Recombinant vector for treating macular degeneration and diabetic retinopathy and pharmaceutical composition thereof

Abstract

The present invention relates to a bicistronic recombinant vector designed to inhibit VEGF signaling and simultaneously activate the Tie2 pathway, and a composition comprising same, wherein Aflibercept (Afb) and COMP-Ang1 (cAng1) are simultaneously expressed using a single vector. Aflibercept is a representative anti-VEGF therapeutic agent and is a recombinant fusion protein containing extracellular domains of VEGFR1 and VEGFR2 fused to the Fc portion of human IgG1. Aflibercept reduces angiogenesis and leakage of blood vessels by inhibiting downstream signaling by binding with high affinity to VEGF-A, VEGF-B, and PlGF. In addition, COMP-Ang1 demonstrates both anti-angiogenic effects (inhibition of CNV) and vascular stabilization (promotion of healthy blood vessels).

Description

Recombinant vector for treating macular degeneration and diabetic retinopathy and the pharmaceutical composition thereof

[0001] The present invention relates to a recombinant vector for treating macular degeneration and diabetic retinopathy, a composition utilizing the same, and a method of use. More specifically, the present invention provides a recombinant vector, a composition for gene therapy, a kit, a reagent composition, a use of the vector, a cell, a culture medium, a method for the simultaneous production of a protein, a pharmaceutical composition for treating macular degeneration and diabetic retinopathy, a method for delivering a protein / gene, and a method for treating macular degeneration and diabetic retinopathy.

[0002] Neovascular diseases constitute a major burden on health because they cause vision loss, chronic inflammation, and tissue damage in various pathological conditions. The underlying mechanism of these diseases is primarily angiogenesis, the formation of new blood vessels within existing vascular structures. Angiogenesis is essential for normal processes such as wound healing and embryonic development. However, if it is excessive or uncontrolled, it can lead to various diseases, including cancer, arthritis, and ophthalmic disorders. In ophthalmology in particular, abnormal angiogenesis is a significant pathogenesis mechanism for both age-related macular degeneration (AMD) and diabetic retinopathy (DR). These conditions are the leading causes of blindness worldwide.

[0003] Macular degeneration is one of the major causes of visual impairment in the elderly population. It is divided into dry and wet (neovascular) forms. Dry macular degeneration is characterized by atrophy of the retinal pigment epithelium (RPE) and the accumulation of drusen, whereas wet macular degeneration involves choroidal neovascularization (CNV), where newly formed blood vessels leak blood and fluid beneath the retina, often leading to acute vision loss. Meanwhile, diabetic retinopathy is a microvascular complication of diabetes and is classified into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). In the early non-proliferative phase, retinal vascular damage and ischemia occur. In progressive proliferative diabetic retinopathy (PDR), abnormal neovascularization grows in response to retinal ischemia, causing vitreous hemorrhage and tractional retinal detachment, which can lead to severe and irreversible vision loss. In other words, both non-proliferative and proliferative diabetic retinopathy can cause diabetic macular edema, characterized by the leakage and accumulation of fluid in the macula, at all stages of progression.

[0004] Various therapeutic strategies have been sought to inhibit abnormal angiogenesis, and the inhibition of Vascular Endothelial Growth Factor (VEGF) and the regulation of the angiopoietin pathway have emerged as key targets. VEGF is the most potent inducer of angiogenesis, promoting the proliferation, migration, and survival of endothelial cells while increasing vascular permeability. VEGF is particularly important in the pathological angiogenesis of ophthalmic diseases. For this reason, anti-VEGF agents have now become the standard treatment for macular degeneration and diabetic macular edema (DME). More specifically, Ang1 (Angiopoietin 1) and Ang2 (Angiopoietin 2) are vascular growth factors that regulate vascular permeability and angiogenesis. In a normal retina, Ang1 and Ang2 maintain a balance through homeostasis. However, in macular degeneration and diabetic retinopathy, Ang2 increases, which has been found to be correlated with a decrease in pericyte coverage and an increase in apoptosis.

[0005] A number of investigational drugs are being attempted based on such reaction mechanisms. In addition to such investigational drugs, for example, Patent Document 1 discloses a VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) antibody used for the treatment of macular degeneration and cancer. In this document, an antibody is disclosed that specifically binds to VEGFR2, which is overexpressed in vascular endothelial cells, by having the antibody or its antigen-binding fragment possess a specific sequence structure.

[0006] However, all macular degeneration treatments currently in clinical trials, including the invention described in Patent Document 1, have limitations in that they employ a mechanism of inhibiting angiogenesis that targets VEGF directly. Similarly, Patent Document 2 provides a method for treating neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR), comprising the steps of administering anti-hVEGF treatment and steroid treatment, wherein the anti-hVEGF treatment comprises administering a therapeutically effective amount of a recombinant viral vector containing a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the eye of a subject, and the steroid treatment comprises administering a therapeutically effective amount of steroid to the eye of a subject. However, this method is considerably cumbersome in that steroids must be administered separately.

[0007] Therefore, there is still a strong demand for new therapeutic approaches with higher treatment efficiency for macular degeneration and diabetic retinopathy.

[0008] [Prior Art Literature]

[0009] Patent Document 1: Republic of Korea Patent Publication 10-2017-0064086

[0010] Patent Document 2: Republic of Korea Patent Publication 10-2025-0099772

[0011] The objective of the present invention is to provide a novel recombinant vector capable of simultaneously expressing Aflibercept (Afb) and Ang1, i.e., COMP-Ang1 (cAng1), which are candidate substances for the treatment of macular degeneration and diabetic retinopathy, in a single recombinant vector, unlike conventional approaches for treating macular degeneration and diabetic retinopathy, as well as a composition utilizing the same and a method of use. More specifically, the present invention aims to solve the problem by providing a recombinant vector, a composition for gene therapy, a kit, a reagent composition, a use of the vector, a cell, a culture medium, a method for the simultaneous production of proteins, a pharmaceutical composition for treating macular degeneration and diabetic retinopathy, a method for delivering proteins / genes, and a method for treating macular degeneration and diabetic retinopathy. Accordingly, Aflibercept and Ang1 are simultaneously expressed or act upon each other through a single treatment, thereby enabling excellent therapeutic efficacy and anti-angiogenesis efficacy. Furthermore, by establishing a system capable of simultaneously treating macular degeneration and diabetic retinopathy, the efficiency of the therapeutic approach can be enhanced.

[0012] Conventional anti-VEGF monotherapy for neovascular age-related macular degeneration (AMD) and diabetic retinopathy (DR) has limited application due to incomplete therapeutic response, drug resistance, and the high burden of frequent intravitreal injections. While extensive research is currently underway to improve treatment outcomes, the need for more effective strategies remains significant. To address these limitations, the present invention provides a recombinant vector system co-expressing aflibercept (Afb) and COMP-Ang1 (cAng1), and a pharmaceutical composition for treating macular degeneration and diabetic retinopathy utilizing the same. Specifically, the present invention is designed to inhibit VEGF signaling while simultaneously activating the Tie2 pathway. In vitro characterization using a conditioned medium containing secreted Afb / cAng1 showed that the treatment effectively inhibited VEGF-induced VEGFR2 phosphorylation to below baseline levels while potently activating Tie2. As a result, the supernatant significantly inhibited endothelial cell migration and tubule formation, and restored VE-cadherin expression impaired by VEGF stimulation or hyperglycemic conditions. These findings were further demonstrated in a microfluidic 3D angiogenesis chip (MAC) model that mimics physiological interstitial flow. In this complex microenvironment, Afb / cAng1 demonstrated superior efficacy in reducing neovascularization area and vascular permeability compared to Eylea, the commercial standard for aflibercept (Afb). The active ingredient of Eylea is aflibercept.

[0013] In summary, the present invention demonstrates that a dual-targeting strategy of Afb / cAng1 exerts synergistic effects of anti-angiogenesis and barrier stabilization, and consequently provides a promising gene therapy for macular degeneration and diabetic retinopathy.

[0014] More specifically, the present invention relates to the following.

[0015] One aspect of the present invention provides a recombinant vector that co-expresses the Afb gene and the cAng1 gene under the control of a single promoter.

[0016] In addition, according to one embodiment of the present invention, the recombinant vector is an adeno-associated virus (AAV)-based vector.

[0017] In addition, according to one embodiment of the present invention, the vector further comprises one or more of an IRES sequence and a 2A sequence (2A sequence).

[0018] In addition, according to one embodiment of the present invention, the single promoter is selected from the group consisting of an inducible promoter (hypoxia, drug-induced), a universal promoter, a CMV promoter, a CAG promoter, a CB7 promoter, a CBh promoter, an EF1α promoter, and a retina-specific promoter.

[0019] In addition, according to one embodiment of the present invention, the vector comprises a nucleotide sequence having 80% or more sequence identity with respect to the nucleotide sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4 within a range that does not impair the therapeutic efficacy of the present invention.

[0020] In addition, according to one embodiment of the present invention, the recombinant vector includes a nucleotide sequence in which a mutation of an amino acid is caused through a mutation of a nucleotide for the sequences of SEQ ID NOs 1 to 4, the binding free energy (ΔG) of the resulting protein variant is calculated by applying the MM-GBSA methodology, and the change in energy (ΔΔG) relative to the wild type (i.e., before the mutation is introduced) is calculated and the value is about 10.0 or less.

[0021] In addition, according to one embodiment of the present invention, the recombinant vector, through nucleotide mutation for the sequence of SEQ ID NO. 4, modifies PRO 288 to ASN, MET 248 to PHE, PRO 288 to TRP, ALA 257 to VAL, PRO 260 to ARG, PRO 288 to PHE, PRO 260 to PHE, LEU 277 to TRP, LYS 281 to ARG, ASP 256 to GLU, LEU 277 to TYR, SER 289 to ARG, GLY 275 to ARG, ALA 257 to ASP*, LEU 277 to PHE, PRO 288 to TYR, MET 248 to LEU, and LYS 276 It includes a nucleotide sequence that expresses a COMP-Ang1(cAng1) protein containing one or more of the variants in which TYR or GLY 275 is substituted with LYS.

[0022] In addition, according to one embodiment of the present invention, the vector has a FLAG-tag sequence added to the Afb gene or the cAng1 gene.

[0023] In addition, according to one embodiment of the present invention, the vector comprises one or more of a regulatory element and a bovine growth hormone (bGH) polyadenylation signal.

[0024] In addition, according to one embodiment of the present invention, the regulatory element is a posttranscriptional regulatory element.

[0025] Additionally, according to one embodiment of the present invention, the recombinant vector of the present invention comprises one or more inverted terminal repeat (ITR) sequences.

[0026] Another aspect of the present invention provides a gene therapy composition for treating macular degeneration and diabetic retinopathy, comprising a recombinant vector of the present invention; and a pharmaceutically acceptable carrier.

[0027] In addition, according to one embodiment of the present invention, the composition inhibits VEGF signaling and simultaneously activates the Tie2 pathway.

[0028] In addition, according to one embodiment of the present invention, the composition comprises a recombinant vector of the present invention of approximately 1.0 × 10 per eye. 10 Up to about 5.0 × 10 11 It is administered at a dose equal to the number of genome copies.

[0029] In addition, according to one embodiment of the present invention, the composition is administered to an individual in need by injecting it into the subretinal, choroid, or vitreous cavity within the retina.

[0030] Another aspect of the present invention provides a kit comprising a gene therapy composition and administration instructions of the present invention.

[0031] Furthermore, another aspect of the present invention provides an experimental reagent composition for the prevention or inhibition of macular degeneration and diabetic retinopathy comprising a recombinant vector of the present invention.

[0032] Additionally, one aspect of the present invention provides a use of the recombinant vector of the present invention for use in the preparation of a pharmaceutical composition that prevents or inhibits macular degeneration and diabetic retinopathy.

[0033] In addition, one aspect of the present invention provides a cell transfected with the recombinant vector of the present invention.

[0034] Furthermore, one aspect of the present invention provides a culture medium in which cells transfected with the recombinant vector of the present invention are cultured.

[0035] In addition, one aspect of the present invention provides a method for the simultaneous production of an aflibercept protein and a COMP-Ang1(cAng1) protein, comprising the steps of: introducing the recombinant vector of the present invention into a host cell; culturing the host cell to simultaneously express an aflibercept protein and a COMP-Ang1(cAng1) protein; and obtaining the aflibercept protein and the COMP-Ang1(cAng1) protein from the supernatant of a culture medium.

[0036] In addition, one aspect of the present invention provides a pharmaceutical composition for treating macular degeneration and diabetic retinopathy, comprising: an aflibercept protein and a COMP-Ang1(cAng1) protein prepared by the method for the simultaneous preparation of an aflibercept protein and a COMP-Ang1(cAng1) protein of the present invention; and one or more pharmaceutical additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, binders, disintegrants, lubricants, and any combination thereof.

[0037] In addition, one aspect of the present invention provides a method for delivering an aflibercept protein and a COMP-Ang1 (cAng1) protein or a gene encoding them to an individual in need, comprising the step of administering an effective amount of the recombinant vector of the present invention, the gene therapy composition of the present invention, or the pharmaceutical composition for treating macular degeneration and diabetic retinopathy to an individual.

[0038] In addition, one aspect of the present invention provides a method for treating macular degeneration and diabetic retinopathy, comprising the step of administering an effective amount of the recombinant vector of the present invention, the gene therapy composition of the present invention, or the pharmaceutical composition for treating macular degeneration and diabetic retinopathy to an individual.

[0039] According to the present invention, by providing a recombinant vector for treating macular degeneration and diabetic retinopathy and a composition thereof for gene therapy, macular degeneration and diabetic retinopathy can be fundamentally treated by blocking causative factors at the early stages of the pathogenesis of said macular degeneration and diabetic retinopathy. Furthermore, by enabling aflibercept and Ang1 to be simultaneously expressed or act upon by a single treatment, excellent therapeutic efficacy and anti-angiogenesis efficacy can be demonstrated. In addition, by establishing a system capable of simultaneously treating macular degeneration and diabetic retinopathy, the efficiency of the therapeutic approach can be enhanced.

[0040] (A) of FIG. 1a shows the structure of the gene of the bicistrone recombinant AAV vector (rAAV8-Afb / cAng1) of the present invention that simultaneously expresses Afb and cAng1 according to one embodiment of the present invention. Here, an IRES is interposed between the Afb gene and the cAng1 gene.

[0041] Figure 1a (B) illustrates the process in which individual proteins of Afb and cAng1 are simultaneously produced by the binding of additional ribosomes by an intermediate IRES to the mRNA according to the vector gene of (A).

[0042] Figure 1b (A) shows the structure of the gene of the bicistrone recombinant AAV vector (rAAV8-Afb / cAng1) of the present invention that simultaneously expresses Afb and cAng1 according to one embodiment of the present invention. Here, the 2A sequence is interposed between the Afb gene and the cAng1 gene.

[0043] Figure 1b (B) illustrates the process in which individual proteins of Afb and cAng1 are produced simultaneously, even though only one ribosome is bound to the mRNA according to the vector gene of (A) above, by the intermediate 2A sequence.

[0044] FIG. 2a shows the structures of the vector of the present invention, a vector expressing only Afb, and a vector expressing only cAng1, respectively.

[0045] Figure 2b shows the results of Western blot analysis of the culture supernatant of HEK293T cells transfected with the vector of Figure 2 (A).

[0046] Figure 2c shows the results of a Western blot analysis regarding VEGFR2 phosphorylation in human umbilical vein endothelial cells (HUVEC; produced by Lonza) after VEGF stimulation.

[0047] Figure 2d shows the results of the immunoprecipitation analysis of Tie2 phosphorylation.

[0048] Figure 2e shows the quantification graph of the Western blot analysis results (Figure 2c) regarding VEGFR2 phosphorylation in HUVECs after VEGF stimulation. Results are presented as the mean of three independent experiments. *P < 0.05. ***P < 0.001, ****P < 0.0001, ns: not significant.

[0049] Figure 2f shows a quantification graph of the results of the immunoprecipitation analysis (Figure 2d) of Tie2 phosphorylation.

[0050] Figure 3a shows a representative image of HUVEC migration performed under VEGF stimulation, illustrating the inhibitory effect of the vector system (Afb / cAng1 treatment) of the present invention on VEGF-induced endothelial cell migration and tubule formation.

[0051] FIG. 3b shows the inhibitory effect of the vector system (Afb / cAng1 treatment) of the present invention on VEGF-induced endothelial cell migration and tubule formation, and shows a representative image of tubule formation performed under VEGF stimulation.

[0052] FIG. 3c shows the results of a quantitative analysis of the number of migrated cells as a graph regarding the inhibitory effect of the vector system (Afb / cAng1) of the present invention on VEGF-induced endothelial cell migration and tube formation.

[0053] FIG. 3d relates to the inhibitory effect of the vector system (Afb / cAng1 treatment) of the present invention on VEGF-induced endothelial cell migration and tubule formation, showing the results of a quantitative analysis of tubule formation parameters: number of nodes, number of master junctions, and total tubule length. *P < 0.05, **P < 0.01, ***P < 0.001.

[0054] Figure 4a shows the inhibition of endothelial permeability and the recovery of VE-cadherin expression under VEGF stimulation and hyperglycemic conditions, and the results of Western blot analysis of VE-cadherin expression in HUVECs after VEGF stimulation.

[0055] Figure 4b relates to the inhibition of endothelial permeability and the recovery of VE-cadherin expression under VEGF stimulation and hyperglycemic conditions, and shows a quantification graph of the Western blot analysis results of VE-cadherin expression in HUVECs after VEGF stimulation (Figure 4a). *P < 0.05, **P < 0.01 ***P < 0.001, ****P < 0.0001, ns: not significant.

[0056] Figure 4c shows the results of the evaluation of endothelial permeability using a trans-well assay.

[0057] Figure 4d relates to the inhibition of endothelial permeability and the recovery of VE-cadherin expression under VEGF stimulation and hyperglycemic conditions, showing the results of Western blot analysis of VE-cadherin expression in HUVECs exposed to hyperglycemic conditions (30 mM D-glucose).

[0058] [Correction pursuant to Rule 91 28.04.2026]<Deleted>

[0059] Figure 5a relates to the evaluation of anti-angiogenic efficacy and vascular barrier integrity using a microfluidic 3D angiogenesis chip (MAC), and shows a schematic diagram and timeline of the microfluidic 3D angiogenesis chip (MAC) platform. Here, interstitial flow was induced by maintaining a hydrostatic gradient between endothelial cells and fibroblast channels.

[0060] Figure 5b shows the evaluation of anti-angiogenic efficacy and vascular barrier integrity using a microfluidic 3D angiogenesis chip (MAC), and is a representative fluorescent image of the vascular morphology on day 5. The blood vessels were visualized through fluorescent staining.

[0061] Figure 5c relates to the evaluation of anti-angiogenic efficacy and vascular barrier integrity using a microfluidic 3D angiogenesis chip (MAC), showing fluorescence images according to each treatment component.

[0062] Figure 5d relates to the evaluation of anti-angiogenic efficacy and vascular barrier integrity using a microfluidic 3D angiogenesis chip (MAC), showing the results of a quantitative analysis of angiogenic parameters including vascular area, average vascular length, and thickness.

[0063] Figure 5e relates to the evaluation of anti-angiogenic efficacy and vascular barrier integrity using a microfluidic 3D angiogenesis chip (MAC), showing representative images for each administered component through vascular permeability analysis using fluorescence tracer diffusion.

[0064] Figure 5f relates to the evaluation of anti-angiogenic efficacy and vascular barrier integrity using a microfluidic 3D angiogenesis chip (MAC), showing the results of quantifying vascular leakage through vascular permeability analysis using fluorescence tracer diffusion. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0065] Figure 6a relates to the production, characterization, and cytotoxicity evaluation of the rAAV8-Afb / cAng1 viral vector, and the presence of three capsid proteins (VP1, VP2, and VP3) by silver staining of the purified rAAV8-Afb / cAng1, rAAV8-Afb, rAAV8-cAng1, and rAAV8-tD tomato vectors demonstrates a high degree of vector purity.

[0066] Figure 6b relates to the production, characterization, and cytotoxicity evaluation of the rAAV8-Afb / cAng1 viral vector and shows the results of Western blot analysis on the supernatant from HEK293T cells transfected with rAAV8-Afb / cAng1 72 hours after transduction.

[0067] Figure 6c relates to the production, characterization, and cytotoxicity evaluation of the rAAV8-Afb / cAng1 viral vector, wherein rAAV8-Afb / cAng1 (MOI 1 x 10⁻⁶ 5 and 8 x 10 5 ) Shows the results of cell viability analysis (CCK assay) in HUVEC cells on days 1 and 5 after transduction.

[0068] Figure 6d relates to the production, characterization, and cytotoxicity evaluation of the rAAV8-Afb / cAng1 viral vector, wherein rAAV8-Afb / cAng1 (MOI 1 x 10⁻⁶ 5 and 8 x 10 5 ) Shows the results of cell viability analysis (CCK assay) in ARPE-19 cells on days 1 and 5 after transduction.

[0069] All technical terms used herein shall be used in the sense generally understood by those skilled in the art in the relevant field of the present invention, unless otherwise defined. Furthermore, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of the present invention. Additionally, numerical values ​​described herein are deemed to include the meaning of "approximately" unless explicitly stated otherwise. The contents of all publications cited as references in this specification are incorporated into this specification in their entirety by reference.

[0070] In this specification, the terms “about” or “approximately” may be interpreted to mean a value or range within 10%, 5%, 4%, 3%, 2%, or 1% above or below a given value or range.

[0071] (Recombination vector)

[0072] One aspect of the present invention provides a recombinant vector capable of simultaneously expressing the Afb gene and the cAng1 gene under the control of a single promoter. The Afb gene and the cAng1 gene may be present in a singular or plural within a single vector. The order of these genes within the recombinant vector of the present invention is not limited as long as it does not impede the purpose of the present invention.

[0073] The Afb gene, which is one of the key components of the vector of the present invention, expresses the aflibercept protein, and aflibercept (Afb) is a representative anti-VEGF therapeutic agent. It is a recombinant fusion protein containing the extracellular domains of VEGFR1 and VEGFR2 fused to the Fc portion of human IgG1. Through its unique structure as described above, aflibercept can bind with high affinity to VEGF-A, VEGF-B, and PlGF, and by blocking the action of VEGF, it inhibits downstream signaling, thereby reducing angiogenesis and leakage. Anti-VEGF drugs are administered via intravitreal injection (IVT), and vision has improved in many patients. However, the simple administration of aflibercept has limitations, such as the need for lifelong injections, side effects associated with VEGF inhibition, and inappropriate response or resistance in some patients. Due to these limitations, there is a critical demand for new therapeutic strategies that provide sustained efficacy while reducing the burden of treatment.

[0074] In addition, the cAng1 gene, which is another key component of the vector of the present invention, expresses a variant of the Angiopoietin-1 protein. Angiopoietin-1 (Ang1) and Angiopoietin-2 (Ang2) bind to Tie2, a receptor on endothelial cells. While Ang1 activates Tie2 signaling to stabilize blood vessels, Ang2 generally antagonizes Tie2 signaling, destabilizes vascular structures, and promotes angiogenesis, particularly under inflammatory or hypoxic conditions. COMP-Ang1 (cAng1) employed in the present invention is an engineered variant of Ang1 developed for therapeutic purposes. This is because native Ang1 is difficult to use due to its complex structure, low solubility, and short half-life. cAng1 enhances stability and promotes Tie2 activation by incorporating the coiled-coil domain of the cartilage oligomer matrix protein (COMP). cAng1 exhibits both anti-angiogenic effects (inhibition of CNV) and vascular stabilization (promotion of healthy blood vessels). This dual activity of cAng1 differs from conventional anti-VEGF drugs; consequently, cAng1 strengthens endothelial junctions and reduces permeability to prevent leakage.

[0075] The recombinant vector employed in the present invention is not particularly limited as long as it is capable of simultaneously expressing the Afb gene and the cAng1 gene, and various types such as viral vectors, bacterial vectors, yeast vectors, insect cell vectors, and mammalian cell vectors may be used; however, it is preferably an AAV-based viral vector, more preferably an AAV2, AAV5, or AAV8-based viral vector, and most preferably an AAV8-based viral vector. More preferably, the vector of the present invention possesses targeting for retinal cells.

[0076] According to one embodiment of the present invention, the recombinant vector may further include one or more of the IRES sequence and the 2A sequence (2A sequence). The order of these sequences within the recombinant vector of the present invention is not limited as long as it does not impede the purpose of the present invention.

[0077] The above IRES sequence is a sequence relating to the Internal Ribosome Entry Site (IRES), a special RNA element that allows ribosomes to directly bind to the internal region of mRNA and initiate protein translation (synthesis) without relying on the 5' cap structure of eukaryotic mRNA, and acts as a mechanism for efficiently synthesizing proteins when cap-dependent translation is inhibited, mainly in situations of viral infection or stress. When the vector of the present invention includes the IRES sequence, if the aflibercept gene as the first gene and the cAng1 gene as the second gene are arranged in sequence, it is preferable that the IRES sequence be located at the beginning of the second gene or placed in the middle of the first gene and the second gene.

[0078] The above 2A sequence is a short amino acid sequence used to independently produce multiple proteins during the process of translating a single protein. When translating from RNA to protein, the presence of this sequence allows ribosomes to perform 'ribosomal skipping' without physically detaching from the end of the 2A sequence while producing the protein. Accordingly, although gene expression begins with a single mRNA, the result can be two separate proteins produced in equal amounts. Examples of 2A sequences include P2A, T2A, E2A, F2A, etc., derived from porcine viruses, and can be applied without limitation in the present invention.

[0079] The single promoter employed in the vector of the present invention is not particularly limited as long as it is capable of expressing the Afb gene and the cAng1 gene. Examples include inducible promoters (hypoxia, drug-induced), universal promoters, CMV promoters, CAG promoters, CB7 promoters, CBh promoters, EF1α promoters, and retina-specific promoters, preferably CBh promoters, EF1α promoters, and retina-specific promoters, and more preferably CBh promoters. The CBh promoter (Chicken β-actin hybrid promoter) is a mini version of the CBA promoter and is a universal promoter of about 798 bp in size that combines the regulatory element of the chicken-actin promoter with a mouse virus (MVM) intron to reduce size while maintaining potent expression.

[0080] In addition, according to one embodiment of the present invention, the vector comprises having at least about 80%, preferably at least about 90%, more preferably at least about 93%, even more preferably at least about 95%, and most preferably about 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO. 1, within a range that does not impair the therapeutic efficacy of the present invention. SEQ ID NO. 1 is a nucleotide sequence comprising the Afb gene of SEQ ID NO. 3, the cAng1 gene of SEQ ID NO. 4, and an internal ribosome entry site (IRES) inserted between them.

[0081] In addition, according to one embodiment of the present invention, the vector comprises having at least about 80%, preferably at least about 90%, more preferably at least about 93%, even more preferably at least about 95%, and most preferably about 100% sequence identity with respect to the nucleotide sequence of SEQ ID NO. 2, within a range that does not impair the therapeutic efficacy of the present invention. SEQ ID NO. 2 is a nucleotide sequence comprising the Afb gene of SEQ ID NO. 3, the cAng1 gene of SEQ ID NO. 4, and an internal 2A sequence inserted between them.

[0082] In addition, according to one embodiment of the present invention, the Afb gene of SEQ ID NO. 3 included in the vector is codon-optimized as being different from a conventional sequence. The vector of the present invention comprises having sequence identity of at least about 80%, preferably at least about 90%, more preferably at least about 93%, even more preferably at least about 95%, and most preferably about 100% with respect to the nucleotide sequence of SEQ ID NO. 3, to a extent that does not impair the therapeutic efficacy of the present invention:

[0083] In addition, according to one embodiment of the present invention, the cAng1 gene of SEQ ID NO. 4 included in the vector is codon-optimized as it is different from the conventional sequence. The vector of the present invention comprises having sequence identity of about 80% or more, preferably about 90% or more, more preferably about 93% or more, even more preferably about 95% or more, and most preferably about 100% with respect to the nucleotide sequence of SEQ ID NO. 4, within a range that does not impair the therapeutic efficacy of the present invention.

[0084] The Afb gene of SEQ ID NO. 3 and the cAng1 gene of SEQ ID NO. 4 are codon-optimized, so they are different from aflibercept or Ang1 in their natural state.

[0085] Even when mutations in nucleotides regarding the sequences of SEQ ID NOs 1 to 4 are caused to result in mutations in the amino acids expressed therein and consequently alter the protein structure, the therapeutic efficacy based on the recombinant vector of the present invention may not be impaired, or may even be improved compared to before the mutation. Accordingly, the present invention includes nucleotide variants having a certain sequence identity with respect to the nucleotide sequences of SEQ ID NOs 1 to 4 as described above. Furthermore, although the above SEQ ID NOs are nucleotide sequences, a protein variant resulting from a mutation in this sequence and a subsequent mutation in the amino acid sequence is also naturally considered to be within the scope of the technical concept of the present invention, as long as its function does not deviate from the scope of the present invention.

[0086] For example, regarding the amino acid sequence of the COMP-Ang1(cAng1) protein represented by SEQ ID NO. 5, the binding free energy (ΔG) of each variant is calculated by applying the MM-GBSA methodology, and the change in energy (ΔΔG) relative to the wild type (i.e., before mutation introduction) is calculated. If the value is approximately 10.0 or less, preferably approximately 5.0 or less, more preferably approximately 2.0 or less, and even more preferably approximately 1.5 or less, it is highly likely that the efficacy of the present invention will be maintained. A lower change in energy (ΔΔG) is advantageous because it indicates improved binding affinity; if it is 0 or less, it can be considered that the efficacy of the present invention is further enhanced compared to the sequence before mutation introduction. Therefore, it is most desirable when the value of the change in energy (ΔΔG) relative to the wild type (i.e., before mutation introduction) is 0 or less.

[0087] For example, in Example 7 described at the end of this specification, a variation was attempted in the amino acid sequence of the COMP-Ang1(cAng1) protein of SEQ ID NO. 5. SEQ ID NO. 1, for expressing a COMP-Ang1(cAng1) protein comprising one or more of the mutations wherein, for example, preferably, PRO 288 is substituted with ASN, MET 248 with PHE, PRO 288 with TRP, ALA 257 with VAL, PRO 260 with ARG, PRO 288 with PHE, PRO 260 with PHE, LEU 277 with TRP, LYS 281 with ARG, ASP 256 with GLU, LEU 277 with TYR, SER 289 with ARG, GLY 275 with ARG, ALA 257 with ASP*, LEU 277 with PHE, PRO 288 with TYR, MET 248 with LEU, LYS 276 with TYR, or GLY 275 with LYS. The variations included variations in nucleotide sequences in 2 or 4. More preferably, the variations may substitute one or more of the following: PRO 288 with ASN, MET 248 with PHE, PRO 288 with TRP, ALA 257 with VAL, PRO 260 with ARG, PRO 288 with PHE, PRO 260 with PHE, LEU 277 with TRP, LYS 281 with ARG, ASP 256 with GLU, LEU 277 with TYR, SER 289 with ARG, GLY 275 with ARG, ALA 257 with ASP*, LEU 277 with PHE, PRO 288 with TYR, or MET 248 with LEU.The above variations may more preferably substitute one or more of PRO 288 with ASN, MET 248 with PHE, PRO 288 with TRP, ALA 257 with VAL, PRO 260 with ARG, PRO 288 with PHE, PRO 260 with PHE, LEU 277 with TRP, LYS 281 with ARG, or ASP 256 with GLU. The above variations may more preferably substitute one or more of PRO 288 with ASN, MET 248 with PHE, PRO 288 with TRP, ALA 257 with VAL, and PRO 260 with ARG, and most preferably substitute PRO 288 with ASN.

[0088] In addition, according to one embodiment of the present invention, the vector may add a FLAG-tag sequence to the Afb gene or the cAng1 gene. The FLAG-tag is a hydrophilic peptide composed of eight amino acids (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) attached to the N-terminus or C-terminus of a protein for detection and purification. However, since its presence may not be appropriate when directly applying the vector of the present invention or a therapeutic composition containing it clinically, it may be preferable not to include the FLAG-tag sequence.

[0089] In addition, according to one embodiment of the present invention, the vector may include a regulatory element to increase the expression efficiency of the protein according to the present invention. Generally, a regulatory element is a non-coding region within a DNA or RNA molecule that increases or decreases the expression (transcription and translation) of a specific gene, and primarily serves as a fine-tuning mechanism where a protein (transcription factor) binds to turn the gene on or off, and includes promoters, enhancers, silencers, etc. Examples of the regulatory elements of the present invention include the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), WPRE 3, HPRE, other PRE sequences, Intron A, S / MARs, Flaviviral RNA elements, etc., but preferably it is WPRE or WPRE 3, and more preferably it is WPRE 3. The above WPRE3 is a posttranscriptional regulatory element that plays a role in increasing mRNA stability and promoting export out of the nucleus, thereby improving protein expression levels. It is not limited to any specific location within the vector of the present invention, but is preferably incorporated downstream of the protein gene to be expressed.

[0090] In addition, according to one embodiment of the present invention, the vector may include a bovine growth hormone (bGH) polyadenylation signal (polyA signal). This includes the 3' untranslated region (UTR) sequence of the bovine growth hormone gene and provides a signal to attach a poly(A) tail to the 3' end of the transcribed mRNA, thereby increasing the stability of the mRNA and increasing the efficiency of outflow to the nucleus and translation.

[0091] According to one embodiment of the present invention, the vector may include one or more Woodchuck hepatitis virus post-transcriptional regulatory factor 3 (WPRE3) and / or bovine growth hormone (bGH) polyadenylation signals.

[0092] According to one embodiment of the present invention, the vector may include one or more inverted terminal repeat (ITR) sequences. The ITR is a repeat sequence of about 145 bp in length located at both ends of the DNA of an adeno-associated virus (AAV) and forms a T-shaped hairpin structure, which can be used to package a recombinant gene expression cassette in the virion of a viral vector.

[0093] (Composition for gene therapy)

[0094] Another aspect of the present invention provides a gene therapy composition for treating macular degeneration and diabetic retinopathy comprising a recombinant vector of the present invention.

[0095] In the present invention, the phrases “pharmacologically effective amount” and “therapeutically effective amount” or simply “effective amount” refer to the amount of a dual-acting molecule effective in producing an intended pharmacological, therapeutic, or prophylactic result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by 20% or more, the therapeutically effective amount of a drug to treat that disease or disorder is the amount required to reduce the said parameter by at least 20%.

[0096] Furthermore, as used herein, “treatment” or “treating” is defined as the application or administration of a therapeutic agent (e.g., a molecule of the present invention) to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line, for the purpose of treating, curing, alleviating, reducing, altering, remedying, improving, enhancing, or affecting a disease or disorder, symptoms of a disease or disorder, or a predisposition to a disease, said patient having a disease or disorder, symptoms of a disease or disorder, or a predisposition to a disease or disorder.

[0097] The composition of the present invention inhibits VEGF signaling and simultaneously activates the Tie2 pathway. As mentioned above, Afb and cAng1 act through different mechanisms. Afb primarily inhibits abnormal angiogenesis by blocking VEGF, while cAng1 stabilizes blood vessels through Tie2 activation. By combining these two approaches, the present invention overcomes the limitations of monotherapy and achieves excellent therapeutic results. In other words, the present invention enables a gene therapy composition to exhibit excellent synergistic therapeutic efficacy through a recombinant vector designed to simultaneously express Afb and cAng1 (Afb / cAng1). Since the recombinant vector enables long-term transgenic gene expression after a single administration, thereby reducing the burden of repeated intravitreal injections, it is a gene delivery platform suitable for ocular indications. In this invention, the synergistic effects of this dual-targeting strategy on angiogenesis and vascular stabilization were demonstrated, and in particular, it was shown that pathological neovascularization can be more effectively inhibited and vascular integrity improved through the simultaneous application of VEGF signaling inhibition and Tie2 signaling activation. This suggests that the recombinant vector-based gene therapy composition of this invention is a promising gene therapy strategy for the treatment of macular degeneration and diabetic retinopathy. In this specification, the meaning of "macular degeneration and diabetic retinopathy" includes cases of "macular degeneration or diabetic retinopathy."

[0098] The gene therapy composition of the present invention is a gene therapy composition for treating macular degeneration and diabetic retinopathy, comprising a recombinant vector that simultaneously expresses the Afb gene and the cAng1 gene of the present invention and a pharmaceutically acceptable carrier. In some cases, the carrier may be omitted.

[0099] The term "pharmaceuticalally acceptable carrier" above may refer to a carrier or diluent that does not irritate the organism and does not impair the biological activity and properties of the injected compound. Here, "pharmaceuticalally acceptable" means that the target of application (prescription) does not possess toxicity beyond an acceptable level without inhibiting the activity of the active ingredient. The carrier of the gene therapy composition may overlap with carriers usable in pharmaceutical compositions, and any type of carrier that is commonly used and pharmaceutically acceptable in the relevant technical field may be used. Non-limiting examples of the above carriers include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. These may be used alone or in a mixture of two or more. The above composition may be prepared into an oral or parenteral formulation according to the route of administration by conventional methods known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient.

[0100] The above-mentioned carrier may be suitable for delivering the recombinant vector of the present invention into vivo. Specifically, the carrier may be selected to be suitable for formulation into a parenteral formulation (e.g., an injectable formulation). For example, the carrier may be selected to be suitable for formulation into an intravenous injection formulation. The carrier may be an aqueous solution, e.g., water or a buffered saline solution.

[0101] The gene therapy composition may be prepared in any formulation according to conventional methods. The gene therapy composition may be formulated in a form suitable for delivering the recombinant vector of the present invention to an individual. The composition may be formulated in an aqueous solution, for example, in water or a buffered saline solution. The pharmaceutical composition may be formulated in an injectable formulation suitable for administration via any suitable route, such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, or intravertebral. The pharmaceutical composition may be prepared as a systemic formulation or a local formulation.

[0102] The above gene therapy composition may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, or surfactants when formulated, similar to pharmaceutical compositions, but may not be limited thereto.

[0103] The gene therapy composition described above is delivered systemically or locally to an individual in need. In one embodiment, it is delivered by injecting into an affected area, more specifically the eye, and even more specifically into the choroid, subretina, vitreous humor, periscle, subconjunctiva, retina, subretinal or vitreous cavity, preferably into the subretinal, choroid, or vitreous cavity within the retina. Due to this injection, the Afb gene and the cAng1 gene are simultaneously expressed around the injection site, producing aflibercept protein and cAng1 protein, which consequently inhibit VEGF signaling and simultaneously activate the Tie2 pathway, thereby alleviating the symptoms of macular degeneration and diabetic retinopathy and ultimately treating them.

[0104] In one embodiment, the gene therapy composition is administered in a volume ranging from 10 to 200 μl, preferably 20 to 100 μl, more preferably 40 to 80 μl, and most preferably 50 to 70 μl.

[0105] Alternatively, in one embodiment, the gene therapy composition is administered based on the number of genome copies administered to the patient's eye. For example, the composition is administered at 1.0 × 10 per eye based on the recombinant vector of the present invention. 10 Up to 5.0 × 10 11 Genome copy number, preferably 5.0 × 10⁶ 10 Up to 2.5 × 10 11 Genome copy number, more preferably 1.0 × 10⁶ 11 Up to 3.0 × 10 11 It is administered at a dose equal to the number of genome copies. In one embodiment, approximately 2.5 × 10⁶ per eye 11 It is desirable to administer a dose equal to the number of genome copies.

[0106] Meanwhile, the present invention relates to a kit comprising the gene therapy composition and administration instructions of the present invention. The kit may include, if necessary, an apparatus or device to assist in the administration of the gene therapy composition of the present invention.

[0107] Furthermore, the present invention relates to an experimental reagent composition for macular degeneration and diabetic retinopathy comprising the recombinant vector of the present invention. The reagent composition may include a buffer solution to maintain the stability and activity of the recombinant vector of the present invention. The reagent composition may be used in experiments related to macular degeneration and diabetic retinopathy by simultaneously expressing the Afb gene and the cAng1 gene, for the purpose of elucidating the pathogenesis of the disease and researching improved therapeutic drugs or treatment methods. The recombinant vector of the present invention and the composition containing it may be freely used in vivo, in vitro, or in vitro.

[0108] In addition, the present invention relates to the use of a recombinant vector of any one of claims 1 to 9 in the manufacture of a pharmaceutical composition for treating macular degeneration and diabetic retinopathy. The pharmaceutical composition may appropriately utilize aflibercept protein and cAng1 protein, which are efficiently produced by appropriately using the recombinant vector of the present invention during manufacturing.

[0109] Furthermore, the present invention comprises cells transfected with the recombinant vector of the present invention. Such cells include, for example, general eukaryotic cells, preferably animal cells, plant cells, insect cells, more preferably mammalian cells including human cells, CHO-K1 cells for protein expression for experimental purposes, human umbilical vein endothelial cells (HUVEC), human retinal photoreceptor cells (cone cells and rod cells), supporting cells, neurons, pigment epithelial cells, etc., but are not limited thereto. Alternatively, cells for expressing the viral vector of the present invention may be, for example, A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. Alternatively, the cells may be human, monkey, mouse, rat, rabbit, or hamster cells. The cells may be treated in vitro and then returned to the body to increase the expression activity of aflibercept protein and cAng1 protein, thereby allowing them to act within the body. Alternatively, the cells may be used in experiments related to macular degeneration and diabetic retinopathy through additional treatment in vitro to elucidate the pathogenesis of the disease and to study improved therapeutic drugs or treatment methods.

[0110] Furthermore, one aspect of the present invention provides a culture medium in which cells transfected with the recombinant vector of the present invention are cultured. Such a culture medium forms the basis of the gene therapy composition of the present invention and comprises a host cell transfected with the vector of the present invention and a medium for culturing said cell. The culture medium comprises components of a medium for cell culture, and may include, for example, basic nutritional components such as amino acids, vitamins, inorganic salts, and glucose, along with serum (e.g., Fetal Bovine Serum), glutamine, antibiotics (e.g., penicillin, streptomycin), buffers (e.g., HEPES, sodium bicarbonate), non-essential amino acids, sodium pyruvate, and other growth factors.

[0111] The present invention also provides a method for the simultaneous production of aflibercept protein and COMP-Ang1 (cAng1) protein. The method comprises the steps of: introducing a recombinant vector into a host cell; culturing the host cell to simultaneously express the aflibercept protein and COMP-Ang1 (cAng1) protein; and obtaining the aflibercept protein and COMP-Ang1 (cAng1) protein from the supernatant of the culture medium. The host cell is not particularly limited as long as it is capable of producing the aflibercept protein and COMP-Ang1 (cAng1) protein expressed by the recombinant vector of the present invention, and may overlap with examples of cells transfected with the recombinant vector of the present invention. In this case, the recombinant vector of the present invention may include a sequence of additional expression switches or expression regulatory functions regarding protein expression for conditional or selective protein expression.

[0112] The present invention also provides a pharmaceutical composition for treating macular degeneration and diabetic retinopathy, comprising: an aflibercept protein and a COMP-Ang1(cAng1) protein prepared by a method for the simultaneous preparation of the aflibercept protein and the COMP-Ang1(cAng1) protein; and one or more pharmaceutical additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, binders, disintegrants, lubricants, and any combination thereof.

[0113] In certain embodiments, the pharmaceutical composition of the present invention may generally comprise molecules and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes saline solutions, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retardants that are compatible with pharmaceutical administration. Additional active compounds may also be included in the composition. In other words, the pharmaceutical composition of the present invention may further comprise pharmaceutical additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, binders, disintegrants, lubricants, and any combination thereof.

[0114] The pharmaceutical composition may be formulated to be compatible with the intended route of administration. Preferably, the composition of the present invention may be a formulation selected from the group consisting of eye drops, artificial tears, eye ointments, tablets, pills, capsules, troches, inhalants, injections, patches, and suppositories.

[0115] Examples of administration routes include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Preferably, it is for parenteral administration.

[0116] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following ingredients: sterile diluents such as water for injection, saline solution, fixing oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate; and tonic-regulating agents such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be placed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0117] Pharmaceutical compositions suitable for injection comprise sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Carriers suitable for intravenous administration comprise physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to facilitate injection. It must be stable under manufacturing and storage conditions and preserved against the action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity may be maintained, for example, by the use of a coating such as lecithin, maintaining the required particle size in the case of dispersion, and the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is desirable to include isotonic agents, such as sugars, polyalcohols like mannitol, sorbitol, and sodium chloride, in the composition. Long-term absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0118] Sterile injectable solutions can be prepared by incorporating the required amount of an active compound into a suitable solvent with one or a combination of the components listed above as needed, and then filtering and sterilizing. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and other necessary components listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which produce any desired additional components from the powder of the active ingredient and a previously sterile filtered solution.

[0119] Oral compositions generally comprise an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically suitable binder and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain any of the following components or compounds of similar properties: a binder, e.g., microcrystalline cellulose, tragacanth gum, or gelatin; an excipient, such as starch or lactose; a disintegrant, such as alginate, primogel, or corn starch; a lubricant, such as magnesium stearate or sterotose; a lubricant, such as colloidal silicon dioxide; a sweetener, such as sucrose or saccharin; or a flavoring, such as peppermint, methyl salicylate, or orange flavoring.

[0120] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, such as a gas like carbon dioxide, for example, or a nebulizer.

[0121] Systemic administration may also be performed by transmucosal or transdermal means. In the case of transmucosal or transdermal administration, a penetrating agent suitable for the barrier to be penetrated is used in the formulation. Such penetrating agents are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be performed using nasal sprays or suppositories. For transdermal administration, the active compound is formulated as an ointment, plaster, gel, or cream as is generally known in the art.

[0122] The compound can also be prepared in the form of suppositories (e.g., with a conventional suppository base such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0123] Data obtained from cell culture analyses and animal studies can be used to formulate various dosages for human use. The dosage of these compounds is preferably ED with little to no toxicity. 50 It is within the circulating concentration range that includes. The dosage may vary within this range depending on the form of administration used and the route of administration used.

[0124] The therapeutically effective amount (i.e., effective dosage) of the composition of the present invention varies depending on the condition of the selected patient. For example, a single dose in the range of about 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or 1000 mg may be administered.

[0125] In some embodiments, a composition of 1 ng / ml to 100 μg / ml, preferably 3 ng / ml to 50 μg / ml, more preferably 5 ng / ml to 500 ng / ml, and particularly preferably 10 ng / ml to 200 ng / ml may be administered. The composition may be administered at least once a day or at least once a week, including once every other day. A person skilled in the art will recognize that certain factors may influence the dose and timing required to effectively treat an individual, including, but not limited to, the severity of the disease or disorder, previous treatment, the individual's general health and / or age, and other pre-existing conditions. Furthermore, treating an individual with a therapeutically effective amount of the molecule of the present invention may include a single treatment or, preferably, a series of treatments.

[0126] The composition of the present invention, depending on the patient's condition, has an administration dose of 5 mg / kg / week to 500 mg / kg / week, for example, 5 mg / kg / week, 10 mg / kg / week, 15 mg / kg / week, 20 mg / kg / week, 25 mg / kg / week, 30 mg / kg / week, 35 mg / kg / week, 40 mg / kg / week, 45 mg / kg / week, 50 mg / kg / week, 55 mg / kg / week, 60 mg / kg / week, 65 mg / kg / week, 70 mg / kg / week, 75 mg / kg / week, 80 mg / kg / week, 85 mg / kg / week, 90 mg / kg / week, 95 mg / kg / week, 100 mg / kg / week, 150 mg / kg / week, 200 mg / kg / week, 250 mg / kg / week, The dosage is within the range of 300 mg / kg / week, 350 mg / kg / week, 400 mg / kg / week, 450 mg / kg / week, and 500 mg / kg / week. In certain embodiments, the dosage of the dual-acting molecule according to the present invention is within the range of 10 mg / kg / week to 200 mg / kg / week, 20 mg / kg / week to 150 mg / kg / week, or 25 mg / kg / week to 100 mg / kg / week. In certain embodiments, the composition of the present invention is administered 1x times per week for 2 weeks to 6 months, e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 26 weeks, 6 months, 8 months, 10 months, or 1 year or more. In a specific embodiment, the composition of the present invention is administered 2x times per week. In another embodiment, the composition of the present invention is administered every other week.

[0127] The composition of the present invention may be formulated into a pharmaceutical composition comprising a pharmacologically effective amount of HAPLN1 protein molecules and a pharmaceutically acceptable carrier. A pharmacologically or therapeutically effective amount refers to an amount effective in producing an intended pharmacological, therapeutic, or prophylactic result. The phrases "pharmacologically effective amount" and "therapeutic effective amount," or simply "effective amount," refer to the amount of a dual-acting molecule effective in producing an intended pharmacological, therapeutic, or prophylactic result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by 20% or more, the therapeutically effective amount of a drug to treat that disease or disorder is the amount required to reduce the said parameter by at least 20%.

[0128] The appropriately formulated pharmaceutical composition of the present invention may be administered by any means known in the art, such as parenteral routes including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal, and local (including buccal and sublingual) administration. In some embodiments, the pharmaceutical composition is administered by intravenous or parenteral infusion or injection.

[0129] Generally, appropriate dosage units of the molecule are in the range of 0.001 to 0.25 mg per kg of body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kg of body weight per day, or in the range of 0.01 to 10 micrograms per kg of body weight per day, or in the range of 0.10 to 5 micrograms per kg of body weight per day, or in the range of 0.1 to 2.5 micrograms per kg of body weight per day. A pharmaceutical composition containing the molecule may be administered once daily. However, the therapeutic agent may also be administered in dosage units comprising 2, 3, 4, 5, 6, or more sub-doses administered at appropriate intervals throughout the day. Dosage units may also be formulated as a single dose over several days, for example, using a conventional sustained-release formulation that provides a continuous and consistent release of the molecule over a period of several days. Sustained-release formulations are well known in the art. In this embodiment, the dosage unit includes a corresponding multiple of the daily dosage.

[0130] The pharmaceutical composition may be included in a kit, container, pack, or dispenser along with administration instructions.

[0131] Alternatively, generally, appropriate dosage units of the molecule are in the range of 0.001 to 0.25 mg per kg of the recipient's body weight per day, or in the range of 0.01 to 20 micrograms per kg of body weight per day, or in the range of 0.01 to 10 micrograms per kg of body weight per day, or in the range of 0.10 to 5 micrograms per kg of body weight per day, or in the range of 0.1 to 2.5 micrograms per kg of body weight per day. A pharmaceutical composition containing the molecule may be administered once daily. However, the therapeutic agent may also be administered in dosage units comprising 2, 3, 4, 5, 6, or more sub-dose units administered at appropriate intervals throughout the day. Dosage units may also be formulated as a single dose over several days, for example, using a conventional sustained-release formulation that provides a continuous and consistent release of the molecule over a period of several days. Sustained-release formulations are well known in the art. In this embodiment, the dosage unit includes a corresponding multiple of the daily dosage.

[0132] Additionally, according to one embodiment of the present invention, the composition may be administered to an individual in need at a single topical dose of 1 ng / ml to 100 μg / ml. Preferably, a composition of 3 ng / ml to 50 μg / ml, more preferably 5 ng / ml to 500 ng / ml, and particularly preferably 10 ng / ml to 200 ng / ml may be administered. The composition may be administered at least once a day or at least once a week, including once every other day. A person skilled in the art will recognize that certain factors may influence the dose and timing required to effectively treat an individual, including, but not limited to, the severity of the disease or disorder, previous treatment, the individual's general health and / or age, and other pre-existing conditions. Furthermore, treating an individual with a therapeutically effective amount of the molecule of the present invention may involve a single treatment or, preferably, a series of treatments.

[0133] In addition, according to one embodiment of the present invention, the dosage of the composition of the present invention may be 5 mg / kg / week to 500 mg / kg / week depending on the patient's condition, for example, 5 mg / kg / week, 10 mg / kg / week, 15 mg / kg / week, 20 mg / kg / week, 25 mg / kg / week, 30 mg / kg / week, 35 mg / kg / week, 40 mg / kg / week, 45 mg / kg / week, 50 mg / kg / week, 55 mg / kg / week, 60 mg / kg / week, 65 mg / kg / week, 70 mg / kg / week, 75 mg / kg / week, 80 mg / kg / week, 85 mg / kg / week, 90 mg / kg / week, 95 mg / kg / week, 100 mg / kg / week, 150 mg / kg / week, 200 mg / kg / week, The dosage is within the range of 250 mg / kg / week, 300 mg / kg / week, 350 mg / kg / week, 400 mg / kg / week, 450 mg / kg / week, and 500 mg / kg / week. In certain embodiments, the dosage of the dual-acting molecule according to the present invention is within the range of 10 mg / kg / week to 200 mg / kg / week, 20 mg / kg / week to 150 mg / kg / week, or 25 mg / kg / week to 100 mg / kg / week. In certain embodiments, the composition of the present invention is administered 1x times per week for 2 weeks to 6 months, e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 26 weeks, 6 months, 8 months, 10 months, or 1 year or more. In certain embodiments, the composition of the present invention is administered 2x times per week. In other embodiments, the composition of the present invention is administered every other week. The therapeutically effective amount (i.e., effective dosage) of the composition of the present invention varies depending on the condition of the selected patient. For example, a single dose in the range of about 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or 1000 mg may be administered.

[0134] Furthermore, one aspect of the present invention provides a method for delivering an aflibercept protein and a COMP-Ang1 (cAng1) protein or a gene encoding them to an individual in need of them, comprising the step of administering an effective amount of the recombinant vector of the present invention, the gene therapy composition of the present invention, or the pharmaceutical composition of the present invention to an individual.

[0135] Furthermore, one aspect of the present invention provides a method for treating macular degeneration and diabetic retinopathy, comprising the step of administering an effective amount of the recombinant vector of the present invention, the gene therapy composition of the present invention, or the pharmaceutical composition of the present invention to an individual. That is, it relates to a method for altering the onset of symptoms of macular degeneration and diabetic retinopathy. These methods may be carried out in vitro or alternatively in vivo by administering, for example, the vector or composition of the present invention to an individual.

[0136] Examples

[0137] The present invention is to be described in more detail through the following examples, but the following examples are merely illustrative for the purpose of explanation and are not intended to limit the scope of the present invention. A person skilled in the art to which the present invention pertains can sufficiently modify or alter the following examples without preserving the technical spirit of the present invention, and such variations or equivalents fall within the scope of the present invention as defined in the appended claims.

[0138] First, the experimental materials and methods used in the embodiments of the present invention will be described.

[0139] cell culture

[0140] Human umbilical vein endothelial cells (HUVEC; manufactured by Lonza) were cultured in Endothelial Growth Medium-2 (EGM-2; manufactured by Lonza) and used between passages 4 and 6. Human lung fibroblasts (LF; manufactured by Lonza) were cultured in Fibroblast Growth Medium-2 (FGM-2; manufactured by Lonza) and used between passages 6 and 8. HEK293 cells (Korean Cell Line Bank) were maintained in Dulbecco Modified Eagle Medium (DMEM; manufactured by Invitrogen) supplemented with 10% fetal bovine serum (FBS). All cells were maintained at 37°C in a humidified incubator containing 5% CO2. To prepare the conditioned medium, HEK293 cells (1 × 10⁶) 6 Cells were seeded into 6-well plates and reached 70% confluence. After 24 hours, the cells were transfected using Lipofectamine 3000 with the Afb-IRES-cAng1 cassette or plasmids encoding individual genes. The culture supernatant was collected 24 hours after transfection, concentrated using an Amicon ultracentrifuge filter, and then used. All experiments were performed in triplicate.

[0141] Western blot analysis

[0142] Cells were lysed using RIPA buffer supplemented with a protease inhibitor (Cell Signaling Technology). Protein concentration was determined using a Bradford assay. Total protein lysate (30 μg) was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked and incubated with the following primary antibodies: anti-VEGFR2, anti-phospho-VEGFR2, anti-Tie2, anti-VE-cadherin, anti-Ang1, and anti-β-actin (all Cell Signaling Technology). After washing three times with TBST, the membrane was incubated with IRDye 800-conjugated goat anti-mouse IgG or IRDye 680-conjugated donkey anti-rabbit IgG secondary antibodies (LI-COR Biosciences). Protein bands were visualized using an Odyssey® Imaging System (LI-COR Biosciences).

[0143] Immunoprecipitation (IP)

[0144] Human umbilical vein endothelial cells (HUVECs) were cultured in 100 mm dishes on EGM-2. At the 80–90% confluence point, cells were serum-deprived on EBM-2 for 6 hours and treated with various concentrations of anti-Tie2 antibodies (hTAAB IgG1, chimeric hTAAB Fab, or humanized variants) for 30 minutes. Cells were washed with cold PBS and lysed in lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor) at 4°C for 60 minutes. The lysate was removed by centrifugation at 15,000 × g for 10 minutes, and protein concentrations were quantified via BCA assay. For Tie2 immunoprecipitation, 500 μg of cell lysate was incubated overnight with 1 μg of anti-Tie2 antibody (AF313; R&D Systems) at 4°C while rotating. The immunocomplex was precipitated for 2 hours using Dynabiz Protein G (Life Technologies). The beads were magnetically separated, washed three times with lysis buffer, and eluted for 10 minutes with 2×SDS sample buffer containing 2-mercaptoethanol at 70°C. The samples were digested on a 4-15% SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane. For phosphorylation analysis, the membrane was blocked with 5% BSA and incubated with mouse anti-phospho-tyrosine antibody (4G10, 1:1000; Millipore) at 4°C for 8 hours, followed by incubation with HRP-conjugated goat anti-mouse IgG (HAF007, 1:5000; R&D Systems). To determine total Tie2 levels, the membrane was stripped, re-blocked, probed with anti-Tie2 antibody (AF313, 1:1000), and then probed with HRP-conjugated donkey anti-goat IgG (HAF109, 1:5000).

[0145] ELISA (Enzyme-Linked Immunosorbent Assay)

[0146] HEK293 cells were seeded into 6-well plates at 70% confluence and incubated overnight. Cells were infected with the rAAV vector at a specified MOI, and culture medium was collected 48 hours after infection. The concentration of aflibercept in the culture supernatant was quantified using an Aflibercept ELISA kit (Abcam) according to the manufacturer's instructions.

[0147] Wound Healing Assay

[0148] To evaluate endothelial cell migration, human umbilical vein endothelial cells (HUVECs) were seeded into 24-well plates and allowed to reach 90% confluence. A scratch was made in the center of the monolayer using a sterile pipette tip. After washing with PBS, the cells were treated with VEGF (80 ng / mL) alone or in combination with the specified conditioned medium. After incubation at 37°C for 12 hours, migration distance was measured using ImageJ software.

[0149] Tube formation analysis

[0150] μ-slide angiogenesis wells (ibidi) were coated with Matrigel (Corning) and incubated at 37°C for 10 minutes. Human umbilical vein endothelial cells (HUVECs) were kept serum-deprived overnight in medium containing 1% FBS, resuspended in VEGF-containing medium (80 ng / mL) with or without conditioned medium, and inoculated onto Matrigel-coated slides. After 6 hours, tubular formation was visualized using an Olympus CKX53 microscope (4× objective lens). Angiogenesis parameters, including the number of nodes, master junctions, master segments, and meshwork, were quantified using the ImageJ angiogenesis analyzer plugin.

[0151] In Vitro Trans-Well Assay

[0152] Human umbilical vein endothelial cells (HUVECs) were inoculated into Collagen I-coated Transwell inserts (0.4 μm pore size; Corning) and cultured for 5 days to form a confluent monolayer. Cells were serum-deprived for 6 hours and treated with VEGF (80 ng / mL) alone or in combination with conditioned medium. Streptavidin-HRP (R&D Systems) was added to the upper chamber for 5 minutes, followed by incubation with TMB substrate for 5 minutes. The amount of HRP permeated into the lower chamber was quantified by measuring absorbance at 450 nm using a microplate reader (Epoch).

[0153] glucose-induced hyperglycemia

[0154] Human umbilical vein endothelial cells (HUVECs) were serum-deprived for 6 hours and then treated with D-glucose (30 mM) for 48 hours with or without conditioned medium derived from plasmid-transfected cells. After treatment, cells were collected and Western blot analysis was performed to evaluate changes in endothelial permeability markers (VE-cadherin expression).

[0155] Fabrication of microfluidic devices and establishment of 3D angiogenesis models

[0156] The microfluidic 3D angiogenesis chip (MAC) was fabricated from polystyrene (PS) via injection molding using an aluminum alloy mold (Al 7075). Injection conditions included a clamping force of 130 tons, a pressure of 68 bar, a cycle time of 30 seconds, and a nozzle temperature of 230°C. A pressure-sensitive adhesive (PSA) film was attached to the bottom of the chip and treated with air plasma (75W, 3 min) to enhance hydrophilicity. Fluid patterning within the MAC utilized spontaneous capillary flow to follow the sequence of central (C), lateral (S), and open (O) channels. Bovine fibrinogen (Sigma-Aldrich) was dissolved in PBS (10 mg / mL) containing aprotin (0.15 U / mL). Cell hydrogel (3 mg / mL fibrin) was loaded into channel C. A cell hydrogel containing pulmonary fibroblasts (LF, 3 × 10^6 cells / mL in 3 mg / mL fibrin) was loaded into channel S. Thrombin (0.5 U / mL) was added immediately before loading. After gelation, HUVECs (1 × 10^6 cells / mL) were inoculated into channel O to generate interstitial flow by hydrostatic pressure difference. For drug treatment, conditioned media containing IRES control, Aflibercept, COMP-Ang1, or Eylea were prepared at specified concentrations. The drugs were administered starting from the initial cell inoculation and replenished every 2 days for a total of 4 days.

[0157] Immunocytochemistry and quantitative morphological analysis

[0158] To visualize vascular structures, samples were fixed with 4% paraformaldehyde for 15 minutes, permeated with 0.2% Triton X-100, and then blocked with 3% BSA. Samples were incubated for 2 days with anti-CD31 (1:200), anti-ZO-1 (1:1000), and DAPI (1:1000). Single-plane and z-stack images were captured using a confocal microscope (Nikon Ti-2). For quantitative analysis, 3D stacks were converted to 2D projections. Images were preprocessed using a custom Python GUI to normalize contrast and minimize noise. Angiogenesis parameters, including bud length, vascular area, endpoints, and branching points, were quantified using segmentation and skeletonization algorithms.

[0159] Real-time monitoring and functional permeability evaluation

[0160] Live imaging was performed using a Celloger Mini Plus system (Curiosis) placed inside a CO2 incubator. To monitor cell response, images were captured at 30-minute intervals starting immediately after inoculation, and videos were compiled using DaVinci Resolve software. To evaluate barrier integrity, a fluorescence-based permeability assay was performed using 70 kDa FITC-dextran (Sigma-Aldrich). The medium was removed, and directional flow was established by adding PBS (30 μL) and FITC-dextran (50 μL) to the lateral and open-channel reservoirs, respectively. Fluorescence images were acquired every 2 seconds for 3 minutes using a confocal microscope (Nikon Eclipse Ti2). Real-time permeability through defined ROIs was quantified using Fiji software.

[0161] Statistical analysis

[0162] Statistical difference evaluation was performed using GraphPad Prism software. Data are expressed as mean ± SEM. Statistical significance was determined using Student's t-test or ANOVA. A P-value less than 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0163] Example 1. Design and Construction of Bicystron rAAV8-Afb / cAng1

[0164] The recombinant AAV vector, which is the main component of the AAV-based gene therapy (rAAV8-Afb / cAng1) of the present invention, was designed to co-express Afb and cAng1 through an internal ribosomal entry site (IRES) under the control of the CBh promoter (see Fig. 1 (A)). More specifically, the AAV-based gene therapy of the present invention is designed to enable the translation of both proteins from a single mRNA transcript by inserting an internal ribosomal entry site (IRES) between the two genes to achieve co-expression of Afb and cAng1 (see Fig. 1 (B)). Considering the limited packaging capacity of AAV, the present invention utilized a compact CBh promoter. Both the Afb and cAng1 genes were codon-optimized, and the flag tag sequence was removed from the cAng1 gene to facilitate future clinical application. To enhance the efficiency and stability of transgenic gene expression, this construct includes Woodchuck hepatitis virus post-transcriptional regulatory factor 3 (WPRE3) and bovine growth hormone (bGH) polyadenylation signals downstream of the code sequence. This vector aims to promote therapeutic efficacy via a single subretinal injection. Plasmid construction and recombinant AAV (RAV) production were performed at Vector Builder (Chicago, IL, USA).

[0165] Meanwhile, for comparison, control vectors expressing only Afb or cAng1 were also constructed (see Fig. 2a). Plasmids containing these vectors were prepared and transfected into HEK293T cells, after which Western blot analysis of the culture supernatant was performed. The results indicated that both proteins were successfully secreted (see Fig. 2b).

[0166] Example 2. Verification of VEGFR2 and Tie2 Phosphorylation Regulation

[0167] The anti-angiogenic activity of the AAV-based gene therapy of the present invention prepared in Example 1 was verified. Specifically, the anti-angiogenic activity of the Afb / cAng1 supernatant of the present invention was investigated. Human umbilical vein endothelial cells (HUVEC) were treated with VEGF in the presence of each supernatant, and the degree of VEGFR2 phosphorylation was analyzed.

[0168] Figures 2c and 2e respectively show the results of Western blot analysis and quantification regarding VEGFR2 phosphorylation in HUVECs after VEGF stimulation. Here, Control represents the culture supernatant after expression of an empty vector as a control, Afb / cAng1 represents the culture supernatant after expression of the vector of the present invention, Afb represents the culture supernatant after expression of the aflibercept gene within the vector, cAng1 represents the culture supernatant after expression of the cAng1 gene within the vector, Eylea represents commercially purchased aflibercept protein, pVEGFR2 represents the protein amount of phosphorylated vascular endothelial growth factor receptor 2 (VEGFR2), and VEGFR2 represents the protein amount of VEGFR2 before phosphorylation. In Figure 2e, Relative band intensity indicates the relative intensity of each band. As can be seen in Figures 2c and 2e, VEGF treatment increased VEGFR2 phosphorylation by approximately 4.7-fold.

[0169] However, the Afb / cAng1 supernatant according to the present invention effectively inhibited this VEGF-induced phosphorylation by 0.4 times, which was much lower than the baseline. In particular, this inhibitory effect was more potent than that of a single protein supernatant (Afb: 0.7 times; cAng1: 3.6 times) and was superior to that of commercial aflibercept (Eylea) (0.86 times) at the same concentration.

[0170] Furthermore, in this embodiment, by evaluating Tie2 activation using immunoprecipitation, it was confirmed whether cAng1 expressed in Afb / cAng1 effectively activates Tie2 (Fig. 2d). Quantifying the results of this immunoprecipitation assay, the supernatant of Afb / cAng1 according to the present invention increased Tie2 phosphorylation by 143.7 times (Fig. 2f). This level was similar to that of cAng1 alone (175.7 times), but the Afb supernatant showed no significant effect. Overall, these results indicate that Afb / cAng1 of the present invention can inhibit VEGFR2 phosphorylation and simultaneously and synergistically enhance Tie2 phosphorylation.

[0171] Example 3. Inhibition of VEGF-induced endothelial cell migration and tube formation

[0172] In this embodiment, whether Afb / cAng1 according to the present invention can inhibit VEGF-stimulated angiogenesis in HUVECs, particularly endothelial cell migration and tubule formation, was investigated by comparing it with Afb or cAng1 monotherapy.

[0173] FIGS. 3a to 3d relate to the inhibitory effects of the vector system (Afb / cAng1 treatment) of the present invention on VEGF-induced endothelial cell migration and tubular formation. First, FIG. 3a shows representative images of HUVEC migration performed under VEGF stimulation. 0h shows cell migration immediately after VEGF stimulation, and 12h shows cell migration at 12 hours after VEGF stimulation. FIG. 3b shows representative images of tubular formation performed under VEGF stimulation. Additionally, FIG. 3c shows the results of a quantitative analysis of the number of migrated cells. Here, wound closure indicates the degree to which the area of ​​the empty space decreases over time as HUVEC cells migrate.

[0174] Experimental results showed that VEGF treatment increased cell migration by 1.48 times. Treatment with Afb or cAng1 alone inhibited the migration of the VEGF-induced cells by approximately 1.0 times, restoring migration to basal levels (Fig. 3a). On the other hand, surprisingly, the Afb / cAng1 treatment of the present invention reduced cell migration to below basal levels (0.9 times), and this inhibitory effect on cell migration was statistically significant as it was superior compared to monotherapy (Fig. 3c).

[0175] Meanwhile, a tubule formation assay was performed to evaluate the effect of Afb / cAng1 of the present invention on VEGF-induced tubule formation. Figure 3d shows the quantitative analysis results of tubule formation parameters based on the number of nodes, master junctions, and total tubule length. First, VEGF treatment significantly increased the number of nodes (2.2-fold), the number of master junctions (3.2-fold), and the total tubule length (1.5-fold). However, surprisingly, treatment with Afb / cAng1 of the present invention effectively suppressed this VEGF-induced increase, reducing them to 1.0-fold, 1.15-fold, and 1.0-fold, respectively, restoring them to near-baseline levels. Treatment with cAng1 alone did not have a significant effect on tubule formation. In other words, it can be seen that the AAV-based gene therapy (Afb / cAng1) of the present invention effectively inhibited EGF-induced tubular formation, showing potent efficacy similar to the Afb monotherapy group, whereas cAng1 monotherapy did not show a significant effect. Collectively, these results indicate that the AAV-based gene therapy of the present invention effectively inhibits the VEGF-induced angiogenesis process in endothelial cells.

[0176] Example 4. Inhibition of VEGF and Hyperglycemia-Induced Endothelial Permeability

[0177] Vascular leakage due to increased endothelial permeability is a major pathological feature of macular degeneration and diabetic retinopathy. Since VEGF is a major mediator of this permeability, this embodiment investigated whether Afb / cAng1 according to the present invention can inhibit VEGF signaling. VE-cadherin is essential for maintaining vascular integrity and is known to decrease in expression upon VEGF stimulation. Accordingly, this embodiment investigated whether Afb / cAng1 according to the present invention can effectively restore VE-cadherin expression reduced by VEGF in HUVECs.

[0178] Figure 4a shows the results of Western blot analysis of VE-cadherin expression in HUVECs after VEGF stimulation, and Figure 4b shows a quantification graph based on the results. Western blot analysis showed that VEGF treatment reduced VE-cadherin expression by 0.5 times. In contrast, Afb / cAng1 treatment according to the present invention restored VE-cadherin expression to pre-VEGF levels (1.1 times) (Figures 4a and 4b). The expression level of VE-cadherin was higher in the Afb / cAng1 group of the present invention than in the Afb-alone treatment group (1.0 times), while the cAng1-alone treatment group showed no effect. In other words, Afb / cAng1 treatment according to the present invention not only restored VE-cadherin expression that was downregulated by VEGF but also increased it to a level exceeding that of the control group.

[0179] Next, a trans-well assay was performed to evaluate endothelial permeability. The results showed that Afb / cAng1 according to the present invention significantly reduced VEGF-induced permeability (2.4-fold) to 1.1-fold (Fig. 4c). In the graph, Relative HRP absorbance represents relative HRP absorbance, and Monolayer permeability represents monolayer permeability. HRP absorbance refers to the value measured by the degree of light absorption of the amount of colored substance produced by the reaction between the HRP enzyme (Horseradish Peroxidase) and the substrate in immunological experiments such as ELISA (Enzyme Immunoassay). The inhibitory effect described above was much stronger than in the case of single protein treatment (Afb: 1.6-fold; cAng1: 1.7-fold).

[0180] Meanwhile, chronic hyperglycemia is a major pathological driver of diabetic retinopathy. Therefore, if the reduction of VE-cadherin caused by hyperglycemia in endothelial cells is alleviated, it suggests a therapeutic effect for diabetic retinopathy. Accordingly, this time, we investigated whether Afb / cAng1 according to the present invention can restore VE-cadherin expression under hyperglycemic conditions.

[0181] [Correction pursuant to Rule 91 28.04.2026] Fig. 4d shows the results of Western blot analysis of VE-cadherin expression in HUVECs exposed to hyperglycemic conditions (30 mM D-glucose) and the quantification graph based thereon. As a result of the experiment, when human umbilical vein endothelial cells (HUVEC; manufactured by Lonza) were treated with 30 mM D-glucose, VE-cadherin expression decreased by approximately 0.7 times compared to the control group. In contrast, treatment with cAng1 or Afb alone restored the expression levels of VE-cadherin to 1.0 times and 0.9 times, respectively. However, treatment with Afb / cAng1 according to the present invention (1.1 times) resulted in significantly higher VE-cadherin expression compared to Afb monotherapy. Taken together, these results suggest that Afb / cAng1 according to the present invention effectively inhibits the increase in endothelial permeability mediated by VEGF and hyperglycemia.

[0182] Example 5. Evaluation of anti-angiogenic efficacy and vascular barrier integrity using a microfluidic 3D angiogenesis chip (MAC)

[0183] To further evaluate the anti-angiogenic efficacy of Afb / cAng1 according to the present invention, a microfluidic-based 3D angiogenesis chip (MAC) platform was utilized. This platform is based on human umbilical vein endothelial cells (HUVEC) and is designed to mimic physiological angiogenesis within a 96-well format, enabling high-throughput drug screening. Each chip utilizes a cell-free hydrogel matrix to separate endothelial and fibroblast channels, enabling real-time visualization and quantitative analysis of angiogenesis.

[0184] Experimental modeling began on Day 0 by establishing a vascularized microenvironment through the seeding of lung fibroblasts and endothelial cells into respective channels. Drug treatment was administered from Day 1 to Day 4. The directional movement and concentration gradient of the therapeutic agent were controlled by maintaining a hydrostatic pressure difference (generated by the median volume difference) between the endothelial and fibroblast channels. Figure 5a shows a schematic diagram and timeline of the microfluidic 3D angiogenesis chip (MAC) platform. Interstitial flow was induced by maintaining a hydrostatic pressure gradient between the endothelial and fibroblast channels. On Day 5, vascular morphology and function were analyzed following fixation and imaging, and Figure 5b shows representative fluorescent images of the vascular morphology. The blood vessels were visualized through fluorescent staining.

[0185] In addition, vascular area, length, and thickness were calculated using automated image processing, including duplication and skeletal analysis. Figures 5c and 5d show the quantitative analysis of angiogenesis parameters, including vascular area, average vascular length, and thickness. Afb / cAng1 according to the present invention significantly reduced vascular area by 0.2 times and average vascular length by 0.4 times compared to the vehicle control group. Although treatment with Afb alone also reduced vascular growth (area by 0.8 times and length by 0.6 times), the combination therapy of Afb / cAng1 according to the present invention showed much greater inhibitory efficacy than treatment with Afb alone. In particular, Afb / cAng1 according to the present invention surpassed the commercial standard aflibercept, Aylea, by reducing vascular area and length by 0.8 times and 0.6 times, respectively, confirming the superior efficacy of the bicistronic approach in a complex 3D microenvironment. The Afb / cAng1 treatment according to the present invention was shown to inhibit angiogenesis most potently compared to the control group and monotherapy groups, and surprisingly, demonstrated superior efficacy even compared to the commercial treatment Eylea. The cAng1 monotherapy group was found to actually worsen symptoms by increasing blood vessel thickness and promoting neovascularization.

[0186] Meanwhile, endothelial barrier integrity was evaluated through a permeability analysis measuring fluorescence leakage through endothelialized channels. Specifically, the effect of Afb / cAng1 according to the present invention on vascular barrier function was evaluated by performing a permeability assay using fluorescence tracer diffusion. Time-lapse imaging was used to track fluorescence leakage in real time, and permeability coefficients were calculated to quantify barrier integrity.

[0187] Quantitative analysis results showed that Afb / cAng1 according to the present invention exhibited the most potent anti-angiogenic effect. Specifically, Figures 5e and 5f show representative images and the results of quantifying vascular leakage based thereon, obtained through vascular permeability analysis using fluorescence tracer diffusion. In Figure 5e, 30 sec, 1 min, and 1 min 30 sec on the left side represent the exposure times to FITC-dextran, respectively. In Figure 5f, the permeability coefficient is a quantitative indicator representing the ease of material transport through blood vessels or cell barriers, and is used to evaluate the stability of the vascular barrier and the degree of leakage. Consistent with in vitro results, the Afb / cAng1 group showed the most significant reduction in vascular leakage. More specifically, Afb / cAng1 of the present invention showed the most potent reduction in vascular leakage, with the permeability coefficient decreasing by 0.2 times compared to the control group. Although monotherapy (Afb: 0.4x; cAng1: 0.4x) and commercial standard Eylea (0.4x) also significantly inhibited fluorescence diffusion compared to the control group, their barrier stabilization efficacy was significantly lower than that of Afb / cAng1 of the present invention. Taken together, these results indicate that Afb / cAng1 of the present invention exerts a strong synergistic effect on both anti-angiogenesis and vascular barrier stabilization.

[0188] Example 6. Production and Characterization of rAAV8-Afb / cAng1

[0189] Next, the recombinant viral vector rAAV8-Afb / cAng1 of the present invention was generated using the AAV8 capsid to evaluate the potential for AAV-mediated delivery of Afb / cAng1. The AAV8 serotype was selected to be suitable for subretinal injection in subsequent in vivo studies. Additionally, rAAV8-td tomato was generated as a control.

[0190] Figures 6a to 6d show the results of the production, characterization, and cytotoxicity evaluation of the rAAV8-Afb / cAng1 viral vector. Silver staining results of the purified rAAV8-Afb / cAng1, rAAV8-Afb, rAAV8-cAng1, and rAAV8-td tomato vectors confirmed high vector purity with the presence of three capsid proteins (VP1, VP2, and VP3) with minimal non-specific bands (Figure 6a).

[0191] Next, HEK293T cells were transfected with rAAV8-Afb / cAng1 to investigate whether the therapeutic vector co-expressed Afb and cAng1. Western blot analysis of the culture supernatant collected 72 hours after transfection revealed that both Afb and cAng1 were co-expressed (2 x 10⁶ 5 and 8 x 10 5 MOI of (Fig. 6b).

[0192] To evaluate cytotoxicity, HUVEC and ARPE-19 cells were 8 x 10 5 Transfection with rAAV8-Afb / cAng1 was performed at the MOI (Figs. 6c and 6d). In the graph, Relative cell viability represents relative cell viability. Cell viability was evaluated using CCK assays on days 1 and 5 after transfection. Transfection with rAAV8-Afb / cAng1 did not have a significant effect on cell proliferation in either cell line compared to rAAV8-td tomato. In other words, no significant cytotoxicity was observed in the case of the present invention compared to the rAAV8-td tomato control. The results are presented as the mean of three independent experiments.

[0193] These results indicate that rAAV8-Afb / cAng1 according to the present invention does not exhibit significant cytotoxicity at the transduction titers used in this study.

[0194] The following facts can be confirmed from the above embodiments.

[0195] Anti-VEGF therapy, represented by Afb, has demonstrated excellent efficacy in inhibiting pathological angiogenesis and has provided significant clinical benefits in ocular vascular diseases, including macular degeneration and diabetic retinopathy. Nevertheless, the lifelong requirement of intravitreal injections places a significant burden on patients, leading to reduced quality of life, discomfort, pain, and an increased risk of infection. These factors inevitably lower treatment adherence and increase the risk of treatment failure. Furthermore, some patients highlight the limitations of current anti-VEGF strategies, failing to achieve an optimal response or developing resistance.

[0196] On the other hand, cAng1 has been reported to improve the stability of newly formed blood vessels by inhibiting VEGF-induced pathological angiogenesis and vascular leakage while simultaneously promoting "non-leakage" angiogenesis. This dual effect suggests that cAng1 not only inhibits abnormal blood vessel growth but also promotes the formation of healthy blood vessels essential for tissue repair.

[0197] However, efficacy as a monotherapy is significant only in early-stage diabetes models and remains limited in advanced disease stages. This implies that potent inhibition of VEGF-induced neovascularization is indispensable for the treatment of advanced or progressive macular degeneration and diabetic retinopathy.

[0198] In this invention, a combination therapy strategy utilizing the bicistrone co-expression of Afb and cAng1 was developed to leverage the strengths of each agent and mitigate their limitations. The experimental results of the above examples demonstrate that the simultaneous administration of Afb and cAng1 according to the present invention overcomes the disadvantages of monotherapy and generates a powerful synergistic effect. First, it was confirmed that Afb / cAng1 according to the present invention effectively inhibits VEGF-induced migration and angiogenesis in human umbilical vein endothelial cells (HUVEC). This finding suggests that Afb / cAng1 strongly inhibits angiogenesis, and this inhibitory effect was significantly superior to that of Afb or cAng1 administered alone. The finding that Afb / cAng1 according to the present invention outperforms Afb at the same concentration implies that cAng1 contributes to this inhibition. This finding is particularly noteworthy in that cAng1 alone showed partial inhibition of migration but did not affect angiogenesis.

[0199] In addition, it was observed that Afb / cAng1 according to the present invention effectively prevents VE-cadherin loss in both VEGF-stimulated and hyperglycemic states, thereby exerting a therapeutic effect against vascular leakage. Increased vascular permeability is a well-known characteristic of pathological angiogenesis and depends heavily on the stability of VE-cadherin, which maintains endothelial cell adhesion. VEGF and hyperglycemia activate Src kinase, inducing phosphorylation and internalization of VE-cadherin, which weakens cell-cell adhesion. As a result, the integrity of the vascular barrier is compromised, promoting leakage and inflammatory cell infiltration. These results suggest that Afb / cAng1 not only inhibits unnecessary neovascularization but also stabilizes the existing vascular barrier.

[0200] The clinical relevance of these molecular findings was further demonstrated through results from a 3D microfluidic chip model. While conventional static 2D cultures often fail to accurately summarize the complex three-dimensional structure and functional dynamics of blood vessels, the human umbilical vein endothelial cell (HUVEC)-based angiogenesis chip used overcomes these limitations. The platform used in this study provides accurate quantitative data on VEGF-induced angiogenesis and vascular stability, offering significant advantages in predicting in vivo drug efficacy. Consistent with previous organ-on-a-chip studies demonstrating that VEGF blockade effectively inhibits neovascularization, the data in this embodiment confirmed the benefits of the strategy of the present invention under physiological flow.

[0201] Based on these facts, it was found that Afb / cAng1 according to the present invention significantly reduced vascular permeability, effectively inhibited neovascularization, and significantly reduced vascular length and area. Surprisingly, in this complex microenvironment, Afb / cAng1 of the present invention demonstrated superior efficacy compared to two monotherapys and the commercial standard aflibercept (Eylea). These results not only confirm the potent anti-angiogenic and barrier-stabilizing effects of the combination strategy of the present invention but also suggest its potential as a more effective therapeutic option than current standard treatments.

[0202] The approach of the present invention differs from recently approved bispecific antibodies, such as pariximab, which simultaneously target VEGF and Ang-2. Particularly in diabetic retinopathy, dual inhibition of VEGF and Ang-2 has been shown to be more effective than blockade of VEGF alone. While pariximab inhibits the destabilizing effects of Ang-2, our approach differs in that it more actively promotes vascular normalization and stability by directly activating Tie2 signaling via cAng1. Furthermore, unlike most AAV gene therapies currently in clinical trials that target only VEGF, the rAAV8-Afb / cAng1 vector of the present invention incorporates this dual-target mechanism within the AAV modality. Such constructs of the present invention not only alleviate the therapeutic burden associated with repeated protein injections but also possess the potential to sustain long-term therapeutic efficacy.

[0203] Clinically, this combinatorial approach can reduce injection frequency, increase patient compliance, and expand treatment options for individuals who do not respond to existing anti-VEGF therapy. Ultimately, the Afb / cAng1 of the present invention presents a new therapeutic paradigm for the treatment of macular degeneration and diabetic retinopathy that maximizes VEGF inhibition while promoting vascular stabilization and regeneration.

[0204] Example 7. Design of variants of the recombinant vector and verification of efficacy

[0205] In order to determine the extent to which mutations are introduced into the nucleotide sequences within the recombinant vector of the present invention to exhibit the effects of the present invention, various variants of the recombinant vector were designed and their efficacy was verified through an in silico process. More specifically, variants were designed to enhance Tie2-Ang1 binding affinity, and results were obtained in the following four steps.

[0206] (1) Step: Key Residue Identification

[0207] As analytical tools, PDBePISA and HotPoint were used to analyze the interface where Tie2 and Ang1 bind. By calculating the solvent exposure area (SASA), blocked surface area (BSA), and binding energy contribution (Potential) as selection criteria, 'hotspot' residues playing a critical role in binding were identified. Consequently, key residues such as ALA172, LEU192, and ASP171 were selected as targets for variant design.

[0208] (2) Step: Candidate variant design and screening (Mutation Design)

[0209] AI-based prediction tools such as ProteinMPNN (Inverse Folding), mCSM-PPI2, and Pythia-PPI were used in parallel, and the AI ​​prediction scores were combined with the researchers' visual inspection to select a total of 20 final variant candidates, including the experimentally verified A172D.

[0210] (3) Step: Molecular Dynamics Simulation (MD Simulation)

[0211] Environment setup: After applying an alternative loop of the monomer structure to reflect physiological conditions (pH=7.4) and optimizing the protonation state of each residue, structural stability was verified by performing three replica simulations of 125 ns per variant using an Amber ff14SB force field.

[0212] (4) Step: Calculation of MM-GBSA Bond Free Energy (Final Scoring)

[0213] 1,000 frames were extracted from the 'tightly binding structure' section of the simulation trajectory, and the MM-GBSA methodology was applied to calculate the binding free energy (ΔG) of each variant. The energy change (ΔΔG) relative to the wild-type was calculated and summarized in Table 1. In this case, a negative ΔΔG indicates an increase in binding affinity relative to the wild-type.

[0214]

[0215] It can be seen that even when mutations in amino acids expressed through nucleotide mutations in the sequences of SEQ ID NOs 1 to 4 are caused and consequently changes in the protein structure are made, the therapeutic efficacy based on the recombinant vector of the present invention is not impaired, or the therapeutic efficacy may actually be improved compared to before the mutation.

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[0260] According to the present invention, by providing a recombinant vector for treating macular degeneration and diabetic retinopathy and a composition for gene therapy thereof, it is possible to fundamentally treat macular degeneration and diabetic retinopathy by blocking causative factors at the early stages of the pathogenesis of said conditions. Therefore, there is a very high potential for application and expansion not only in the medical field but also in related biotechnology industries.

[0261] (Sequence No. 1)

[0262]

[0263] (Sequence No. 2)

[0264]

[0265] (Sequence No. 3)

[0266]

[0267] (Sequence No. 4)

[0268]

[0269] (Sequence No. 5)

[0270]

Claims

1. A recombinant vector that simultaneously expresses the aflibercept (Afb) gene and the COMP-Ang1 (cAng1) gene under the control of a single promoter.

2. The recombinant vector of claim 1, wherein the recombinant vector is an adeno-associated virus (AAV)-based vector.

3. The recombinant vector of claim 1, further comprising one or more of the IRES sequence and the 2A sequence (2A sequence).

4. The recombinant vector of claim 1, wherein the single promoter is selected from the group consisting of an inducible promoter (hypoxia, drug-induced), a universal promoter, a CMV promoter, a CAG promoter, a CB7 promoter, a CBh promoter, an EF1α promoter, and a retina-specific promoter.

5. The recombinant vector of claim 1, wherein the recombinant vector comprises a nucleotide sequence having at least 80% sequence identity with respect to the nucleotide sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4, to a range that does not impair the therapeutic efficacy of the present invention.

6. The recombinant vector of Claim 1, wherein the recombinant vector comprises a nucleotide sequence having a value of 10.0 or less, wherein the binding free energy (ΔG) of the resulting protein variant is calculated by applying the MM-GBSA methodology to induce a mutation of an amino acid expressed through a nucleotide mutation for the sequences of SEQ ID NOs 1 to 4, and the change in energy (ΔΔG) relative to the wild type (i.e., before mutation introduction) is calculated.

7. In Claim 1, the recombinant vector, through nucleotide mutation for the sequence of SEQ ID NO. 1, 2, or 4, in the amino acid sequence of the COMP-Ang1(cAng1) protein of SEQ ID NO. 5, PRO 288 to ASN, MET 248 to PHE, PRO 288 to TRP, ALA 257 to VAL, PRO 260 to ARG, PRO 288 to PHE, PRO 260 to PHE, LEU 277 to TRP, LYS 281 to ARG, ASP 256 to GLU, LEU 277 to TYR, SER 289 to ARG, GLY 275 to ARG, ALA 257 to ASP, LEU 277 to PHE, PRO 288 to TYR, MET 248 to LEU, LYS A recombinant vector comprising a nucleotide sequence that expresses a COMP-Ang1(cAng1) protein including one or more variants in which 276 is substituted with TYR or GLY 275 is substituted with LYS.

8. The recombinant vector of Claim 1, wherein a FLAG-tag sequence is added to the Afb gene or the cAng1 gene.

9. The recombinant vector of Claim 1, further comprising one or more of a regulatory element and a bovine growth hormone (bGH) polyadenylation signal.

10. A recombinant vector according to claim 9, wherein the regulatory element is a posttranscriptional regulatory element.

11. The recombinant vector of claim 1, wherein the recombinant vector comprises one or more inverted terminal repeat (ITR) sequences.

12. A gene therapy composition for treating macular degeneration and diabetic retinopathy, comprising a recombinant vector of any one of claims 1 to 11; and a pharmaceutically acceptable carrier.

13. A gene therapy composition according to claim 12 that inhibits VEGF signaling and simultaneously activates the Tie2 pathway.

14. In claim 12, the composition comprises approximately 1.0 × 10 recombinant vectors per eye. 10 Up to about 5.0 × 10 11 A gene therapy composition that is administered in a dose corresponding to the number of genome copies.

15. A gene therapy composition according to claim 12, wherein the composition is administered to an individual in need by injecting it into the subretinal cavity, choroid, or vitreous cavity within the retina.

16. A kit comprising a gene therapy composition and administration instructions according to any one of claims 12 to 15.

17. An experimental reagent composition for macular degeneration and diabetic retinopathy comprising a recombinant vector of any one of claims 1 to 11.

18. Use of the recombinant vector of any one of claims 1 to 11 in the manufacture of a pharmaceutical composition for treating macular degeneration and diabetic retinopathy.

19. A cell transfected with a recombinant vector of any one of claims 1 to 11.

20. A culture medium in which the cells of claim 19 have been cultured.

21. A step of introducing the recombinant vector of any one of claims 1 to 11 into a host cell; A step of culturing the above host cells to simultaneously express aflibercept protein and COMP-Ang1 (cAng1) protein; and A method for the simultaneous production of aflibercept protein and COMP-Ang1(cAng1) protein, comprising the step of obtaining aflibercept protein and COMP-Ang1(cAng1) protein from the supernatant of a culture medium.

22. A pharmaceutical composition for treating macular degeneration and diabetic retinopathy, comprising: an aflibercept protein and a COMP-Ang1 (cAng1) protein prepared by the method of claim 21; and one or more pharmaceutical additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, binders, disintegrants, lubricants, and any combination thereof.

23. A method for delivering an aflibercept protein and a COMP-Ang1 (cAng1) protein or a gene encoding them to an individual in need of them, comprising the step of administering to an individual an effective amount of a recombinant vector of any one of claims 1 to 11, a composition of any one of claims 12 to 15, or a pharmaceutical composition of claim 22.

24. A method for treating macular degeneration and diabetic retinopathy, comprising the step of administering an effective amount of a vector of any one of claims 1 to 11, a composition of any one of claims 12 to 15, or a pharmaceutical composition of claim 22 to an individual.

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