Adeno-associated virus (aav)-mediated lysyl oxidase gene therapy for keratoconus and constructs thereof

Abstract

This invention discloses an adeno-associated virus-mediated lysyl oxidase gene construct for keratoconus, wherein the construction method of the recombinant AAV LOX vector includes: culturing human corneal fibroblasts, preparing the rAAV LOX vector through transfection, purifying the prepared rAAV LOX, extracting viral DNA, and transducing the rAAV LOX vector. This invention discloses the codon sequence of LOX (SEQ ID NO:3) and its optimized codon sequence (SEQ ID NO:7). This invention can enhance the expression of lysyl oxidase, fibronectin, and connective tissue growth factor, and reduce the expression of the extracellular matrix degradation protein matrix metalloproteinase 9. This adeno-associated virus-mediated LOX gene construct helps improve corneal rigidity, increase collagen expression levels, and enhance collagen cross-linking, and has safety and biocompatibility.

Description

[0001] Priority Statement This application claims priority to provisional patent application No. 202341034428 entitled “Adeno-associated virus (AAV)-mediated lysine oxidase gene therapy for keratoconus and construct thereof”, filed on June 17, 2023, with the Chennai Patent Office, India, the entire contents of which are expressly incorporated herein by reference.

[0002] Preface to the instruction manual The following description is intended to illustrate the present invention and its embodiments: Summary of the Invention Technical Field of the Invention This invention relates to an adeno-associated virus (AAV)-mediated lysine oxidase (LOX) gene construct for the treatment of keratoconus, a corneal disease. More specifically, this invention discloses an AAV-mediated LOX delivery technology that can increase collagen expression levels, thereby enhancing collagen cross-linking. The AAV-mediated lysine oxidase gene construct is safe and biocompatible. Background of the Invention Keratoconus (commonly known as conical keratoconus) is characterized by progressive thinning of the cornea, eventually causing it to bulge forward in a cone shape. Keratoconus can cause symptoms such as glare, photophobia, refractive errors, and blurred vision; in severe cases, it can lead to blindness. The progression of keratoconus can be classified as abortive or suspected keratoconus, mild to moderate keratoconus, and severe keratoconus.

[0004] Common causes of keratoconus include genetic and environmental factors, stress, atopic constitution, inflammation, and eye-rubbing behavior. Excessive eye-rubbing can damage the cornea and may cause corneal ectasia. Pre-existing conditions (including retinitis pigmentosa, Down syndrome, Ehlers-Danlos syndrome, Marfan syndrome, allergies, and asthma) increase the risk of developing keratoconus. Symptoms of keratoconus include blurred vision, metamorphopsia, bilateral asymmetrical irregular astigmatism, glare, photophobia, and corneal opacity. Severe keratoconus can lead to blindness, corneal edema, endokeratosis, and corneal scarring.

[0005] Genetic factors associated with keratoconus are described through several phenomena, including specific gene loci in family studies, gene-gene interactions, genetic heterogeneity, and relevant variants in genome-wide studies. Variations in multiple genes are associated with the development of keratoconus, and these genes are mainly involved in processes such as eye development, corneal formation and structure, extracellular matrix (ECM), inflammation, and cell growth regulation.

[0006] Most gene expression variations are associated with the synthesis of extracellular matrix proteins, including collagen. Collagen is a class of proteins that support and strengthen tissue structures, formed by the assembly of multiple collagen fibrils involving various enzymes. The pathogenesis of keratoconus is associated with changes in the extracellular matrix (ECM); increased activity of ECM-degrading enzymes leads to ECM degradation; dysregulated collagen levels and insufficient production of the collagen fibril maturation enzyme lysyl oxidase (LOX) affect the pathogenesis of keratoconus.

[0007] Current treatment options for keratoconus primarily target the visual distortion symptoms caused by corneal thinning and bulging, including: using soft contact lenses to correct myopia and astigmatism; using rigid gas-permeable contact lenses to correct vision in patients with progressive keratoconus; implanting an integrated implant to flatten the bulging cornea; using corneal cross-linking to stop disease progression; and performing corneal transplantation for patients with advanced keratoconus.

[0008] A Korean patent application (patent number: KR102466887B1) entitled "Optogenetic Visual Restoration Using Chrimson Protein" discloses a composition comprising a vector expressing chrimson fused with Td-Tomato (TdT). This vector system enables highly efficient protein expression for the treatment and prevention of eye diseases. The composition contains Chrimson 88 and Chrimson R, expressed via an adeno-associated virus vector, and can effectively prevent various eye diseases, including glaucoma, cataracts, corneal dystrophy, keratoconus, blinding diseases caused by photoreceptor degeneration, dysfunction, loss, or death, retinal dystrophy, retinitis pigmentosa (RP), retinal degeneration due to photoreceptor loss, macular degeneration (MD), congenital non-progressive night blindness, and age-related macular degeneration. The composition induces retinal ganglion cell responses through photostimulation at intensity below radiation safety limits, and comprises AAV2 and AAV2.7m8 vectors and a promoter.

[0009] International patent application (publication number: WO2010091279A1) entitled "Method and Composition for Treating Neovascularization" discloses a composition comprising a lysyl oxidase for treating ocular neovascularization. The lysyl oxidase can bind to antibodies, including lysyl oxidase (LOX) and LOX 2. This composition and method can prevent neovascularization in age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinopathy of prematurity. The composition comprising a polynucleotide encoding the antibody is introduced into retinal cells of the eye, the polynucleotide encoding the antibody being encapsulated in an adeno-associated virus (AAV) vector system comprising AAV types 2 and 4.

[0010] Although various treatment options exist for keratoconus, these options primarily focus on alleviating the symptoms caused by keratoconus, with the main goal of correcting vision problems resulting from corneal thinning and bulging. Therefore, there is an urgent need for a long-term effective treatment for keratoconus that can effectively regulate collagen synthesis. Invention Overview This invention overcomes the shortcomings of existing technologies and provides an adeno-associated virus (AAV)-mediated gene construct for keratoconus. The invention discloses a method for preparing recombinant adeno-associated virus (rAAV) containing the lysyl oxidase (LOX) gene, wherein the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) vector is transduced into fibroblasts to achieve expression of the lysyl oxidase (LOX) gene. The AAV-mediated lysyl oxidase (LOX) delivery enhances collagen cross-linking, thereby increasing the expression level of lysyl oxidase (LOX).

[0012] According to the present invention, the construction process of the recombinant adeno-associated virus lysine oxidase (rAAV LOX) vector includes the following steps: culturing human corneal stromal lenticule tissue to obtain human corneal fibroblasts, wherein the human corneal stromal lenticule tissue is washed with an antibacterial-antifungal solution and the washed tissue is cut into small pieces; the tissue pieces are placed in a 1:1 mixture of Durbeco modified Eagle medium (DMEM) and F12 nutrient mixture and incubated at 37°C, wherein the mixture contains 10% fetal bovine serum (FBS), 0.3 mg / mL L-glutamine, 0.1 mg / mL streptomycin and 1000 IU / mL penicillin.

[0013] Furthermore, RNA was extracted from cultured human corneal fibroblasts, and the extracted RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) to analyze the gene expression level of lysyl oxidase (LOX). Quantitative polymerase chain reaction (qPCR) was used to analyze the gene expression of lysyl oxidase (LOX): the extracted RNA was reverse transcribed into cDNA using a kit (e.g., a cDNA synthesis kit); protein samples were electrophoresed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expression level of lysyl oxidase (LOX) protein was analyzed using an anti-LOX antibody. The LOX open reading frame was cloned from the cDNA into a construct containing an AAV inverted terminal repeat (ITR) sequence to obtain the AAV.LOX construct.

[0014] In subsequent steps, adenovirus-free triple plasmid transfection was performed using the calcium phosphate method to prepare the recombinant adeno-associated virus (rAAV) LOX vector: The prepared recombinant adeno-associated virus (rAAV) LOX vector was purified, and cells infected with the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) vector were collected and centrifuged to obtain a cell pellet; the cell pellet was resuspended in 10 mM Tris-HCl buffer and then sonicated; DNase I was added to the lysed cell suspension, followed by trypsin and sodium deoxycholate, and incubated at 37°C for 30 minutes; cesium chloride was added to the cell suspension and centrifuged, the supernatant was collected and ultracentrifuged at 46,000 rpm for 40 hours, and the components after ultracentrifugation were collected and analyzed by quantitative PCR.

[0015] Furthermore, slit blot analysis was performed on viral DNA extracted from the recombinant adeno-associated virus (rAAV) LOX vector: Viral DNA from the purified recombinant adeno-associated virus (rAAV) LOX vector was extracted and subjected to slit blot hybridization, with positive signals detected by LOX probe hybridization. Further, the recombinant adeno-associated virus (rAAV) LOX vector was transduced to assess its infectivity; transduction efficiency and infection rate were analyzed using human corneal stromal fibroblasts. Additionally, the recombinant adeno-associated virus (rAAV) LOX vector was processed to analyze collagen polymerization, and the enzymatic activity of LOX was assessed by detecting relative fluorescence intensity.

[0016] This invention offers the following advantages: the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) gene construct exhibits safety, biocompatibility, and high efficiency; the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) gene construct can inhibit extracellular matrix (ECM) degradation induced by the progression of keratoconus. Furthermore, by transducing the endogenous collagen cross-linking enzyme LOX via rAAV, collagen cross-linking is promoted, thereby preventing ECM degradation. In addition, this invention can downregulate the expression of the ECM-degrading enzyme MMP9 and upregulate the expression of ECM-related proteins, including LOX, fibronectin, and CTGF. Simultaneously, this invention can improve collagen gel shrinkage rate and enhance tissue elasticity, thereby improving collagen cross-linking, increasing tissue tensile strength, and corneal rigidity.

[0017] Brief description of the attached figures The above and other features of the present invention will become clearer when the following detailed description is read in conjunction with the accompanying drawings, in which the same reference numerals refer to the same elements.

[0018] Figure 1 The flowchart for constructing the recombinant AAV LOX vector is shown.

[0019] Figure 2This illustrates the LOX expression mediated by rAAV LOX.

[0020] Figure 3 The gel shrinkage rate of rAAV LOX in human fibroblasts is shown.

[0021] Figure 4 The activity of LOX enzyme in human fibroblasts after rAAV LOX transduction is shown.

[0022] Figure 5 The results of immunohistochemical (IHC) staining of rAAV LOX are shown.

[0023] Figure 6 The expression of collagen and lamellar structure after rAAV LOX transduction are shown.

[0024] Figure 7 The gene expression levels of LOX, type I collagen, α-smooth muscle actin (α-SMA), fibronectin, and MMP9 are shown after rAAV LOX transduction.

[0025] Figure 8 The relative gene expression levels of LOX, type I collagen, type IV collagen, and MMP9 in corneal tissue are shown.

[0026] Figure 9 The results of immunofluorescence staining of LOX in corneal tissue are shown.

[0027] Figure 10 The elastic modulus after rAAV LOX transduction is shown.

[0028] Figure 11 This demonstrates the biocompatibility of rAAV LOX with ocular tissue.

[0029] Figure 12A The results of rabbit eye observation using a slit-lamp biological microscope are shown before and 12 weeks after intramatrix injection of AAV.LOX and AAV.eGFP (control group).

[0030] Figure 12B The images show central corneal thickness (CCT) and corneal thrombosis data measured using anterior segment optical coherence tomography (AS-OCT) before and after injection, used to assess changes in corneal thickness.

[0031] Figure 12C The images show the posterior segment structures of the eye, including the retina and optic nerve, analyzed using ophthalmoscopy and polarization-sensitive optical coherence tomography (PS-OCT) before and after injection.

[0032] Figure 12DThe images show in vivo confocal examinations performed using a Heidelberg retinal tomography (HRT) scanner before and after injection to analyze the cornea and intraocular structures.

[0033] Figure 12E The images show corneal endothelial cells examined using corneal endothelial microscopy before and after injection to assess cell density, morphology, and size.

[0034] Figure 12F The study shows that corneal thickness, curvature, and related parameters were assessed by intrastromal measurements 12 weeks after injection to analyze corneal structural integrity and treatment efficacy.

[0035] Detailed description of the invention To clearly and concisely illustrate the technical subject matter claimed in this invention, specific terms used in the specification are defined below.

[0036] The term "adeno-associated virus or AAV" refers to a non-enveloped virus that can be engineered to deliver DNA to target cells.

[0037] The term "gene construct" refers to a vector that modifies genes within cells to treat diseases.

[0038] The term "extracellular matrix (ECM)" refers to the protein network that provides structure and support for cells and tissues.

[0039] This invention discloses an adeno-associated virus (AAV)-mediated gene construct for keratoconus and a method for preparing recombinant AAV containing the lysyl oxidase (LOX) gene. The rAAV LOX vector is transduced into fibroblasts to achieve LOX gene expression; the AAV-mediated LOX delivery upregulates LOX expression levels and downregulates matrix metalloproteinase expression, thereby improving collagen cross-linking.

[0040] Figure 1 A flowchart illustrating the construction of the recombinant AAV LOX vector is shown. The process (100) begins with step (101): culturing human corneal lenticule tissue to obtain human corneal fibroblasts. The human corneal lenticule tissue was washed with an antibacterial-antifungal solution, cut into small pieces, and then placed in a 1:1 mixture of Durbeco Modified Eagle Medium (DMEM) and F12 nutrient mixture, and incubated at 37°C; the mixture was supplemented with 10% fetal bovine serum (FBS), 0.3 mg / mL L-glutamine, 0.1 mg / mL streptomycin, and 1000 IU / mL penicillin.

[0041] Step (102): RNA was extracted from cultured human corneal fibroblasts. The extracted RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) for analysis of lysyl oxidase (LOX) gene expression levels. The extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit, and LOX gene expression was analyzed by quantitative polymerase chain reaction (qPCR). Protein samples were electrophoresed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expression level of LOX protein was analyzed using an anti-LOX antibody. The LOX open reading frame was cloned from the cDNA into a construct containing an AAV inverted terminal repeat (ITR) sequence to obtain the AAV.LOX construct.

[0042] Step (103): Adenovirus-free triple plasmid transfection was performed using the calcium phosphate method to prepare a recombinant adeno-associated virus (rAAV) LOX vector. Step (104): The prepared rAAV LOX vector was purified, rAAV LOX cells were collected and centrifuged at low speed to obtain cell pellet; the cell pellet was resuspended in 10mM Tris-HCl buffer and then sonicated; DNase I was added to the cell suspension, followed by 0.25% trypsin and 10% sodium deoxycholate, and incubated at 37°C for 30 minutes; cesium chloride with a density of 0.6814 g / mL was added to the cell suspension and centrifuged, the supernatant was collected, and ultracentrifuged at 46,000 rpm for 40 hours; the components after ultracentrifugation were collected and analyzed by qPCR.

[0043] Step (105): Slit blot analysis was performed on the viral DNA extracted from the rAAV LOX vector. The purified viral DNA from the rAAV LOX vector was extracted and subjected to slit blot hybridization. Positive signals were detected by LOX probe hybridization. Step (106): The rAAV LOX vector was transduced to assess its infectivity. Transduction efficiency and infection rate were analyzed using human corneal stromal fibroblasts.

[0044] Step (107) involves performing collagen gel shrinkage experiments, human corneal stromal lenticule extraction, and transduction on the rAAV LOX vector. 0.25% trypsin and 0.02% EDTA were added to LOX-transduced human fibroblasts to induce cell detachment; the human fibroblasts were incubated for 48 hours, and collagen polymerization was analyzed.

[0045] Step (108) involves performing collagen gel shrinkage experiments, extraction of human corneal stromal lenses, and transduction on the rAAV LOX vector. Corneal stromal lenses were collected intraoperatively and cultured in DMEM medium supplemented with 10% FBS, glutamine, streptomycin, and penicillin to analyze the collagen gel shrinkage experiment and extraction of human corneal stromal lenses. LOX activity was analyzed using a standard kit and experimental protocol containing β-aminopropionitrile (for specific inhibition of LOX activity), and the activity level of LOX was characterized by relative fluorescence intensity.

[0046] The following examples are used to illustrate various aspects of the present invention, but are not intended to limit or define the scope of protection of the present invention in any way.

[0047] After transducing the rAAV LOX vector into cells, the gene expression level of LOX was detected by various analytical methods, including immunohistochemistry (IHC), histopathology, gene expression, relative gene expression, differential expression, and LOX overexpression.

[0048] Example 1: Expression analysis of LOX gene in fibroblasts Immunohistochemistry (IHC) was performed on rAAV LOX vector-transduced tissues to detect the presence of specific proteins in 4 μm thick tissue sections. An antigen retrieval step was performed first, followed by overnight incubation with LOX primary antibody. After washing away the primary antibody, secondary antibody was added, and the tissues were counterstained with hematoxylin. Staining intensity characterized the expression level of LOX protein. Figure 5 The IHC staining results of rAAV LOX are shown. LOX protein expression was analyzed by IHC staining, which included negative control staining and hematoxylin-eosin staining. IHC staining showed that, compared with the control AAV vector, intrastromal injection of AAV.LOX significantly upregulated the expression level of LOX protein in the corneal stroma.

[0049] Histopathological analysis of the rAAV LOX vector was performed to assess signs of tissue abnormalities, followed by observation using an optical microscope. The rAAV LOX-transduced corneas were embedded in 4 μm thick paraffin, followed by dewaxing and rehydration. Figure 11 The results of rAAVLOX biocompatibility in ocular tissues are shown. The biocompatibility of rAAVLOX-transduced cells in ocular tissues was analyzed by staining with hematoxylin and eosin and observing the cells. The results showed no structural abnormalities.

[0050] Figure 6The image shows collagen expression and lamellar structure after rAAV LOX transduction. The AAV.LOX vector helps improve corneal compactness or density; the results shown indicate increased stromal density, suggesting upregulated gene expression. Enhanced LOX gene expression further contributes to the effective treatment of keratoconus. In addition to upregulating LOX gene expression, rAAV LOX transduction also downregulates matrix metalloproteinase 9 (MMP9) gene expression. Figure 7 The gene expression levels of LOX, type I collagen, α-smooth muscle actin (α-SMA), fibronectin, and MMP9 were shown after rAAV LOX transduction. LOX overexpression downregulated MMP9 expression by 4-fold. Compared with the corresponding control group, the expression of type I collagen in the extracellular matrix proteins was increased by 3-fold, and the expression of fibronectin was increased by 2.5-fold. Figure 8 The relative gene expression levels of LOX, type I collagen, type IV collagen, and MMP9 in corneal tissue are shown. The results indicate that the relative gene expression levels of LOX, type I collagen, and type IV collagen are significantly enhanced, while the relative gene expression level of MMP9 is significantly reduced. The enhanced gene expression of ECM proteins LOX, type I collagen, and type IV collagen, and the reduced relative gene expression of MMP9, can effectively inhibit the degradation of ECM proteins under pathological conditions of keratoconus.

[0051] rAAV LOX carrier was applied topically to the cornea of ​​mice. Figure 9 Immunofluorescence staining results of LOX in corneal tissue are shown, which characterize the expression intensity of LOX in the cornea. Differential expression analysis of ECM proteins showed that the expression of the ECM degrading enzyme MMP9 was significantly decreased, while the expression of type I and type IV collagen was significantly increased. Figure 10 The elastic modulus of the cornea transduced by rAAVLOX is shown. Transduction of the rAAVLOX gene enhances corneal strength by increasing the elastic modulus. After adding rAAVLOX, the elastic modulus increased by 5 to 10 times, indicating that the tensile strength of the ECM can be restored through collagen cross-linking.

[0052] A corneal capsule is created using a laser, and rAAV LOX carrier is injected into the stroma within the capsule at a concentration of 5 × 10¹. 0 Multiple of infection (MOI). Subsequently, the cornea with margins was excised and cultured in DMEM F12 medium supplemented with penicillin and streptomycin. Figure 2The results of rAAV LOX-mediated LOX expression are shown. LOX expression was extremely low in stromal cells injected with the control group AAV vector. At concentrations of 50 k MOI, 25 k MOI, and 10 k MOI, LOX transduction significantly increased the expression levels of other ECM proteins, such as fibronectin and connective tissue growth factor (CTGF). Western blot analysis showed significantly enhanced expression of ECM proteins including LOX, fibronectin, and CTGF.

[0053] Figure 3 The gel shrinkage rate of the rAAV LOX vector in human fibroblasts is shown. LOX transduction of corneal cells significantly improved cell shrinkage ability. The results illustrate the gel shrinkage rates of primary corneal fibroblasts from healthy donors and corneal fibroblasts from keratoconus patients; at concentrations of 50 kMOI, 25 kMOI, and 10 kMOI, the average gel shrinkage rate for both cell types reached 75%. Collagen gel shrinkage experiments showed that transduction with the AAV.LOX vector significantly increased the collagen gel shrinkage rate of human corneal fibroblasts.

[0054] rAAV LOX enhances LOX activity in a dose-dependent manner. In healthy donor primary corneal fibroblasts and corneal fibroblasts from keratoconus patients, rAAV LOX transduction enhances the secretion of LOX enzymes in corneal epithelial cultures, thereby increasing enzyme activity. Figure 4 The activity of LOX enzyme in human fibroblasts after rAAV LOX transduction is shown.

[0055] This invention discloses a lysyl oxidase (LOX) sequence for rAAV vector expression and a codon-optimized lysyl oxidase (LOX) sequence. The LOX open reading frame for AAV vector expression comprises: a 111-base-pair AAV2 5' inverted terminal repeat (ITR) and a 116-base-pair AAV2 3' inverted terminal repeat (ITR) as shown in SEQ ID NO:1; a 527-base-pair cytomegalovirus (CMV) promoter as shown in SEQ ID NO:2; a 1254-base-pair LOX open reading frame (ORF) as shown in SEQ ID NO:3; and a 192-base-pair polyadenylate sequence as shown in SEQ ID NO:4. In addition, the codon-optimized LOX for AAV vector expression comprises: a 111-base-pair AAV2 5'ITR and a 116-base-pair AAV2 3'ITR as shown in SEQ ID NO:5; a 527-base-pair CMV promoter as shown in SEQ ID NO:6; a 1254-base-pair codon-optimized LOX as shown in SEQ ID NO:7; and a 192-base-pair polyadenylate sequence as shown in SEQ ID NO:8.

[0056] Example 1: Study on viscoelastic properties of rabbit corneal stroma after intrastromal injection of AAV.LOX virus According to some embodiments of the present invention, the viscoelastic properties of the cornea were determined by injecting AAV.LOX virus into the corneal stroma of a rabbit eye. The procedure for delivering AAV.LOX virus into the rabbit cornea is as follows: 30 μL of viral solution (including AAV.LOX virus sample and AAV-.eGFP control virus) was injected into the corneal stroma, and the rabbits were monitored for 12 weeks; imaging was performed weekly for the first 6 weeks, and then every two weeks until the 12th week. The imaging techniques used for weekly and bi-weekly imaging included: slit-lamp biological microscopy, anterior segment optical coherence tomography (AS-OCT) for measuring central corneal thickness (CCT) and corneal thoracoscopic data, corneal and intraocular structural analysis using a Heidelberg retinal tomography (HRT) scanner, corneal endothelial microscopy, and fundus microscopy with polarization-sensitive optical coherence tomography (PS-OCT).

[0057] Figure 12A The results of rabbit eyes observed using a slit-lamp biomicroscopy before and 12 weeks after intrastromal injection of AAV.LOX and AAV-eGFP (control group) are shown. Slit-lamp biomicroscopy examination showed that the corneas injected with both AAV.LOX and AAV.eGFP maintained normal anatomical structure and transparency; intrastromal delivery of AAV.LOX and AAV.eGFP did not have any negative impact on corneal morphology, nor did it cause any clinically significant adverse reactions to corneal physiological function. Observations revealed no signs of intraocular inflammation, hyperemia, increased ocular discharge, corneal or conjunctival edema, or infection in rabbit eyes during the entire 12-week period, as observed by subjective clinical assessment.

[0058] Figure 12B This image shows central corneal thickness (CCT) and corneal thrombosis data measured using anterior segment optical coherence tomography (AS-OCT) before and after intrastromal injection of AAV.LOX and AAV.eGFP (control group), used to assess changes in corneal thickness. Figure 12B As shown, the AS-OCT examination used for CCT and corneal thickness measurement involved measuring the corneal thickness in the central region using optical coherence tomography; the results showed that in both the AAV.LOX and AAV.eGFP groups, the central and overall corneal thickness increased significantly from week 1 to week 3 after viral delivery, and the corneal thickness began to decrease around week 4 and remained stable throughout the 12-week study period.

[0059] Figure 12CThe image shows the results of ophthalmoscopy and polarization-sensitive optical coherence tomography (PS-OCT) analysis of posterior ocular structures, including the retina and optic nerve, before and after intrastromal injection of AAV.LOX and AAV.eGFP (control group). Figure 12C As shown, fundus and polarization-sensitive optical coherence tomography (PS-OCT) scans were performed to evaluate the posterior segments, including the retina and optic nerve. This evaluation helped determine any potential effects on retinal structure following intrastromal delivery of the carrier. No changes in retinal sublayer thickness were detected throughout the monitoring period of multiple follow-up evaluations; the results indicate that administration of AAV.LOX and AAV.eGFP to the rabbit cornea did not adversely affect retinal morphology or impair optic nerve integrity.

[0060] Figure 12D This image shows the results of in vivo confocal examination using a Heidelberg retinal tomography (HRT) scanner before and after intrastromal injection of AAV.LOX and AAV.eGFP (control group) to analyze corneal and intraocular structures. In vivo confocal examination using a Heidelberg retinal tomography (HRT) was used to assess corneal and intraocular structures before and after AAV.LOX and AAV.eGFP injection. Detailed analysis showed that the cornea remained normal throughout the 12-week period. Specifically, the superficial epithelial cells were flat and healthy, indicating no abnormalities on the corneal surface; the distribution of stromal keratinocytes was consistent with normal physiological conditions, indicating that the integrity of the stromal structure and cell arrangement were maintained; the endothelial cell morphology was normal (essential for maintaining corneal transparency and overall health); and no signs of stromal fibroblast transformation were observed during the 12-week follow-up period.

[0061] Figure 12E The results show the corneal endothelial cells examined by corneal endothelial microscopy before and after intrastromal injection of AAV.LOX and AAV.eGFP (control group), assessing cell density, morphology, and size. The results indicate that endothelial cells maintained normal morphology and stable cell density throughout the 12-week monitoring period. Figure 12E As shown, this indicates that no significant loss or proliferation of endothelial cells occurred after injection.

[0062] Figure 12FThis study presents the results of intrastromal measurements 12 weeks after intrastromal injection of AAV.LOX and AAV.eGFP (control group) to assess corneal thickness, curvature, and related parameters, used to analyze corneal structural integrity and treatment efficacy. Uniaxial tensile testing was used to analyze corneal structural integrity and treatment efficacy. The results showed that, compared with the control group, intrastromal injection of AAV.LOX significantly improved the mechanical properties of the rabbit cornea; corneal tensile strength was also significantly increased after AAV.LOX injection. This significant improvement clearly indicates enhanced collagen cross-linking within the corneal stroma, resulting in improved tissue integrity and strength, making it suitable for treating pathological functional impairment of keratoconus.

[0063] This invention discloses an adeno-associated virus (AAV)-mediated lysyl oxidase gene therapy for keratoconus and its construct, which can inhibit ECM degradation caused by the progression of keratoconus. Insufficient collagen fibrillary cross-linking leads to ECM degradation, ultimately causing corneal thinning; by transducing the endogenous collagen cross-linking enzyme LOX via rAAV, collagen cross-linking can be promoted, thereby preventing ECM degradation. This invention can also downregulate the expression of the ECM-degrading enzyme MMP9 and upregulate the expression of ECM proteins including LOX, fibronectin, and CTGF; simultaneously, it increases collagen gel shrinkage rate and enhances elasticity, thereby improving collagen cross-linking, tensile strength, and corneal rigidity. This invention is safe, biocompatible, and highly effective.

[0064] According to one embodiment of the present invention, the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) vector can be used to treat corneal ectasia, wherein the lysyl oxidase (LOX) protein enhances the extracellular matrix (ECM) structure through biocrosslinking; by stabilizing corneal convexity and thinning, the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) vector can be used to treat keratoconus. Furthermore, the transgene of the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) vector can be packaged in AAV capsids of different serotypes, suitable for various functional studies or human clinical applications. Alternatively, the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) vector can be used in scenarios requiring enhancement of the ECM in tissues such as the cornea and skin.

Claims

1. An adeno-associated virus (AAV)-mediated lysyl oxidase (LOX) gene therapy vector, said vector comprising the lysyl oxidase (LOX) gene, characterized in that, The gene therapy vector can express lysine oxidase (LOX), which acts as a biocrosslinker in cells and tissues.

2. A method for constructing a recombinant adeno-associated virus lysine oxidase (rAAV LOX) vector, the method comprising the following steps: a. Human corneal stromal lenticule tissue was washed with an antibacterial-antifungal solution and cultured to obtain human corneal fibroblasts; b. RNA was extracted from cultured fibroblasts, and the extracted RNA was reverse transcribed into cDNA. The open reading frame of lysyl oxidase (LOX) was then isolated and cloned from the adeno-associated virus vector backbone. c. Recombinant adeno-associated virus (rAAV) lysine oxidase (LOX) vector was prepared by transfection with three plasmids without adenovirus using the calcium phosphate method; d. The prepared recombinant adeno-associated virus (rAAV) lysine oxidase (LOX) vector was purified, and viral DNA was extracted from the purified rAAV LOX vector; e. Slit blot analysis was performed on viral DNA extracted from the recombinant adeno-associated virus (rAAV) lysyl oxidase (LOX) vector, and the infectivity was assessed by transducing the recombinant adeno-associated virus (rAAV) lysyl oxidase (LOX) vector. f. The recombinant adeno-associated virus (rAAV) lysyl oxidase (LOX) vector was subjected to collagen gel shrinkage experiments, human corneal stromal lens extraction and transduction to construct a recombinant adeno-associated virus (rAAV) carrying the lysyl oxidase (LOX) gene.

3. The method according to claim 2, characterized in that, The lysyl oxidase (LOX) open reading frame from the cDNA was cloned into a construct containing an AAV inverted terminal repeat (ITR), the lysyl oxidase (LOX) sequence was codon optimized, and all optimized sequences were cloned into a vector backbone containing an AAV inverted terminal repeat (ITR) construct.

4. The method according to claim 2, characterized in that, Slit blot analysis was performed on viral DNA extracted from the rAAV lysyl oxidase (LOX) vector; viral DNA was extracted from the purified rAAV lysyl oxidase (LOX) vector and verified by slit blot hybridization to demonstrate its effective packaging in the AAV capsid.

5. The method according to claim 2, characterized in that, Collagen gel shrinkage experiment, human corneal stromal lenticule extraction and transduction were performed on the recombinant adeno-associated virus lysyl oxidase (rAAV LOX) vector. After transduction, transducing LOX into human fibroblasts can induce significantly stronger collagen contraction, which is associated with stronger stretching function.

6. The method according to claim 2, characterized in that, The recombinant adeno-associated virus lysine oxidase (rAAVLOX) vector enhances collagen cross-linking by transducing the endogenous collagen cross-linking enzyme lysine oxidase (LOX) via rAAV, thereby inhibiting the degradation of the extracellular matrix (ECM).

7. The method according to claim 2, characterized in that, The expression of lysyl oxidase (LOX) in cells and tissues can effectively improve the tensile strength of tissues and corneal rigidity.

8. The method according to claim 2, characterized in that, The recombinant adeno-associated virus lysyl oxidase (rAAVLOX) vector can promote the upregulation of the expression of lysyl oxidase (LOX), fibronectin, and connective tissue growth factor (CTGF).

9. The method according to claim 2, characterized in that, The recombinant adeno-associated virus lysyl oxidase (rAAVLOX) vector can inhibit the expression of matrix metalloproteinase 9 (MMP9), a protein associated with extracellular matrix (ECM) degradation.

10. The method according to claim 2, characterized in that, The recombinant AAV-mediated LOX gene construct is biocompatible.

11. The method according to claim 2, characterized in that, The recombinant AAV-mediated LOX gene construct can be used to treat at least one corneal complication, including keratosis by enhancing the extracellular matrix structure through biocrosslinking; and keratoconus by stabilizing corneal protrusion and thinning.

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