ScAAV vector for efficiently targeting aqueous humor outflow functional cells as well as screening method and application of ScAAV vector

Abstract

The invention discloses an ScAAV vector for efficiently targeting aqueous humor outflow functional cells as well as a screening method and application of the ScAAV vector. The ScAAV vector with the dominant serotype and the promoter is selected through the screening method, target cells can be efficiently infected, therapeutic genes are efficiently expressed to form protein to induce the target cells, the shape of the target cells is shrunk, intraocular aqueous humor outflow is promoted, and intraocular pressure is effectively reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a ScAAV vector that efficiently targets aqueous humor outflow functional cells, and a screening method and application thereof. Background Art

[0002] Glaucoma is an eye disease caused by excessive intraocular pressure (IOP), which leads to retinal nerve damage and visual field loss. If the intraocular pressure is extremely high for a long time, it may seriously affect vision and even cause blindness. Lowering IOP is currently the only treatment strategy that has been proven to slow the progression of the disease. The main determinant of IOP is the balance between the production and drainage of aqueous humor, among which obstructed drainage is generally regarded as one of the main reasons for the dangerous increase in IOP. Existing studies have found that trabecular meshwork cells are key tissues for maintaining IOP, and their permeability largely determines the fluidity of aqueous humor. Therefore, improving the problem of trabecular meshwork cell dysfunction through gene therapy is currently a very hot research field.

[0003] Junkai Tan et al. found that C3 gene expression can induce morphological changes in trabecular meshwork cells, and the cells become contracted and rounded. In vivo rat experiments also proved that C3 gene expression can reduce the intraocular pressure of rats (Junkai Tan, et al. Invest Ophthalmol Vis Sci. 2018; 59: 4937–4944.).

[0004] Laura Rodriguez-Estevez et al. discussed in detail the optimization of transduction efficiency of adeno-associated virus (AAV) vectors in human glaucoma gene therapy and their reversed cell entry characteristics (Laura Rodriguez-Estevez, et al. Gene Therapy (2020) 27:127–142). They explored the cell entry mechanism of AAV vectors, which is crucial for designing more effective gene therapy vectors. The results showed that the choice of AAV vector has a great influence on the transduction efficiency in trabecular meshwork cells.

[0005] Steven J. Gray et al. found that the 800-bp CBh promoter provides strong, long-term, and ubiquitous central nervous system expression, while the MeCP2 promoter allows an additional 570-bp packaging capacity. Its expression in the central nervous system is low and mainly neuron-specific, similar to the MeCP2 transcription factor (Steven J. Gray, et al. HUMAN GENE THERAPY 22:1143–1153 (September 2011)). This study provides two new promoter options for gene therapy using AAV vectors, which will help achieve effective standards and expression of larger therapeutic genes.

[0006] Chinese patent invention CN111088264B discloses an adeno-associated virus vector carrying a C3 gene expression cassette and its application, which provides a recombinant adeno-associated virus vector carrying a Clostridium botulinum C3 gene expression cassette. In vivo experiments have shown that the recombinant adeno-associated virus vector can be efficiently introduced into trabecular meshwork cells via intracameral injection, and continuously and stably expresses and produces C3 protein, increases the permeability of trabecular channels, reduces intraocular pressure, and provides a new option for the treatment of glaucoma. At the same time, the C3 gene expression cassette includes a type 2 double-stranded AAV vector, and also includes promoters CAM, CH3L1 and MGP, and experiments have proven that the CH3L1 and MGP promoters regulate the expression of C3 protein in the treatment of glaucoma. Effectiveness and safety.

[0007] Based on the above patents or literature, the present invention intends to provide a ScAAV vector with high infection efficiency and efficient and specific expression of regulatory genes, so that it can specifically target key cells involved in the outflow function of aqueous humor and promote aqueous humor outflow by delivering therapeutic genes to reduce intraocular pressure. Summary of the invention

[0008] The purpose of the present invention is to overcome at least one of the shortcomings of the prior art. The present invention provides a ScAAV vector that efficiently targets aqueous humor outflow functional cells and a screening method and application thereof. The ScAAV vector with a dominant serotype and promoter is selected by the screening method of the present invention, which can efficiently infect the target cells and efficiently express the therapeutic gene to form proteins to induce the target cells to shrink their shape, promote the outflow of aqueous humor in the eye, and effectively reduce the intraocular pressure.

[0009] The technical solution adopted by the present invention is:

[0010] In one aspect, the present invention provides a ScAAV vector that efficiently targets aqueous humor outflow functional cells, wherein the ScAAV vector is of AAV2.7m8 serotype, and the aqueous humor outflow functional cells are trabecular meshwork cells.

[0011] In a feasible embodiment, the ScAAV vector contains the promoter RSPO4 of the nucleotide sequence shown in SEQ ID NO.8.

[0012] In another aspect, the screening method comprises a screening method for vector serotypes and / or a screening method for promoters, wherein the screening method for serotypes is:

[0013] (1) Construction of plasmids for different serotypes;

[0014] (2) packaging the virus, using a ScAAV vector containing a promoter CAG and a reporter gene EGFP as a delivery plasmid, and transfecting the different serotype plasmids and the AAV-helper plasmid into AAV cells for packaging, respectively, using a transfection reagent to obtain transfected AAV cells of different serotypes;

[0015] (3) Serotype screening: AAV cells transfected with different serotypes of the aforementioned vectors with different physical titers were used to infect trabecular meshwork cells and 293T cells, respectively. EGFP expression was observed under a fluorescence microscope and flow cytometry was performed. The vector with the dominant serotype was selected based on the expression ratio of EGFP-positive cells.

[0016] In a feasible embodiment, the screening method of the promoter is:

[0017] (1) Selection and design of promoters: select proteins that are specifically expressed in cells that play a major role in the filtration of aqueous humor outflow, and design corresponding promoter sequences;

[0018] (2) construct plasmids;

[0019] (3) packaging the virus, using a ScAAV vector containing promoters Chi3l1, NEB, Pdpn, RSPO4 and Gaussia luciferase reporter gene Gluc as a delivery plasmid, transfecting the plasmid with the dominant serotype and AAV-helper plasmid into AAV cells through a transfection reagent for packaging, and obtaining transfected AAV cells;

[0020] (4) Promoter screening: using the transfected AAV cells to infect trabecular meshwork cells and 293T cells, respectively, and detecting the expression of Gluc, thereby screening out promoters that regulate gene expression in trabecular meshwork cells but do not regulate gene expression in 293T cells.

[0021] In a feasible embodiment, the serotype plasmid in (1) includes at least pAAV-RC2 and pAAV2.7m8.

[0022] In a feasible embodiment, the plasmid in (2) includes at least ScAAV-RSPO4-Gluc.

[0023] In a feasible embodiment, the physical titer of the AAV cells after transfection in (4) is 1E5.

[0024] In another aspect, the above-mentioned vector is used to target and infect aqueous humor outflow functional cells.

[0025] The beneficial effects of the present invention are:

[0026] (1) In some examples of the present invention, the preferred serotype AAV2.7m8 is obtained through a serotype screening method. This serotype has high infectivity and can effectively improve the targeted infection efficiency of the ScAAV vector.

[0027] (2) In some examples of the present invention, the preferred promoter RSPO4 is selected through a promoter screening method. This promoter can specifically regulate the expression of genes in cells with outflow function effectively and safely, and can also induce the contraction of trabecular meshwork cells, thereby more effectively promoting the outflow of aqueous humor.

[0028] (3) The methods of some examples of the present invention provide serotype screening methods and promoter screening methods that have the advantages of simple operation and efficient screening, and can accurately screen out serotypes and promoters that meet the purpose of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the structure of the pAAV-RC2 vector. (AAV-2rep, the element encodes the protein required for type 2 virus replication. cap VP1, the element encodes three structural proteins of type 2 VP1, VP2, and VP3 through alternative splicing and translation from different start codons, which are responsible for forming the shell of the virus particle. f1 ori, the f1 phage single-stranded DNA replication initiation sequence. AmpR, the element encodes the ampicillin resistance gene. ori, the high copy number ColE1 / pMB1 / pBR322 / pUC replication initiation sequence)

[0030] Figure 2This is a schematic diagram of the structure of the pAAV2.Y3 vector. (AAV-2rep, the element encodes the protein required for type 2 virus replication. cap VP1, the element encodes the three structural proteins of type 2 VP1, VP2, and VP3 through alternative splicing and translation from different start codons, among which the amino acid F at sites 444, 500, and 730 of the CAP protein mutates to Y to form the viral particle shell of serotype AAV2.Y3. f1 ori, f1 bacteriophage replication initiation sequence. AmpR, the element encodes the ampicillin resistance gene. ori, the high copy number ColE1 / pMB1 / pBR322 / pUC replication initiation sequence)

[0031] Figure 3 This is a schematic diagram of the structure of the pAAV2.7m8 vector. (AAV-2rep, element encodes proteins required for type 2 virus replication. cap VP1, element encodes three structural proteins of type 2 VP1, VP2, and VP3 through alternative splicing and translation from different start codons. Among them, RGN 587 A sequence is inserted between the RQA peptide segment to become RGNLALGETTRPARQA, forming the viral particle shell of serotype AAV2.7m8. f1 ori, f1 phage replication initiation sequence. AmpR, element encoding ampicillin resistance gene. ori, high copy number ColE1 / pMB1 / pBR322 / pUC replication initiation sequence)

[0032] Figure 4 This is a schematic diagram of the structure of the ScAAV-ChI3l1-Gluc vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. ChI3l1 promoter, a tissue-specific promoter, regulates the transcription of mRNA of downstream genes in specific cells. hGLuc, a gene for encoding secreted Gaussia luciferase. WPRE, a hepatitis virus woodchuck posttranscriptional regulatory element, enhances the expression of the target gene. SV40 late poly(A) signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0033] Figure 5This is a schematic diagram of the structure of the ScAAV-MYOC-Gluc vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. MYOC promoter, a predicted tissue-specific promoter, regulates the transcription of mRNA of downstream genes in specific cells. hGLuc, a gene for encoding secreted Gaussia luciferase. WPRE, a hepatitis virus woodchuck posttranscriptional regulatory element, enhances the expression of the target gene. SV40 late poly(A) signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0034] Figure 6 This is a schematic diagram of the structure of the ScAAV-NEB-Gluc vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. NEB promoter, a predicted tissue-specific promoter, regulates the transcription of mRNA of downstream genes in specific cells. hGLuc, a gene for encoding secreted Gaussia luciferase. WPRE, a hepatitis virus woodchuck posttranscriptional regulatory element, enhances the expression of the target gene. SV40 late poly(A) signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0035] Figure 7This is a schematic diagram of the structure of the ScAAV-PDPN-Gluc vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. PDPN promoter, a predicted tissue-specific promoter, regulates the transcription of mRNA of downstream genes in specific cells. hGLuc, a gene for encoding secreted Gaussia luciferase. WPRE, a hepatitis virus woodchuck posttranscriptional regulatory element, enhances the expression of the target gene. SV40 late poly(A) signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0036] Figure 8 This is a schematic diagram of the structure of the ScAAV-RSPO4-Gluc vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. RSPO4 promoter, a predicted tissue-specific promoter, regulates the transcription of mRNA of downstream genes in specific cells. hGLuc, a gene for encoding secreted Gaussia luciferase. WPRE, a hepatitis virus woodchuck posttranscriptional regulatory element, enhances the expression of the target gene. SV40 latepoly(A)signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0037] Fig. 9This is a schematic diagram of the structure of the ScAAV-ChI3l1-C3 vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. ChI3l1 promoter, a tissue-specific promoter, regulates the transcription of mRNA of downstream genes in specific cells. C3, is used to encode the C3 gene of Clostridium botulinum, which can inactivate Rho protein, promote changes in cell morphology, and thereby increase the outflow of aqueous humor to treat glaucoma. WPRE, the hepatitis virus woodchuck post-transcriptional regulatory element, enhances the expression of the target gene. SV40 late poly(A) signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0038] Fig.10 This is a schematic diagram of the structure of the ScAAV-PDPN-C3 vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. PDPN promoter, a predicted tissue-specific promoter, regulates the transcription of mRNA of downstream genes in specific cells. C3, is used to encode the C3 gene of Clostridium botulinum, which can inactivate Rho protein, promote changes in cell morphology, and thereby increase the outflow of aqueous humor and treat glaucoma. WPRE, hepatitis virus woodchuck post-transcriptional regulatory element, enhances the expression of the target gene. SV40 late poly(A) signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0039] Fig.11This is a schematic diagram of the structure of the ScAAV-RSPO4-C3 vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. RSPO4 promoter, a predicted tissue-specific promoter, regulates the transcription of mRNA of downstream genes in specific cells. C3, used to encode the C3 gene of Clostridium botulinum, which can inactivate Rho protein, promote changes in cell morphology, and thereby increase the outflow of aqueous humor to treat glaucoma. WPRE, hepatitis virus woodchuck post-transcriptional regulatory element, enhances the expression of the target gene. SV40 late poly(A) signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0040] Fig.12 This is a schematic diagram of the structure of the ScAAV-CAG-GFP vector. (ITR, inverted terminal repeat sequence, serves as the origin of viral replication and provides a packaging signal. In this patent, the 5'ITR terminal trs, i.e., the terminal resolution site sequence GTTGGC, is missing in all vectors, resulting in the AAV vector not being cut at this position during viral packaging and replication, but continuing to extend, thereby producing a dsDNA with complementary left and right ends, i.e., the scAAV vector. CAG promoter, a strong constitutive promoter, contains the enhancer sequence of the CMV virus promoter, the chicken β-actin promoter, and the rabbit β-Globin splicing acceptor, regulating the transcription of downstream gene mRNA. GFP, an element is used to encode green fluorescent protein. WPRE, a hepatitis virus woodchuck post-transcriptional regulatory element, enhances the expression of the target gene. SV40 late poly(A) signal, acts as a transcription termination, regulating the addition of a polyA tail to the end of the transcribed mRNA.)

[0041] Fig.13 These are pictures of human trabecular meshwork cells infected with three serotypes (AAV2, AAV2.Y3, AAV2.7m8) and a control group (Ctr) at different physical titers (5E5, 5E4, 5E3) under fluorescence microscope.

[0042] Fig.14 These are pictures of 293T cells infected with three serotypes (AAV2, AAV2.Y3, AAV2.7m8) and a control group (Ctr) at different physical titers (5E5, 5E4, 5E3) under fluorescence microscope.

[0043] Fig.15 This is a flow cytometric analysis of human trabecular meshwork cells infected with three serotypes (AAV2, AAV2.Y3, AAV2.7m8) and a control group (Ctr) at different physical titers (5E5, 5E4, 5E3).

[0044] Fig.16 This is a flow cytometry graph of 293T cells infected with three serotypes (AAV2, AAV2.Y3, AAV2.7m8) and a control group (Ctr) at different physical titers (5E5, 5E4, 5E3).

[0045] Fig.17 This is a statistical graph of relative light units (RLU) obtained by using a Gaussia luciferase reporter gene assay kit (Biyuntian, RG072S) to detect Gluc in trabecular meshwork cells driven by different promoters (NEB, PDPN, MYOC, RSPO4, Chi3L1).

[0046] Fig.18 This is a statistical graph of relative light units (RLU) obtained by using a Gaussia luciferase reporter gene assay kit (Biyuntian, RG072S) to detect Gluc in 293T cells driven by different promoters (NEB, PDPN, MYOC, RSPO4, Chi3L1).

[0047] Fig.19 These are images of cell morphology changes at 40x and 100x magnifications under a microscope after different AAV vectors (AAV2.7M8-ChI3l1-C3, AAV2.7M8-PDPN-C3, AAV2.7M8-RSPO4-C3, AAV2.7M8-CAG-GFP, and control group Ctr) infected trabecular meshwork cells.

[0048] Fig. 20 Yes Fig.19 After the proteins of the cells were collected, Western Blot experiments were performed to detect the expression level of RhoA protein in the above cells. Among them, "GAPDH 38kDa" is glyceraldehyde-3-phosphate dehydrogenase with a molecular weight of about 38kDa, which is used to evaluate the efficiency and consistency of protein extraction and Western Blot experiments, and "RhoA 20kDa" is RhoA protein with a molecular weight of about 20kDa. The presence and intensity of the bands reflect the expression level of RhoA protein in the cells.

[0049] Fig.21 yes Fig. 20 Bar graph showing the relative expression level of RhoA protein in each cell. DETAILED DESCRIPTION

[0050] The present invention provides a ScAAV vector and a screening method thereof that efficiently targets aqueous humor outflow functional cells (trabecular meshwork cells). Preferably, the ScAAV vector used in the present invention is a specific serotype AAV2.7m8, which contains a specific promoter RSPO4. The screening method includes two main parts: serotype screening and promoter screening. Serotype screening is carried out by constructing different serotype plasmids, packaging viruses, and using ScAAV vectors containing CAG promoters and EGFP reporter genes to conduct infection experiments. Finally, the dominant serotype AAV2.7m8 with high infectivity is selected by fluorescence microscopy observation and flow detection in trabecular meshwork cells and 293T cells. Promoter screening is to select the promoter sequence corresponding to the protein specifically expressed in aqueous humor outflow functional cells, and after constructing the plasmid, use the ScAAV vector containing the candidate promoter and the Gluc reporter gene to conduct an infection experiment to screen out the promoter RSPO4 that can effectively regulate gene expression in trabecular meshwork cells but not in 293T cells. This technical solution not only improves the targeted infection efficiency of ScAAV vectors, but also ensures the effective and safe expression of genes in target cells, thereby inducing trabecular meshwork cell contraction and promoting aqueous humor outflow. In addition, the screening method provided is simple and efficient, and can accurately screen out serotypes and promoters that meet the requirements.

[0051] The technical solution of the present invention is further explained below in conjunction with examples.

[0052] 1. Serum type screening example

[0053] 1. Construction of plasmids of different serotypes:

[0054] 1.1 Order plasmid: Order pAAV-RC2 plasmid with catalog number PPLKZ1001-2 from Public Protein / Plasmid Library (Plasmid and shared library, Nanjing Jingmai Biotechnology Co., Ltd., hereinafter referred to as PPL) Figure 1 ), which contains the REP and CAP genes of AAV, and its CAP gene (SEQ ID NO 1) was used as the basis to construct pAAV2.Y3 ( Figure 2 )、pAAV2-7m8( Figure 3 ).

[0055] 1.2 Construction of pAAV2.Y3: Based on the CAP gene in the above-mentioned pAAV-RC2 plasmid, the F (phenylalanine) was mutated to Y (tyrosine) at the 444, 500, and 730 sites of the CAP protein by site-directed mutagenesis technology, thereby constructing the pAAV2.Y3 plasmid. The sequence of the mutated CAP gene is SEQ ID NO 2.

[0056] 1.3 Construction of pAAV2.7m8: Also based on the CAP gene in the above-mentioned pAAV-RC2 plasmid, a sequence was inserted between the AAV2 CAP gene RGN587RQA to become RGNLALGETTRPARQA, thereby constructing the pAAV2.7m8 plasmid. The sequence of the CAP gene after insertion is SEQ ID NO 3.

[0057] 2. Packaging virus:

[0058] After constructing plasmids of different serotypes, the next step is to package the virus. The specific steps are as follows:

[0059] 2.1 Preparation of delivery plasmid: Amplify and purify the ScAAV vector (SEQID NO 4) containing the strong constitutive promoter CAG and the reporter gene EGFP to ensure that the quantity and quality of the delivery plasmid meet the requirements of subsequent experiments. The plasmid can express the green fluorescent protein EGFP, which is convenient for subsequent observation of the virus infection effect.

[0060] 2.2 Transfection of AAV cells: After the delivery plasmid, plasmids of different serotypes (pAAV2.Y3, pAAV2.7m8) and AAV-helper plasmid (provided by PPL) were mixed, AAV293 cells were transfected using PEI 40k transfection reagent (Servicebio, G1802). AAV293 cells are a commonly used cell line for packaging AAV viruses.

[0061] 2.3 Virus packaging and harvesting: After an appropriate time of transfection, the cell culture supernatant containing the packaged AAV virus particles is collected. Cell debris and other impurities are removed by centrifugation, filtration, etc. to obtain a purified virus solution.

[0062] 3. Serotype screening:

[0063] In order to determine which serotype of AAV virus has better infection efficiency and expression effect, a serotype screening experiment is required. The specific steps are as follows:

[0064] 3.1 Detection of virus physical titer: Use qPCR technology to detect the physical titer of the purified virus solution, that is, the number of virus particles.

[0065] 3.2 Infected cells: Three virus solutions with different titers (5E5, 5E4, 5E3 MOI) were selected to infect HTMC (human trabecular meshwork cells) and 293T cells respectively. HTMC cells are the target cell line used to evaluate the infection efficiency and expression effect of the virus; 293T cells are used as a control cell line to eliminate possible errors in the virus itself.

[0066] 3.3 Observation of EGFP expression: 72 hours after infection, the expression of EGFP in HTMC and 293T cells was observed using a fluorescence microscope. By observing the fluorescence intensity and distribution, the infection efficiency and expression effect of the virus can be preliminarily determined. Fig.13 These are pictures of human trabecular meshwork cells infected with three serotypes (AAV2, AAV2.Y3, AAV2.7m8) and a control group (Ctr) at different physical titers (5E5, 5E4, 5E3) under a fluorescence microscope. Fig.14 The following are pictures of three serotypes (AAV2, AAV2.Y3, AAV2.7m8) and a control group (Ctr) infected 293T cells at different physical titers (5E5, 5E4, 5E3) under a fluorescence microscope. According to the observation results, the AAV2.7m8 serotype vector showed better infection efficiency for both trabecular meshwork cells and 293T cells at each physical titer.

[0067] 3.4 Flow cytometry: Collect infected cells and perform flow cytometry. Flow cytometry can accurately measure the proportion of EGFP-positive cells, thereby more accurately evaluating the infection efficiency and expression effect of different serotypes of viruses. Fig.15 This is a flow cytometry image of three serotypes (AAV2, AAV2.Y3, AAV2.7m8) and a control group (Ctr) after infection of human trabecular meshwork cells at different physical titers (5E5, 5E4, 5E3). Fig.16 This is a flow cytometry graph of 293T cells infected with three serotypes (AAV2, AAV2.Y3, AAV2.7m8) and a control group (Ctr) at different physical titers (5E5, 5E4, 5E3). Among them, the green part is the cell marked by green fluorescent protein GFP. According to the flow cytometry results, the GFP-positive cell rate of AAV2.7m8 transduced HTMC cells at the same physical titer is higher than that of other serotypes (MOI=5E5: 78.3%>43.4%>43.1%; MOI=5E4: 64.9%>23.9%>24.4%; MOI=5E3: 37.7%>9.15%>11.2%). The GFP-positive cell rate of all serotypes transduced 293T cells at the same physical titer is not much different, and the positive rate is also above 80% when MOI=5E3, indicating that there are no major problems and errors in the virus itself. It is concluded that the serotype screening results are reliable, and AAV2.7m8 has the highest delivery efficiency for HTMC transduction, confirming that it is the optimal serotype.

[0068] 3.5 Determine the dominant serotype: According to the expression ratio of EGFP-positive cells, determine the serotype with the highest infection efficiency and expression effect. In this experiment, based on the above-mentioned fluorescence microscopy observation and flow cytometry results, AAV2.7m8 was finally selected as the dominant serotype for subsequent research. This result is not only based on the infection effect of HTMC cells, but also excludes possible errors in the virus itself through the control experiment of 293T cells.

[0069] 2. Promoter Screening Example

[0070] 1. Selection and design of promoter:

[0071] In order to further explore the gene expression regulation mechanism of key cell groups in the process of human aqueous humor outflow, we first referred to the research results published by Van Zyl et al., which used single-cell transcriptomics technology to conduct a detailed analysis of multiple cell types involved in the process of human aqueous humor outflow. Five proteins specifically expressed in the cell groups that play the main filtering function in the process of aqueous humor outflow were selected from this article, which are NEB (a protein closely related to the cytoskeleton structure), PDPN (a marker protein for platelet-derived growth factor receptor-positive cells), MYOC (myosin closely related to the pathogenesis of glaucoma), RSPO4 (an important Wnt signaling pathway regulator) and Chi3L1 (an enzyme related to inflammatory response and tissue remodeling).

[0072] Next, we used the UCSC genome browser to extract the first 2,000 base pairs (bp) upstream of the transcription start site (TSS) of each selected protein gene. This region usually contains the core elements and regulatory sequences of the promoter. Subsequently, we conducted a detailed analysis of these sequences, including the precise positioning of the TSS, the identification of CpG islands (these regions are often rich in methylation sites and are closely related to the regulation of gene expression), and the comparison of conserved regions between species, in order to discover potential promoter sequences. At the same time, we also consulted a large number of relevant promoter research literature to obtain more experimental evidence and theoretical support.

[0073] Based on the above analysis, we finally designed four new promoter sequences, corresponding to NEB (SEQ ID NO: 5), PDPN (SEQ ID NO: 6), MYOC (SEQ ID NO: 7) and RSPO4 (SEQ ID NO: 8) genes. In addition, we also used the previously reported Chi3L1 promoter (SEQ ID NO: 9) as the control group of this experiment. These promoter sequences will be used to construct a series of vectors to evaluate their ability to regulate the specific expression of downstream genes in subsequent screening experiments.

[0074] In order to screen out promoters that can specifically regulate downstream gene expression in cells with filtration functions such as trabecular meshwork cells, we used the secreted luciferase reporter gene GLuc as a screening indicator. By delivering vectors containing different promoters, we can intuitively monitor the expression of the GLuc gene in these cells, thereby evaluating the promoter activity.

[0075] 2. Construction of plasmid:

[0076] According to the experimental design, we constructed five ScAAV vectors containing different promoters and GLuc reporter genes, namely ScAAV-ChI3l1-Gluc( Figure 4 )、ScAAV-MYOC-Gluc( Figure 5 )、ScAAV-NEB-Gluc( Figure 6 )、ScAAV-PDPN-Gluc( Figure 7 ) and ScAAV-RSPO4-Gluc( Figure 8 ).

[0077] 3. Virus packaging and titer detection:

[0078] In the virus packaging stage, we selected the above-mentioned dominant serotype AAV2.7m8 as the virus vector and prepared ScAAV vectors containing different promoter-GLuc reporter gene combinations through the standard virus packaging process. Subsequently, we used appropriate detection methods to accurately measure the physical titer of the virus to ensure the accuracy and reliability of subsequent experiments.

[0079] 4. Screening and verification of promoters:

[0080] In the promoter screening stage, we selected ScAAV vectors with an MOI (multiplicity of infection) of 1E5 for cell delivery. 24 hours after delivery, we replaced the culture medium of HTMC (human trabecular meshwork cells) with fresh medium to eliminate possible interfering factors. 72 hours after delivery, we collected the cell supernatant and used the Gaussia luciferase reporter gene assay kit (Biyuntian, RG072S) to quantitatively detect the expression of GLuc. Fig.17 This is a statistical graph of relative light units (RLU) obtained by using a Gaussia luciferase reporter gene assay kit (Biyuntian, RG072S) in trabecular meshwork cells driven by different promoters (NEB, PDPN, MYOC, RSPO4, Chi3L1). Fig.18This is a statistical graph of relative light units (RLU) obtained by using a Gaussia luciferase reporter gene assay kit (Biyuntian, RG072S) for Gluc in 293T cells driven by different promoters (NEB, PDPN, MYOC, RSPO4, Chi3L1). By comparing the expression of different promoters in different cell types (such as HTMC and 293T cells), we screened out promoters that can efficiently regulate GLuc expression in HTMC but not regulate or low regulate in 293T cells. Finally, the RSPO4 promoter was determined as the best choice for this experiment. This promoter showed extremely high specific regulatory ability in HTMC, providing strong support for subsequent gene therapy research and clinical applications.

[0081] III. Example of in vitro functional verification of C3 protein delivered by ScAAV containing dominant serotype and promoter

[0082] 1. Construction of delivery plasmid:

[0083] In order to verify the effects of different promoters in regulating C3 protein expression and its effects on cell function, we first constructed three ScAAV delivery plasmids containing different promoters and C3 genes. Specifically, we selected three promoters that performed well in the previous promoter screening experiment - Chi3L1 (as a control group), PDPN and RSPO4, and fused them with the C3 protein coding sequence to construct three delivery plasmids: ScAAV-ChI3l1-C3, ScAAV-PDPN-C3 and ScAAV-RSPO4-C3.

[0084] 2. Virus packaging and titer detection:

[0085] During the viral packaging stage, we used serotype AAV2.7m8 as a vector, which is widely recognized for its efficient transduction ability of ocular cells. Through the standard viral packaging process, we successfully packaged ScAAV plasmids containing different promoters and C3 genes into viral particles. Subsequently, we used an accurate titer detection method to measure the physical titer of the virus to ensure the accuracy and reliability of subsequent experiments.

[0086] 3. C3 protein delivery and cell function verification experiments:

[0087] In order to verify the function of C3 protein in HTMC (human trabecular meshwork cells) and its effect on cell morphology and RhoA expression, we selected an AAV titer with an MOI (multiplicity of infection) of 1E5 to infect HTMC. 72 hours after infection, we used a microscope to observe the morphological changes of the cells in detail and recorded the relevant image data. Fig.19The following are cell morphology changes at 40x and 100x magnifications under a microscope after different AAV vectors (ScAAV-ChI3l1-C3, ScAAV-PDPN-C3, ScAAV-RSPO4-C3, ScAAV-CAG-GFP, and control group Ctr) infected trabecular meshwork cells. Through observation, we found that the expression of C3 protein mediated by the RSPO4 promoter significantly induced the morphological contraction of HTMCs, indicating that C3 protein had a significant effect on cell morphology under the regulation of the RSPO4 promoter.

[0088] To further verify the effect of C3 protein on cell function, we extracted the infected cell proteins and detected the expression level of cellular RhoA by Western Blot technology. Fig. 20 Yes Fig.19 After the proteins of the cells were collected, Western Blot experiments were performed to detect the expression level of RhoA protein in the above cells. Among them, "GAPDH 38kDa" is a glyceraldehyde-3-phosphate dehydrogenase with a molecular weight of about 38kDa, which is used to evaluate the efficiency and consistency of protein extraction and Western Blot experiments, and "RhoA 20kDa" is a RhoA protein with a molecular weight of about 20kDa. The presence and intensity of the band reflect the expression level of RhoA protein in the cells. According to the Western Blot results, compared with the control group, the expression of RhoA in all experimental groups increased in the early stage of C3 protein infection. However, with the increase of C3 delivery effect and further contraction of cell morphology, we found that the expression level of RhoA began to decrease significantly. This finding shows that the expression of C3 protein is not only closely related to changes in cell morphology, but may also affect the physiological function of cells by regulating the expression of RhoA.

[0089] In summary, through a series of experimental steps such as constructing delivery plasmids, virus packaging, cell infection and functional verification, we have successfully verified the expression of C3 protein mediated by RSPO4 promoter in HTMC and its effect on cell morphology and RhoA expression. This research result provides an important experimental basis for a deeper understanding of the function of C3 protein and its application in the treatment of eye diseases.

[0090] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

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Claims

1. A ScAAV vector that efficiently targets aqueous humor outflow functional cells, characterized in that: The ScAAV vector is of AAV2.7m8 serotype, and the aqueous humor outflow functional cells are trabecular meshwork cells.

2. The carrier according to claim 1, characterized in that The ScAAV vector contains the promoter RSPO4 with the nucleotide sequence shown in SEQ ID NO.

8.

3. A method for screening the vector according to claim 1 or 2, characterized in that: The screening method includes a screening method for vector serotypes and / or a screening method for promoters, wherein the screening method for serotypes is: (1) Construction of plasmids for different serotypes; (2) packaging the virus, using a ScAAV vector containing a promoter CAG and a reporter gene EGFP as a delivery plasmid, and transfecting the different serotype plasmids and the AAV-helper plasmid into AAV cells for packaging, respectively, using a transfection reagent to obtain transfected AAV cells of different serotypes; (3) Serotype screening: AAV cells transfected with different serotypes of the aforementioned vectors with different physical titers were used to infect trabecular meshwork cells and 293T cells, respectively. EGFP expression was observed under a fluorescence microscope and flow cytometry was performed. The vector with the dominant serotype was selected based on the expression ratio of EGFP-positive cells.

4. The carrier according to claim 3, characterized in that The screening method of the promoter is: (1) Selection and design of promoters: select proteins that are specifically expressed in cells that play a major role in the filtration of aqueous humor outflow, and design corresponding promoter sequences; (2) construct plasmids; (3) packaging the virus, using a ScAAV vector containing promoters Chi3l1, NEB, Pdpn, RSPO4 and Gaussia luciferase reporter gene Gluc as a delivery plasmid, transfecting the plasmid with the dominant serotype and AAV-helper plasmid into AAV cells through a transfection reagent for packaging, and obtaining transfected AAV cells; (4) Promoter screening: using the transfected AAV cells to infect trabecular meshwork cells and 293T cells, respectively, and detecting the expression of Gluc, thereby screening out promoters that regulate gene expression in trabecular meshwork cells but do not regulate gene expression in 293T cells.

5. The carrier according to claim 3, characterized in that The serotype plasmids in (1) include at least pAAV-RC2 and pAAV2.7m8.

6. The method according to claim 4, characterized in that The plasmid in (2) includes at least ScAAV-RSPO4-Gluc.

7. The method according to claim 4, characterized in that The physical titer of the AAV cells after transfection in (4) is 1E5.

8. Use of the carrier according to claim 1 or 2, characterized in that: Used to target and infect aqueous humor outflow functional cells.

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