 mol% sterol (preferably cholesterol), and about 1.5 mol% to about 2 mol% PEG lipid.

[0048] According to the present disclosure, the immunogenic composition may further comprise a pharmaceutically acceptable additive and / or carrier. Pharmaceutically acceptable additives and / or carriers include, but are not limited to, water, salts, pH adjusters, isotonicity agents, stabilizers, preservatives, and dispersants. The immunogenic composition is suitable for administration to humans and animals. The administration route may be, for example, parenteral administration, which may be local administration such as intramuscular administration, subcutaneous administration, or intradermal administration, or systemic administration such as intravenous administration.

[0049] In one aspect, there is provided the use of messenger RNA (mRNA) encoding LRG1 or an immunogenic fragment thereof in the manufacture of an immunogenic composition for use in the methods of the disclosure. In one aspect, there is provided the use of an immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or an immunogenic fragment thereof in the manufacture of an immunogenic composition for use in the methods of the disclosure.

[0050] In one embodiment, messenger RNA (mRNA) encoding LRG1 or an immunogenic fragment thereof for use in the methods of the disclosure is provided.

[0051] Materials and Methods (1) Animals Spontaneous subretinal neovascularization model mice, very low density lipoprotein receptor knockout mice, Vldlr - / - were obtained from the Jackson Laboratory and bred to generate experimental mice aged 8–12 weeks. C57BL / 6J, laser-induced choroidal neovascularization (CNV) model mice, 8 weeks old, were obtained from Japan SLC Co., Ltd. All mice were housed at 21–25°C, provided with water and food ad libitum, and handled in accordance with the guidelines of the ARVO statement for the use of animals in ophthalmic and vision research and the guidelines of the Yokohama University Animal Center.

[0052] To examine the efficacy of LRG1 mRNA vaccination, C57BL / 6J mice were used. They were randomly divided into two groups, each with n = 5-6: control (untreated negative control) and LGR1 mRNA-vaccinated group. - / - Mice were randomly assigned to three groups: a control group (untreated negative control), an LRG1 mRNA-administered group, and an anti-VEGFR2 antibody DC101-administered group (positive control).

[0053] (2) Preparation of lipid nanoparticles encapsulating LRG1 mRNA. Template DNA was prepared by PCR using Q5™ High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA). The primer pair and plasmid DNA used for PCR had the sequences shown in SEQ ID NOs: 1 and 2, and SEQ ID NO: 3. mRNA was prepared using ARCA or PureCap analogs. 100% N1-methyl-pseudouridine-modified ARCA mRNA was prepared using the MEGAscript T7 Transcription kit (Thermo Fisher Scientific) according to the manufacturer's instructions. PureCap mRNA was prepared as previously described (Nature communications, 14, 2657 (2023)). Briefly, in vitro transcription of mRNA was performed by incubating 10 U / μL T7 polymerase, 10 ng / μL DNA template, 2 mM NTPs, 5 mM dithiothreitol, and 4 mM DiPureCap at 37°C for 2 hours, followed by DNase I treatment (final concentration: 0.1 μL / unit) at 37°C for 15 minutes. In the IVT reaction, all uridines were modified with N1-methyl-pseudouridine. The mRNA containing PureCap was then purified by HPLC analysis, the hydrophobic tag was removed by light irradiation, and HPLC purification was performed. The resulting mRNA is called PureCap mRNA. LNPs were prepared by mixing the ionic lipid ALC-0315, phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-lipid ALC-0159 (molar ratio: 46.3:9.4:42.7:1.6) with mRNA at a [amino group (N) in ALC-0315] / [phosphate group (P) in mRNA] ratio of 6 using a Nanoassembly Spark (Precision NanoSystems, Vancouver, BC, Canada). For the laser-induced CNV model, ARCA mRNA or PureCap mRNA was added using a Vldlr. - / - PureCap mRNA was used as a model.

[0054] PureCap mRNA has a purity of over 99% after purification. PureCap is a technology that introduces a hydrophobic tag into the cap structure, allowing it to be separated from mRNA without a cap at its end by reverse-phase chromatography. The hydrophobic tag is then removed by light irradiation, allowing mRNA with a cap structure (e.g., Cap 0 structure, Cap 1 structure, and Cap 2 structure) to be purified with a purity of typically over 99%. In this way, in this example, LRG1 mRNA with a Cap 0 structure was purified with a purity of over 99% (see Figures 1A to 1C).

[0055] Effect of LRG-1 Vaccination on the Laser-Induced CNV Model (3) Drug Administration. ARCA mRNA-treated mice received intramuscular (IM) injections of LRG1 mRNA-encapsulated lipid nanoparticles (containing 5 μg / shot mRNA) on days 0 and 14 (see Figure 2). Laser induction of CNV was performed on day 14, and fluorescein angiography (FFA) and eye enucleation were performed 7 days after CNV induction. As a positive control, the anti-VEGFR2 antibody DC101 was administered at a dose of 50 mg / kg body weight (see Figure 3). PureCap mRNA-treated mice received the LRG1 mRNA-encapsulated LNP-treated group. The vaccine (5 μg) was administered intramuscularly on days 0 and 14. As a positive control, the anti-VEGFR2 antibody DC101 was administered intraperitoneally at 50 mg / kg on day 18. Seven days after CNV induction, fluorescein angiography (FFA), eye enucleation, and CNV size were quantified (see Figure 11).

[0056] (4) Laser-Induced Choroidal Neovascularization: C57BL / 6 mice were anesthetized with isoflurane inhalation (Viatris). Midrefrin P solution (Nitto Medic) was topically administered to the right eye to dilate the pupil. The mouse was placed on the platform of a Micron III system (Phoenix Laboratories), and Scopisol solution was instilled into the right eye. CNV was induced by four laser photocoagulation sessions around the optic disc, between major retinal vessels, using a slightly modified Lambert method with a power output of 240–360 mW and a duration of 5 ms. An effective laser spot was confirmed by the formation of vapor bubbles immediately after laser photocoagulation, without any bleeding or hemorrhage. Only mice with intact eyes without capillary rupture or structural abnormalities were randomly assigned to groups after laser irradiation and used for subsequent experiments.

[0057] (5) Fluorescein Angiography (FA). C57BL / 6J mice were anesthetized with isoflurane inhalation (Viatris) in a sealed chamber, and their pupils were dilated with midrefrine P topical solution (Nitto Medic). The mice received 0.1 mL of 10% fluorescein (Novartis) diluted with saline (Otsuka Pharmaceutical) intravenously via the tail vein and were placed on the platform of a Micron III system (Phoenix Laboratories). FA images obtained using the Micron III system were imported into Image J software and analyzed using Image J software (National Institutes of Health [NIH]; Bethesda, MD, USA). Signal intensity (brightness) within the CNV leakage was measured and integrated at each laser irradiation site.

[0058] (6) Flat Mount and Immunofluorescence. Eyeballs were enucleated and immediately fixed in 4% paraformaldehyde phosphate buffer (Nacalai Tesque) for approximately 90 minutes. After rinsing with PBS, the eyeballs were cleaned from extraocular material (muscle, fat, conjunctival tissue attached to the sclera, and optic nerve) under a stereomicroscope (Leica M125). The anterior segment (cornea, lens, and ciliary body) and retina were carefully excised. The remaining posterior cup (sclera-choroid-RPE complex) was incubated in blocking solution (0.5% BSA + 0.5% Triton X-100 in PBS) on a cooling mixer block (Thermomixer C, Eppendorf) at 4°C for 4 hours. The sclera-choroid-RPE complex was then incubated overnight on a 4°C cooling mixer block in a blocking solution containing Griffonia simplicifolia isolectin GS-IB4, Alexa Fluor 488 conjugate (Invitrogen, 1:100) and anti-Iba1 / AIF antibody clone 20A12.1, Alexa Fluor 555 conjugate (Sigma-Aldrich, 1:200). The eyecup was washed with PBS (three times, 10 min each, at room temperature), and the stained sclera-choroid-RPE complex was flattened by making four to five radial cuts between the laser lesions. The sample was mounted in ProLong Diamond Antifade Mountant (Molecular Probes) and coverslipped. FITC-labeled isolectin binds to vascular endothelial cells, while Iba1 binds to microglial cells and macrophages. Micrographs were taken using a fluorescence microscope (Keyence BZ-X710).

[0059] (7) Measurement of CNV area in flat-mount images To determine the CNV area on choroidal flat-mount microscopic images, the integrated intensity area was measured for each antibody using Image J Software, and the background-subtracted corrected total cell fluorescence (CTCF) was calculated (mean, ± SEM).

[0060] (8) Immunofluorescence Staining. Whole eyecup cryosections (10 μm) were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes and blocked with 10% blocking solution (Blocking One, Nacalai Tesque) for 1 hour at room temperature. The sections were incubated with monoclonal anti-glial fibrillary acidic protein (GFAP) clone GA-5 (1:400, G3893, Sigma) for 1 hour, washed three times with PBS, and then incubated with secondary antibody (1:1000, A-11001, Invitrogen) and DAPI (1:1000) for 1 hour. After washing three times with PBS, the slides were coverslipped with ProLong Diamond Antifade Mountant (Molecular Probes). Photomicrographs were taken using a Keyence BZ-X710 fluorescence microscope.

[0061] Effect of LRG1 mRNA vaccination on VLDLR knockout mice (9) After the first FA imaging, Vldlr - / - Lipid nanoparticles encapsulating 5 μg (0.1 mL) of LRG1 mRNA were intramuscularly injected into the LRG1 mRNA group. The second injection was performed 14 days after the first injection. (10) FA of VLDLR KO mice. To evaluate the leakage area before treatment (day 1) and after treatment (day 21 or 28), FA was performed and analyzed using Image J software. The color channels were split, blue and red were removed, and the green image was used for measurement. After applying Invert, the image was processed for local contrast enhancement (CLAHE). At 300% digital magnification, the maximum fluorescein swelling was outlined using the freehand selection tool, and its area was measured in pixels.

[0062] (11) RNA isolation. Eyeballs were enucleated, washed with PBS, and immediately immersed in RNAlater Stabilization Solution (Invitrogen). Eyeballs were dissected under a stereomicroscope (Leica M125), and the retina, RPE, choroid, and sclera were homogenized in 1 mL of Trizol reagent (Invitrogen) using a pestle (BioMasher II Homogenizer). Total RNA was extracted using the PureLink RNA Mini Kit (Ambion, Life Technologies) according to the manufacturer's instructions and stored at -80°C. RNA concentration was measured by UV absorbance using a Nanodrop spectrophotometer (Nanodrop C, ThermoScientific), and RNA integrity was estimated by the OD 260 / 280 ratio.

[0063] (12) Quantitative PCR of Angiogenesis and Inflammation Targets. For cDNA synthesis, 1 μg of total RNA was added to 5x PrimeScript RT Master Mix (Takara) and nuclease-free water (Ambion) in a total reaction volume of 20 μl. Reverse transcription was performed at 37°C for 15 minutes, 85°C for 5 seconds, and the final reaction at 4°C. The reaction mix was SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions. 20 ng of cDNA was used in duplicate for each reaction. Mouse-specific primer sequences are listed in the table. The programmed thermal cycling protocol was 95°C for 3 minutes, with 40 cycles of PCR cycles consisting of 95°C for 10 seconds, 55°C for 30 seconds, and melt curve analysis at 65°C for 10 seconds (Bio-Rad CFX96). Quantitative gene expression was calculated using the comparative Ct method. Target gene expression was normalized to the relative expression of the GAPDH internal control gene (fold change = 2). - △△ Ct law).

[0064]

[0065] (13) Statistical Analysis All data were expressed as the mean (standard error of the mean (SEM)). Statistical analysis was performed using a two-tailed Student's t-test for comparison of two groups, with Bonferroni correction for multiple comparisons. A confidence level of p ≤ 0.05 was considered statistically significant.

[0066] Results: LRG1 messenger RNA (mRNA) was expressed in vivo by encapsulating it in lipid nanoparticles (LNPs). Lipid nanoparticles were prepared by mixing ionizable lipids, phospholipids, cholesterol, and PEG-lipids with mRNA in a microfluidic device. The LNPs were injected intramuscularly to express LRG1 in vivo and potentially induce antibodies against LRG1 in the subject.

[0067] The above LNPs were administered intramuscularly to Vldlr knockout mice (i.e., a model of retinal angiomatous proliferation and macular telangiectasia, a type of age-related macular degeneration) and a laser-induced choroidal neovascularization (CNV) model using the treatment regimen shown in Figure 2. Fluorescein angiography (FA) was used to measure the area of ​​blood vessel leakage of the fluorescent substance fluorescein. A reduction in the leakage area indicates a therapeutic and preventive effect on AMD.

[0068] The LRG1 mRNA administered in Figures 3 and 4 was ARCA mRNA, while the LRG1 mRNA in the LRG1 vaccine administered in Figure 11 was PureCap mRNA. As shown in Figure 3, the area of ​​fluorescein leakage from blood vessels was significantly reduced in the LRG1 mRNA (ARCA mRNA)-encapsulated LNP-administered group compared to the negative control. As shown in Figure 3, the leakage area decreased over time. Photographs obtained by fluorescein fundus angiography are shown in Figure 4. As shown in Figure 4, angiogenesis was suppressed in the LRG1 mRNA (ARCA mRNA)-encapsulated LNP-administered group. Furthermore, as shown in Figures 11A and 11B, the area of ​​fluorescein leakage from blood vessels was significantly reduced in the LRG1 mRNA (PureCap mRNA)-encapsulated LNP-administered group compared to the negative control. CNV size was significantly reduced in the LRG1 mRNA (PureCap mRNA)-encapsulated LNP-administered group compared to the negative control.

[0069] The expression of various factors in RNA isolated from the eyeballs was measured by quantitative PCR, and the results are shown in Figure 5. As shown in Figure 5, significant decreases in the mRNA expression of Pecam1 (CD31), VEGFR1 (Flt1), VEGFR2, and IL6 were observed. In the LRG1 mRNA (ARCA mRNA)-encapsulated LNP group, in addition to simply reducing CD31-expressing cells (i.e., vascular endothelial cells) and angiogenesis, the expression of the above-mentioned angiogenesis-inducing factors was also suppressed. Administration of a vaccine containing LRG1 mRNA may be useful in inhibiting angiogenesis in various settings.

[0070] Furthermore, the results of evaluating laser-induced CNV using choroidal flat-mount images are shown in Figure 6. In the LRG1 mRNA (ARCA mRNA)-encapsulated LNP-administered group, the fluorescence intensity of isolectin and IbaI was significantly reduced, indicating a decrease in CNV size and a decrease in microglial cell migration, respectively. These decreases in the LRG1 mRNA-encapsulated LNP-administered group were greater than those in the anti-VEGFR2 antibody-administered group.

[0071] Furthermore, as shown in Figure 7, hematoxylin and eosin (H&E) staining of fundus tissue sections clearly showed a reduction in CNV area in the LRG1 mRNA (ARCA mRNA)-encapsulated LNP-treated group. Also, as shown in Figure 7, a clear reduction in the CD31 vascular endothelial cell staining area was observed in the LRG1 mRNA (ARCA mRNA)-encapsulated LNP-treated group, indicating that angiogenesis was inhibited in the LRG1 mRNA (ARCA mRNA)-encapsulated LNP-treated group. As shown in Figure 8, flat-mount images also showed a clear reduction in CNV area in the LRG1 mRNA (ARCA mRNA)-encapsulated LNP-treated group, and this reduction was more pronounced than in the anti-VEGFR2 antibody-administered group.

[0072] LRG1 mRNA (ARCA mRNA)-encapsulated LNP or VEGFR2 mRNA-encapsulated LNP was intramuscularly administered to a mouse model of laser-induced CNV. Fluorescein angiography was used to observe fluorescent leakage, and the results are shown in Figure 9. As shown in Figure 9, only the LRG1 mRNA-encapsulated LNP group showed a reduction in leakage; the VEGFR2 mRNA-encapsulated LNP group did not. Antibodies against VEGFR2 were found to have a leakage-reducing effect, but the VEGFR2 mRNA-encapsulated LNP group did not, resulting in contradictory results.

[0073] According to the schedule shown in Figure 10A, LRG1 mRNA (ARCA mRNA)-encapsulated LNP was administered intramuscularly to laser-induced CNV model mice on day 0, followed by another intramuscular boost on day 14. Figure 10B shows the time course of antibody levels in mouse blood after administration. An increase in LRG1-specific antibodies was observed on days 21 and 28, and high antibody levels were maintained even 28 days after the initial immunization.

[0074] Safety was evaluated by H&E staining one day after administration of LRG1 mRNA (PureCap mRNA). Figure 12 shows the results of H&E staining in the liver, lungs, kidneys, spleen, heart, and muscle. Safety was evaluated by H&E staining 21 days after administration of LRG1 mRNA (PureCap mRNA). Figure 12 shows the results of H&E staining in the liver, lungs, kidneys, spleen, and heart. In addition, when the values ​​of AST, ALT, LDH, BUN, and CRE were compared with those of the untreated control group 21 days after administration, no significant increases were observed in any of the indices. Therefore, the safety of LRG1 mRNA (PureCap mRNA) administration was suggested.

[0075] These results suggest that LRG1 is a suitable target for antibody induction and neutralization by vaccines. Administration of the LRG1 mRNA vaccine to non-affected tissues induces LRG1-specific antibodies in the body, inhibiting angiogenesis in the affected area. The LRG1 mRNA vaccine inhibited choroidal neovascularization (CNV) in AMD. Previous treatments have involved intravitreal administration, which is frequent and invasive. However, the LRG1 mRNA vaccine can be administered to non-target tissues to induce LRG1-specific antibodies, potentially achieving systemic efficacy. Furthermore, because antibodies are continuously produced by plasma cells, production is expected to be sustained over the long term, potentially resulting in long-lasting LRG1 inhibitory effects. Therefore, this vaccine may be effective in treating angiogenesis in any target tissue accessible to antibodies, particularly in inhibiting choroidal neovascularization. Treatment of age-related macular degeneration has traditionally been achieved by administering drugs into the vitreous, but according to the present disclosure, treatment may be achieved by administering drugs via routes other than intravitreal administration, such as intradermal / subcutaneous or intramuscular administration.

Claims

1. An immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or a fragment thereof as an immunogen for use in a method for treating angiogenesis in a target tissue of a subject, the method comprising administering to the subject an immunogenic composition comprising messenger RNA (mRNA) encoding LRG1 or an immunogenic fragment thereof, causing LRG1 or the fragment to be expressed in cells of a tissue other than the target tissue, and inducing specific antibodies against LRG1 or the fragment in the subject, and inhibiting LRG1 with the antibodies.

2. The composition of claim 1, wherein the subject has retinopathy and the target tissue is the retina.

3. The composition according to claim 1 or 2, wherein the retinopathy is age-related macular degeneration.

4. The composition according to any one of claims 1 to 3, wherein the administration is intramuscular.

5. A composition according to any one of claims 1 to 4, wherein the mRNA comprises one or more modified nucleic acids.

6. A composition described in any one of claims 1 to 5, wherein the mRNA has a Cap2 structure.

7. A composition described in any one of claims 1 to 6, wherein the purity of mRNA having a Cap 2 structure relative to total mRNA is more than 90%.

8. A composition described in any one of claims 1 to 7, wherein the mRNA is encapsulated in a lipid nanoparticle.

9. The composition of claim 8, wherein the lipid nanoparticles comprise an ionizable lipid, a phospholipid, cholesterol, and a PEG-lipid.

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