Enhancers and promoters for ocular gene therapy and uses thereof

Abstract

The invention relates to the technical field of biology. Specifically, the invention relates to an enhancer and a promoter for eye gene therapy, and a recombinant adeno-associated virus vector containing the enhancer and the promoter. The invention also relates to a pharmaceutical composition comprising the recombinant adeno-associated virus vector and its use in the treatment of glaucoma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to enhancers and promoters for ocular gene therapy, and recombinant adeno-associated viral vectors containing the same. The present invention also relates to pharmaceutical compositions containing the recombinant adeno-associated viral vectors and their use in the treatment of glaucoma. Background Art

[0002] Glaucoma is characterized by optic nerve atrophy and visual field loss, with elevated intraocular pressure as the primary risk factor. Trabecular meshwork cells are the primary target cells for unclogging aqueous humor outflow, lowering intraocular pressure, and treating glaucoma. Rho, a "molecular switch," is a key protein regulating the actin cytoskeleton and related signaling pathways. The exoenzyme C3 transferase (C3), isolated from Clostridium botulinum, can modify Rho through adenosine diphosphate (ADP)-ribosyltransferase, inactivating it and thereby increasing aqueous humor outflow.

[0003] Under the control of universal promoters (e.g., CAM promoter, CMV promoter, etc.), recombinant AAV2 carrying the C3 gene can infect trabecular meshwork cells, effectively degrading the actin structure within the trabecular meshwork cells and changing the morphology of the trabecular meshwork cells. However, because universal promoters are not tissue-specific, in vivo transduction can infect corneal endothelial cells and lead to overexpression of the C3 protein, causing the side effect of corneal edema.

[0004] CN 111088264B discloses promoters derived from chitinase 3-like protein 1 (CH3L1) and matrix Gla protein (MGP), and discloses that the CH3L1 and MGP promoters, compared to the universal promoter (CAM), can specifically infect trabecular meshwork cells and express C3. Jun-Kai Tan et al. (2023, doi:10.18240 / ijo.2023.08.03) reported that adding an enhancer sequence (eMGP) to an existing C3 expression vector regulated by the MGP promoter can help improve the expression efficiency of the target gene.

[0005] However, there is still an urgent need in this field for new trabecular meshwork cell-specific promoters to further improve the specific expression level of target genes in trabecular meshwork cells to meet the needs of glaucoma treatment. SUMMARY OF THE INVENTION

[0006] To solve the above problems, the inventors further designed and screened enhancers based on the existing MGP and eMGP promoters, with the aim of specifically enhancing the transcriptional activity of the target gene in trabecular meshwork cells. After in-depth research, the inventors finally obtained new enhancer fragments RE055, RE071, RE082, RE083, and RE089. As shown in the examples, the promoter constructed using the enhancer sequence of the present invention can drive significant strong expression of C3 protein in human trabecular meshwork cells. The enhancer and promoter of the present invention provide a useful technical means for effectively solving the problem of low expression of exogenous genes in gene therapy. Thus, the inventors established the enhancer, promoter, and their application in gene therapy, especially in the treatment of ophthalmic diseases.

[0007] In a first aspect, the present disclosure provides an enhancer polynucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 1-5, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the polynucleotide can be used to enhance transcription of a gene of interest in a cell, and preferably can be used to increase expression of the gene of interest in the cell. In some embodiments, the cell is a trabecular meshwork cell, particularly a human trabecular meshwork cell.

[0008] In a second aspect, the present disclosure provides a promoter polynucleotide capable of enhancing transcription, wherein the polynucleotide comprises a promoter sequence operably linked to an enhancer sequence of the present invention. In some embodiments, the promoter polynucleotide can be used to enhance transcription of a target gene operably linked thereto in trabecular meshwork cells, and preferably can be used to increase the expression of the target gene in the cells. In some embodiments, the promoter polynucleotide comprises:

[0009] a. an enhancer sequence, wherein the enhancer sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-5 or a nucleotide sequence having at least about 90% identity thereto, for example, a nucleotide sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto, and

[0010] b. A promoter sequence expressed in trabecular meshwork cells, for example, the promoter sequence of the MGP gene shown in SEQ ID NO: 16 or the promoter sequence of the human chitinase 3-like protein 1 gene shown in SEQ ID NO: 23, or a promoter sequence having at least about 90% identity thereto, for example, a promoter sequence having about 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0011] In some embodiments, the promoter polynucleotide further comprises an operably linked second enhancer sequence, such as the enhancer nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence at least about 90% identical thereto, for example, about 95%, 96%, 97%, 98%, 99% or more identical thereto.

[0012] In a third aspect, the present invention provides a gene expression cassette comprising an operably linked:

[0013] a. the promoter polynucleotide of the second aspect; and

[0014] b. A gene of interest, such as a nucleotide sequence encoding an exoenzyme C3 transferase, for example, a nucleotide sequence encoding an exoenzyme C3 transferase as set forth in SEQ ID NO: 18, or a nucleotide sequence having at least about 90% identity (e.g., about 95%, 96%, 97%, 98%, 99% or greater identity) to SEQ ID NO: 18. In some embodiments, the gene of interest comprises the nucleotide sequence of SEQ ID NO: 19, or a nucleotide sequence having about 90% identity (e.g., about 95%, 96%, 97%, 98%, 99% or greater identity) thereof and encoding an exoenzyme C3 transferase. Preferably, in some embodiments, the gene expression cassette is an exoenzyme C3 transferase gene expression cassette.

[0015] In a fourth aspect, the present invention provides a nucleic acid expression vector and a host cell comprising the expression vector, wherein the nucleic acid expression vector comprises the enhancer polynucleotide described in the first aspect, the promoter polynucleotide of the second aspect, or the gene expression cassette of the third aspect.

[0016] In the fifth aspect, the present invention provides an adeno-associated viral vector, which comprises the enhancer polynucleotide described in the first aspect, the promoter polynucleotide of the second aspect, the gene expression cassette of the third aspect, or the nucleic acid expression vector described in the fourth aspect.

[0017] In some embodiments, the adeno-associated virus vector of the present invention is a recombinant adeno-associated virus (rAAV) vector, for example, a recombinant AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11 or AAV12 vector, preferably, a recombinant AAV2 vector.

[0018] In some embodiments, the adeno-associated viral vector of the present invention has a genome that can self-complement to form a double-stranded DNA molecule. In some embodiments, the adeno-associated viral vector of the present invention is a ssAAV or scAAV vector.

[0019] In the sixth aspect, the present invention provides a pharmaceutical composition comprising the enhancer polynucleotide of the first aspect, the promoter polynucleotide of the second aspect, the gene expression cassette of the third aspect, the nucleic acid expression vector of the fourth aspect, or the adeno-associated virus vector of the fifth aspect.

[0020] In the seventh aspect, the present invention provides the use of the enhancer polynucleotide according to the first aspect, the promoter polynucleotide according to the second aspect, the gene expression cassette according to the third aspect, the nucleic acid expression vector according to the fourth aspect, or the adeno-associated virus vector according to the fifth aspect, or the pharmaceutical composition according to the fifth aspect for alleviating or treating glaucoma, or for use in the preparation of a medicament for alleviating or treating glaucoma.

[0021] In some embodiments, the glaucoma is primary open-angle glaucoma and primary angle-closure glaucoma.

[0022] In some embodiments, the nucleic acid expression vector or adeno-associated viral vector of the present invention is administered by intracameral injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Combined with the following Figure 1 When read together, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the present invention, the drawings show the presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0024] Figure 1 Shown is the electrophoresis diagram of the enhancer fragment library obtained by PCR using the pUC57 library as a template.

[0025] Figure 2A A schematic diagram of the AAV backbone plasmid pAAV-MGP-Gluc is shown. In the figure, "MGP" represents the promoter of the matrix glial protein (MGP) gene; "Gluc" represents the secreted luciferase coding sequence from Gaussia.

[0026] Figure 2B Schematic diagram of the RE-MGP-Gluc construct in an AAV plasmid vector. "RE" in the figure represents the enhancer segment of the present invention. "Gluc" in the figure represents the coding sequence for the secreted luciferase Gluc operably linked to MGP; "polyA" represents the polyadenylation signal sequence.

[0027] Figure 3Comparison of expression of the MGP promoter and the RE-MGP promoter in trabecular meshwork cells (HTMCs) is shown. The left panel shows luciferase (Glu) activity detected in the cell supernatant 72 hours after infection of HTMC cells with recombinant AAV viruses containing different promoter sequences. The right panel shows values ​​normalized to the luciferase activity obtained from the MGP promoter.

[0028] Figure 4 The figure shows the construct RE-eMGP-Gluc, generated by inserting the enhancer fragments RE055, RE071, RE082, RE083, and RE089 of the present invention between the enhancer portion of the eMGP promoter and the MGP promoter. "Gluc" in the figure indicates the coding sequence for the secreted luciferase Gluc operably linked to MGP; "polyA" indicates the polyadenylation signal sequence.

[0029] Figure 5 The expression of eMGP promoter and RE-eMGP promoter in trabecular meshwork cells (HTMC) is shown. Figure 4 The left side of the figure shows the luciferase (Gluc) activity detected in the cell supernatant 72 hours after HTMC cells were infected with recombinant AAV viruses containing different promoter sequences. The right side of the figure shows the values ​​normalized to the luciferase activity obtained from the MGP promoter.

[0030] Figure 6 Schematic diagram of the RE-eMGP-C3 construct and the RE-MGP-C3 construct contained in the constructed AAV vector. In the figure, "C3" represents the coding region sequence of the human codon-optimized synthetic Clostridium botulinum C3 gene.

[0031] Figure 7 The expression of C3 protein driven by eMGP promoter and RE-eMGP promoter in trabecular meshwork cells (HTMC) is compared. Figure 4 The left side of the figure shows the relative expression levels of C3 protein detected after infection of HTMC cells with recombinant AAV viruses containing different promoter sequences. The right side of the figure shows the values ​​normalized to the expression levels obtained with the eMGP promoter. DETAILED DESCRIPTION

[0032] Unless otherwise defined hereinafter, all technical and scientific terms used in this specification have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0033] definition

[0034] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0035] As used herein, the terms "comprise" or "comprising" are meant to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0036] The term "operably linked" refers to a relationship between two or more polynucleotide (e.g., DNA) segments that allows them to function in the intended manner. The term refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Some transcriptional regulatory sequences (e.g., enhancers) do not need to be physically adjacent to or in close proximity to the target gene sequence whose transcription they enhance. Enhancers can function in a manner that is independent of the enhancer's orientation, position, and spatial distance relative to the promoter and the target gene to be transcribed.

[0037] Calculation of sequence identity between sequences is performed as follows:

[0038] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0039] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http: / / www.gcg.com), which has been integrated into the GAP program in the GCG software package. The Blossum 62 matrix or the PAM250 matrix is ​​used, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 is used, and a length weight of 1, 2, 3, 4, 5, or 6 is used. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com). A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0040] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0041] Additionally or alternatively, one can further use the nucleic acid sequences and protein sequences described herein as a "query sequence" to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0042] As used herein, the term "conservative amino acid substitution" or "conservative amino acid substitution" refers to changing, replacing or substituting an amino acid for a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size), as known to those skilled in the art.

[0043] The term "regulatory sequence" or "expression control sequence" refers to a nucleic acid sequence that induces, inhibits or otherwise controls the transcription of a protein encoding a nucleic acid sequence to which it is operably linked. Regulatory sequences can be, for example, enhancer sequences, intron sequences, and promoter sequences.

[0044] The term "viral vector" or "viral vector" is intended to refer to a non-wild-type recombinant viral particle that serves as a gene delivery vehicle and comprises a recombinant viral genome packaged within a viral (e.g., AAV) capsid. A specific type of viral vector may be a "recombinant adeno-associated viral vector" or "rAAV vector." The recombinant viral genome packaged in a viral vector is also referred to herein as a "vector genome (vg)."

[0045] As used herein, the term "AAV vector" refers to a highly efficient exogenous gene transfer tool that has been transformed from wild-type AAV viruses as people gain a better understanding of the AAV virus life cycle and its related molecular biological mechanisms. The modified AAV vector genome may only contain the ITR sequence of the AAV virus and the expression cassette carrying the exogenous gene to be transferred. The Rep and Cap proteins required for AAV virus packaging can be provided in trans by other exogenous plasmids or host cells, thereby reducing the potential harm caused by packaging the rep and cap genes into the AAV vector. Furthermore, the AAV virus itself is not pathogenic, which makes the AAV vector recognized as one of the safest viral vectors. The traditional AAV virus is ssAAV (single-stranded AAV) in which both ITRs are unmutated. Deleting the D sequence and trs sequence in the ITR sequence on one side of the AAV virus can make the genome carried by the packaged recombinant AAV virus vector self-complementary and form a double strand, so that the packaged virus becomes scAAV (self-complementary AAV) virus, the so-called double-stranded AAV virus. The packaging capacity of scAAV viral vectors is smaller, only half of that of ssAAV viral vectors, at approximately 2.2kb-2.5kb, but the transduction efficiency after infection of cells is higher. There are many AAV virus serotypes, and different serotypes have different tissue infection tropisms. Therefore, the use of AAV vectors can transport exogenous genes to specific organs and tissues (Wu Z et al., Mol Ther. 2006; 14(3): 316-327). In addition, the physical and chemical properties of AAV vectors are stable, and they show strong tolerance to acid, alkali and high temperature (Gruntman AM et al., Hum Gene Ther Methods. 2015; 26(2): 71-76), making it easy to develop biological products with high stability. In terms of preparation, the existing technology has a relatively mature packaging system for AAV vectors, which can facilitate the large-scale production of AAV vectors. Due to the above characteristics, AAV vectors have gradually become a widely used tool for gene therapy. Currently, the frequency of use of AAV in viral vectors used for gene therapy of eye diseases has reached nearly half.

[0046] The term "vector genome (vg)" refers to the nucleic acid sequence packaged in the rAAV capsid to form the rAAV vector. The vector genome may include regulatory sequences that direct the expression of a functional protein of interest. In one embodiment, the vector genome comprises at least AAV2 5'ITR from 5' to 3' direction, an enhancer sequence according to the present invention, a promoter sequence expressed in trabecular meshwork cells, a nucleic acid sequence encoding a functional exoenzyme C3 transferase, and AAV2 3'ITR. ITRs from AAVs of different sources other than AAV2 can also be selected.

[0047] The term "MOI" (multiplicity of infection) refers to the ratio of virus to cell number at the time of infection. For AAV viruses, the number of vector genomes (vg) is used to express the number of viruses.

[0048] The term "exoenzyme C3 transferase" (C3) is a mono ADP ribosyltransferase (ART) that catalyzes the transfer of an ADP ribose moiety from NAD(+) to a Rho GTPase, thereby causing the Rho GTPase to be ribosylated and lose its function. An example of a C3 protein is the C3 protein from Clostridium botulinum and its functional homologs, such as species homologs. Another example of a C3 protein is a protein having the amino acid sequence of SEQ ID NO: 18. It has been reported that the C3 protein can specifically act on Rho GTPase (guanosine triphosphatases, Rho GTPases, Rho), an important regulatory protein of the actin cytoskeleton and myosin stability, and by modifying Rho, causing Rho to be ribosylated and lose its function, thereby increasing the drainage of aqueous humor and thereby reducing intraocular pressure.

[0049] The term "Rho GTPase" refers to an important component of the Ras superfamily, which primarily includes three subfamilies: Rho (RhoA, RhoB, and RhoC), Rac (Rac1, Rac2, and Rac3), and Cdc42 (Cdc42Hs and G25K). Rho is activated upon binding to GTP, which in turn activates downstream molecules. When the bound GTP is hydrolyzed to GDP, it becomes inactive again.

[0050] The term "Rho" refers to one of the major members of the Rho GTPase family, which plays a key role in biological processes such as the regulation of actin cytoskeletal organization, microtubule dynamics, gene transcription, tumor transformation, and cell cycle progression. Rho-associated coiled-coil kinase (ROCK) belongs to the AGC family of serine / threonine protein kinases and includes two isoforms: ROCK1 (also known as ROCKβ or p160-ROCK) and ROCK2 (also known as ROCKα or p164-ROCK), located on chromosome 18 (18q11.1) and chromosome 2 (2p24), respectively. These are classic Rho downstream effectors, primarily involved in actin-myosin contraction and the dynamic regulation of the actin cytoskeleton. Activated Rho binds to and activates ROCK, forming the "Rho / ROCK signaling pathway," which participates in life activities such as cell morphogenesis, movement, division, polarity, proliferation, migration, and adhesion. It also has a significant impact on the occurrence and development of various diseases, including glaucoma, cardiovascular disease, respiratory disease, kidney disease, blood system disease, nervous system disease, and autoimmune disease. It has been reported that the inhibitor of Rho downstream molecule ROCK, H-1152P, and the Rho kinase inhibitor Fasudil can promote the survival of retinal ganglion cells (RGCs) in the optic nerve crush injury model and excitotoxic injury model, respectively, and have a neuroprotective effect. Therefore, inhibiting Rho and its downstream molecule ROCK may become an important approach for neuroprotection in the treatment of glaucoma.

[0051] The term "treatment" refers to clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

[0052] The following describes various aspects of the present invention. It should be understood that, unless there is an obvious contradiction, the features described in each aspect can be combined with each other without limitation, and these combinations are all within the consideration of the present invention.

[0053] enhancer

[0054] In the first aspect of the present invention, the disclosure provides an enhancer polynucleotide. An enhancer is a non-coding sequence that can activate the expression of a target gene transcribed by RNA polymerase. It is known in the art that the role of an enhancer can be unaffected by the direction, distance and position of the enhancer relative to the promoter and the target gene. For example, the enhancer can be located upstream of the proximal side of the target gene (e.g., approximately -200bp to -1000bp), but it can also play a role at a position farther upstream from the target gene, even up to one million base pairs away. The length of the enhancer is not particularly limited, and multiple enhancers can be clustered together to play a role together. The enhancer sequence can also be fused with a filler sequence without affecting its function, and the filler sequence includes, for example, one or more restriction sites (polylinkers, multiple cloning sites, one or more cloning sites), one or more linker sequences, one or more recombination sites or a combination thereof.

[0055] In some embodiments according to the first aspect of the present invention, the enhancer polynucleotide of the present invention comprises a nucleotide sequence selected from SEQ ID NOs: 1-5, or a nucleotide sequence having at least 95% identity thereto. In some embodiments, the polynucleotide can be used to enhance the transcription of a target gene in a cell, and preferably can be used to increase the expression of the target gene in the cell. In some embodiments, the cell is a trabecular meshwork cell, in particular a human trabecular meshwork cell. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence having at least about 90% identity thereto, for example, a nucleotide sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 2, or a nucleotide sequence having at least about 90% identity thereto, for example, a nucleotide sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having at least about 90% identity thereto, e.g., about 95%, 96%, 97%, 98%, 99% or more identity thereto. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 4, or a nucleotide sequence having at least about 90% identity thereto, e.g., about 95%, 96%, 97%, 98%, 99% or more identity thereto. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 5, or a nucleotide sequence having at least about 90% identity thereto, e.g., about 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0056] In some preferred embodiments, the enhancer polynucleotide according to the present invention has a nucleotide sequence selected from SEQ ID NOs: 1-5.

[0057] In some embodiments, the enhancer polynucleotide according to the present invention comprises a combination of multiple enhancer sequences, for example, a combination of two, three, or four enhancer sequences according to the present invention; or a combination of an enhancer sequence according to the present invention and a second enhancer sequence, for example, a sequence of SEQ ID NO: 17.

[0058] Those skilled in the art can select the appropriate enhancer sequence length and combination form suitable for the construction of the target gene expression cassette as needed. In some cases, considering the capacity of the delivery vector, it may be more advantageous to use a short enhancer sequence. In other cases, considering the required expression level of the target gene in the target tissue, an enhancer sequence with the required strength can be selected. For example, for a target gene with a certain toxicity, sometimes an overly strong enhancer will cause the AAV virus to be unable to be packaged. At this time, it can be considered to appropriately select an enhancer or enhancer combination with a slightly lower strength to balance the therapeutic effect and toxic side effects.

[0059] Since the effect of enhancers is non-directional, it is understood that the present invention also encompasses enhancer polynucleotides comprising the reverse complementary sequence of the above-mentioned enhancer sequences of the present invention.

[0060] promoter

[0061] In a second aspect according to the present invention, the present disclosure provides a promoter polynucleotide comprising an enhancer sequence and a promoter sequence of the present invention. As known in the art, a promoter is generally a DNA sequence located upstream of a gene to be transcribed (i.e., the 5' region of the gene). The promoter plays a role in controlling (e.g., stimulating) gene expression. Therefore, it is understood by those skilled in the art that the expression "promoter" refers to a polynucleotide molecule comprising a nucleotide sequence capable of driving an operably linked gene to be expressed in a cell. The expression "minimal promoter" refers to the minimum (shortest) nucleotide sequence required for driving the expression of a gene to which it is operably linked in a promoter nucleotide sequence, sometimes also referred to as a core promoter. See, for example: Luo et al. (1992) "Characterization of a minimal promoter element required for Transcription of the mouse type 11 / 3 regulatory subunit (RIIB) of CAMP-dependent protein kinase", The Journal of Biological Chemistry, 267(34):24738-47; and Hershko et al. (2001) "The mouse Snrpn minimal promoter and its human orthologue: activity and imprinting", Genes to Cells, 6:967-75. The minimal sequence required to initiate transcription of an operably linked downstream gene sequence in transfected cells and methods for identifying the same are known in the art. For example, the SV40 minimal promoter. Therefore, the present invention not only contemplates promoter polynucleotides comprising a minimal promoter sequence operably linked to an enhancer sequence of the present invention; it also contemplates promoter polynucleotides comprising a partial or full-length promoter sequence operably linked to an enhancer sequence of the present invention. For example, the promoter sequence may comprise the minimal promoter sequence of the MGP promoter, or a partial or full-length sequence of the MGP promoter. As will be appreciated by those skilled in the art, the position and orientation of an enhancer relative to a promoter can vary without affecting its effect of enhancing transcription. Thus, the present disclosure contemplates not only isolated polynucleotides comprising an enhancer of the present invention upstream of a promoter, but also isolated polynucleotides comprising an enhancer of the present invention interposed between two segments of a promoter.

[0062] In some embodiments according to the second aspect of the present invention, the promoter polynucleotide according to the present invention can be used to enhance the transcription of the target gene operably linked thereto in trabecular meshwork cells, and preferably can be used to increase the expression level of the target gene in the cells. In some embodiments, the promoter polynucleotide comprises an operably linked:

[0063] a. an enhancer sequence, wherein the enhancer sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-5 or a nucleotide sequence having at least about 90% identity thereto, for example, a nucleotide sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto, and

[0064] b. A promoter sequence expressed in trabecular meshwork cells, for example, the promoter sequence of the MGP gene shown in SEQ ID NO: 16 or the promoter sequence of the human chitinase 3-like protein 1 gene shown in SEQ ID NO: 23, or a promoter sequence having at least about 90% identity thereto, for example, a promoter sequence having about 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0065] In some embodiments, the promoter polynucleotide further comprises an operably linked second enhancer sequence, such as the enhancer nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence at least about 90% identical thereto, for example, about 95%, 96%, 97%, 98%, 99% or more identical thereto.

[0066] As demonstrated in the Examples of this application, upon combining the enhancer of the present invention with the MGP promoter comprising the sequence shown in SEQ ID NO: 16 or the eMGP promoter comprising the sequence shown in SEQ ID NO: 16 and the sequence shown in SEQ ID NO: 17, the resulting promoter (also referred to herein as RE-MGP or RE-eMGP promoter) exhibits a synergistically enhanced transcription initiation effect. Therefore, in some preferred embodiments, the present invention provides a promoter polynucleotide comprising: (a) an enhancer sequence selected from SEQ ID NOs: 1-5; and (b) the promoter sequence shown in SEQ ID NO: 16, and preferably (c) a second enhancer sequence shown in SEQ ID NO: 17, operably linked.

[0067] In some preferred embodiments, the present disclosure also provides such an isolated promoter polynucleotide, which comprises a nucleotide sequence selected from SEQ ID NOs: 6-15, or a nucleotide sequence having at least about 90% identity thereto, for example, a nucleotide sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0068] Expression Box

[0069] In a third aspect, the present invention provides an expression cassette for expressing an exogenous gene of interest, particularly an expression cassette for specifically expressing an exogenous gene of interest in trabecular meshwork cells, the expression cassette comprising an exogenous gene of interest to be specifically expressed in trabecular meshwork cells and a promoter polynucleotide that directs its expression. In some embodiments, the promoter polynucleotide comprises an enhancer sequence as set forth in any one of SEQ ID NOs: 1-5, or an enhancer sequence having at least about 90% identity thereto, for example, an enhancer sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto, wherein the promoter polynucleotide can be used to enhance transcription of the gene of interest in trabecular meshwork cells, and preferably ultimately increase the expression level of the gene of interest in the cells. In some embodiments, the promoter polynucleotide comprises a promoter polynucleotide according to any embodiment of the second aspect of the present invention.

[0070] The exogenous target gene that can be specifically expressed in trabecular meshwork cells according to the present invention is not particularly limited. Exogenous target genes that can be introduced into trabecular meshwork cells to produce therapeutic proteins or nucleic acid molecules, or to produce proteins or nucleic acid molecules that silence or edit pathogenic genes, are all contemplated by the present invention. In some preferred embodiments, the exogenous gene used in the expression cassette of the present invention is an exoenzyme C3 transferase gene. Therefore, in some embodiments, the present invention also provides an exoenzyme C3 transferase gene expression cassette, comprising a nucleotide sequence encoding an exoenzyme C3 transferase operably linked to an enhancer polynucleotide according to the first aspect of the present invention or a promoter polynucleotide according to the second aspect of the present invention, thereby directing the nucleotide sequence encoding the exoenzyme C3 transferase to be specifically expressed in trabecular meshwork cells. In some embodiments, the nucleotide sequence encoding the exoenzyme C3 transferase is a nucleotide sequence encoding an exoenzyme C3 transferase as shown in SEQ ID NO: 18 or having at least about 90% identity (e.g., about 95%, 96%, 97%, 98%, 99% or more identity) to SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the exoenzyme C3 transferase comprises a nucleotide sequence selected from the group consisting of:

[0071] (i) the nucleotide sequence encoding exoenzyme C3 transferase as shown in SEQ ID NO: 19;

[0072] (ii) a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 19;

[0073] (iii) a nucleotide sequence that encodes the same exoenzyme C3 transferase as the nucleotide sequence of (i) or (ii), but differs from the nucleotide sequence of (i) or (ii) due to the degeneracy of the genetic code; or

[0074] (iv) a sequence that is at least 70%, 80%, or 90% identical (e.g., at least 95%, 96%, 97%, 98%, 99% or more identical) to the nucleotide sequence of (i), (ii), or (iii).

[0075] In some embodiments, the nucleotide sequence encoding the exoenzyme C3 transferase comprises the nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence at least about 90% identical thereto (e.g., about 95%, 96%, 97%, 98%, 99% or more identical).

[0076] In some embodiments, the promoter polynucleotide directing the expression of exoenzyme C3 transferase comprises a promoter sequence selected from any one of SEQ ID NOs: 6-15, preferably selected from any one of SEQ ID NOs: 11-15, or a promoter sequence having at least about 90% identity thereto, for example, a promoter sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0077] As will be appreciated by those skilled in the art, in some cases, the expression cassette according to the present invention may also optionally contain other regulatory sequences that facilitate expression of the gene of interest. A variety of regulatory sequences useful for gene expression in mammalian cells are known in the art. In some embodiments, the expression cassette according to the present invention comprises the following elements operably linked to one another in the direction of transcription:

[0078] - any promoter polynucleotide according to the present invention,

[0079] - optionally, a Kozak sequence,

[0080] - a polynucleotide encoding a gene of interest, for example, a nucleotide sequence encoding exoenzyme C3 transferase, preferably a human codon-optimized exoenzyme C3 transferase coding sequence, more preferably the sequence of SEQ ID NO: 19,

[0081] - Optionally, a transcription terminator, such as a polyA signal sequence, preferably selected from the group consisting of the SV40 late polyA sequence, the rabbit β-globin polyA sequence, the bovine growth hormone polyA sequence, or any variants thereof.

[0082] The Kozak sequence used in the present invention may be a consensus sequence defined as GCCRCC, wherein R is a purine (ie, A or G), and wherein the sequence is located upstream of the start codon. In a preferred embodiment, the Kozak sequence used in the expression cassette according to the present invention has a 5'-GCCACC-3' sequence.

[0083] Nucleic acid expression vector

[0084] In a fourth aspect, the present invention provides a nucleic acid expression vector comprising an expression cassette of the present invention and a host cell comprising the same. In some embodiments, the vector is a plasmid (e.g., a plasmid for the production of recombinant viral particles). In other embodiments, the vector is a viral vector, such as a recombinant AAV vector or a baculovirus vector. In some embodiments, the genome of the recombinant AAV vector is single-stranded (e.g., single-stranded DNA). In some embodiments, the genome of the recombinant AAV vector is self-complementary. In further embodiments, the vector is a baculovirus vector (e.g., Autographa californica nuclear polyhedrosis virus (AcNPV) vector). In some embodiments, the present invention also provides a host cell comprising the vector, such as a mammalian cell or an insect cell. In some embodiments, the cell can be used to produce a recombinant AAV virus.

[0085] viral vectors

[0086] In a fifth aspect, the present invention provides a viral vector, which is an artificial recombinant viral particle, wherein a replication-defective viral genome sequence comprising an expression cassette encoding an exogenous target gene specifically expressed in trabecular meshwork cells (for example, an expression cassette of exoenzyme C3 transferase) is packaged in a viral capsid or envelope, such that the recombinant viral particle cannot produce progeny virions but retains the ability to infect target cells.

[0087] In one embodiment, the genomic sequence of the viral vector does not contain genes encoding enzymes required for viral replication, and therefore, the use of viral vectors in gene therapy is considered safe because in the absence of enzymes required for viral replication, replication and infection of progeny virions cannot occur.

[0088] The recombinant viral vector of the present invention can be a recombinant adeno-associated virus (AAV), adenovirus, bocavirus, AAV / bocavirus hybrid, herpes simplex virus or lentivirus.

[0089] The packaging cell line for producing a recombinant viral vector (e.g., a recombinant AAV vector) can be a prokaryotic cell or a eukaryotic cell (e.g., a human cell, an insect cell, or a yeast cell) containing exogenous DNA introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, and protoplast fusion). Packaging cell line cells include, but are not limited to, E. coli cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK293 cells, hepatocytes, kidney cells, glial cells, or stem cells.

[0090] In a preferred embodiment, the viral vector is a recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and a vector genome packaged therein. In some cases, the rAAV vector is used to treat or alleviate glaucoma. As an example, the vector genome comprises at least an AAV 5' inverted terminal repeat (ITR) or an AAV 5'ΔITR, an enhancer sequence selected from SEQ ID NOs: 1-5 or an enhancer sequence having at least about 90% identity thereto, a promoter sequence expressed in trabecular meshwork cells, a nucleic acid sequence encoding an exogenous protein of interest (e.g., exoenzyme C3 transferase), and an AAV 3'ITR or AAV 3'ΔITR. ITR is a genetic element responsible for the replication and packaging of the genome during vector production and is the only viral cis element required for the production of rAAV. ITRs from AAVs of different sources can be selected, and in some cases, the ITRs can be from an AAV different from the capsid of the viral particle. ΔITR is an ITR with the D sequence and the terminal melting site trs deleted. It can make the genome carried by the packaged recombinant adeno-associated virus vector self-complementary and form a double strand, significantly improving the transduction efficiency of AAV vectors in vivo and in vitro (Wang Z et al., Gene Ther. 2003; 10(26): 2105-2111; McCarty DM et al., Gene Ther. 2003; 10(26): 2112-2118). The double-stranded AAV virus that can be packaged using the combination of ΔITR and ITR is the so-called scAAV (self-complementary AAV). It is different from ssAAV (single-stranded AAV) in which both ITRs are not mutated.

[0091] Unless otherwise indicated, the AAV capsids, ITRs, and other AAV components described herein can be readily selected from any AAV, including but not limited to AAVs of serotypes generally identified as AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11, or AAV12, or combinations thereof. In some embodiments, the AAV capsid is an AAV2 capsid or a variant thereof. Herein, the capsid protein of the rAAV vector is designated by a number or a combination of numbers and letters following the term "AAV" in the name of the rAAV vector. Herein, an AAV capsid "variant" refers to any AAV capsid sequence derived from a known AAV capsid sequence, including those AAV capsid sequences with conservative amino acid substitutions, and sequences having at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity to the amino acid or nucleic acid sequence of the AAV capsid.

[0092] In some embodiments, a rAAV is provided that comprises an AAV serotype 2 (AAV2) capsid; and a vector genome comprising the following nucleotide sequence operably linked:

[0093] a. an enhancer sequence selected from SEQ ID NOs: 1-5 or an enhancer sequence having at least about 90% identity thereto, for example, an enhancer sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto;

[0094] b. a promoter sequence expressed in trabecular meshwork cells, e.g., the promoter sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 23, or a promoter sequence having at least about 90% identity thereto, e.g., a promoter sequence having about 95%, 96%, 97%, 98%, 99% or more identity thereto; and

[0095] c. A nucleotide sequence encoding an exogenous protein of interest (e.g., an exoenzyme C3 transferase), for example, a nucleotide sequence encoding an exoenzyme C3 transferase as set forth in SEQ ID NO: 18 or having at least about 90% identity (e.g., about 95%, 96%, 97%, 98%, 99% or higher identity) to SEQ ID NO: 18.

[0096] In some embodiments, the vector genome comprises a promoter polynucleotide according to any embodiment of the second aspect of the present invention, which is operably linked to the target gene, for example, a promoter polynucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 6-15, in particular selected from SEQ ID NOs: 11-15.

[0097] In some embodiments, the rAAV vector is a ssAAV vector. In other embodiments, the rAAV vector is a scAAV vector.

[0098] The recombinant adeno-associated virus (AAV) vector of the present invention can be produced using known techniques. Such methods involve culturing packaging cells, which contain a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein, which is flanked by AAV inverted terminal repeats (ITRs) or ΔITRs; and sufficient auxiliary functions to allow the expression cassette to be packaged into the AAV capsid protein. Currently commonly used AAV vector packaging systems mainly include a three-plasmid co-transfection system, a system in which adenovirus is used as a helper virus, a packaging system in which herpes simplex virus (Herpes simplex virus type 1, HSV1) is used as a helper virus, and a baculovirus-based packaging system. Each packaging system has its own characteristics, and those skilled in the art can make appropriate choices as needed for the production of rAAV vectors according to the present invention. In addition, other methods for producing rAAV known in the art, for example, by yeast production, can also be used.

[0099] In some aspects, the present invention also provides packaging cells for producing rAAV vectors of the present invention, which contain a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein, which is flanked by AAV inverted terminal repeats (ITRs) or ΔITRs; and sufficient helper functions to allow packaging of the expression cassette into AAV capsid proteins.

[0100] Methods and uses

[0101] Glaucoma is an eye disease characterized by pathologically elevated intraocular pressure, optic disc atrophy, visual field loss, and decreased vision. It is the world's leading irreversible cause of blindness. According to the World Health Organization (WHO), there were approximately 76 million glaucoma patients worldwide in 2020, and the number is expected to exceed 110 million by 2040. This large prevalence of glaucoma imposes a significant economic and social burden on society.

[0102] Glaucoma can be broadly divided into two main categories, open-angle glaucoma and closed-angle glaucoma. When referring to glaucoma, the "angle" refers to the space between the iris and cornea through which fluid (aqueous humor) must flow to exit the eye through the trabecular meshwork. Therefore, regulating the contractility of the trabecular meshwork can treat elevated intraocular pressure in glaucoma patients.

[0103] Gene therapy, an emerging therapeutic approach developed alongside molecular and cell biology, aims to achieve therapeutic goals by modifying the body's genes through the introduction of therapeutic genes, silencing, or editing disease-causing genes. The eye is an ideal site for gene therapy because its relatively small size allows for low gene delivery doses. As a relatively independent organ, the eye possesses immune privileges that can, to a certain extent, reduce inflammation and immune responses induced by exogenous substances. Furthermore, localized ocular administration is convenient, effectively preventing interference with gene delivery caused by the complex physiological environment of the body.

[0104] The viral vector of the present invention, as a gene drug, can be injected intraocularly, for example, intracameral injection, to specifically express an exogenous target protein (e.g., exoenzyme C3 transferase) in the trabecular meshwork to alleviate or treat glaucoma. Intraocular injection of the viral vector of the present invention can specifically and efficiently express the exogenous target protein (e.g., exoenzyme C3 transferase) in the patient's trabecular meshwork cells, thereby achieving the technical effects of lowering intraocular pressure and reducing side effects.

[0105] Therefore, in a sixth aspect, the present invention provides a pharmaceutical composition comprising the enhancer polynucleotide of the first aspect, the promoter polynucleotide of the second aspect, the gene expression cassette of the third aspect, the nucleic acid expression vector of the fourth aspect, or the adeno-associated viral vector of the fifth aspect. In some embodiments, the present invention provides a pharmaceutical composition comprising the recombinant AAV viral vector of the present invention. The pharmaceutical composition of the present invention preferably comprises a pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutical composition of the present invention can be formulated into any suitable formulation.

[0106] In the seventh aspect, the present invention provides the use of the enhancer polynucleotide according to the first aspect, the promoter polynucleotide according to the second aspect, the gene expression cassette according to the third aspect, the nucleic acid expression vector according to the fourth aspect, or the adeno-associated virus vector according to the fifth aspect, or the pharmaceutical composition according to the fifth aspect for alleviating or treating glaucoma, or for use in the preparation of a medicament for alleviating or treating glaucoma.

[0107] Example

[0108] Material

[0109] The pHelper plasmid is derived from the AAV Helper Free System (Agilent Technologies, USA). This plasmid contains the adenovirus-derived helper genes E2A, E4, and VARNA required for co-transfection of three plasmids into HEK293 cells to produce recombinant AAV virus.

[0110] The pAAV-R2C2 plasmid was derived from the AAV Helper Free System (Agilent Technologies, USA). To clarify the function of the pAAV-RC plasmid, the pAAV-RC plasmid was renamed the pAAV-R2C2 plasmid. The pAAV-R2C2 plasmid contains the complete AAV2 rep and cap genes, providing the four Rep proteins (Rep78, Rep68, Rep52, and Rep40) and AAV2 capsid protein required for packaging recombinant AAV2 virus during co-transfection with the three plasmids.

[0111] Example 1. Construction of enhancer library

[0112] Oligo primers are 5' phosphorylated. After annealing the designed oligo primer pairs to form double-stranded structures, the oligos were randomly combined and embedded into the pUC57 backbone after Hpa I digestion using T4 ligation. The library was then transformed into a plate. The sequences of the primer pairs used for the oligos are shown in Table 1:

[0113] Table 1: Primer pair sequences used by Oligo

[0114]

[0115] Using the pUC57 library as a template, PCR was performed to obtain fragments of different sizes, such as Figure 1 The fragment was recovered and connected to the pAAV-MGP-Gluc plasmid backbone by homologous recombination. The specific operation was as follows:

[0116] 1. PCR amplification: Use the pUC57 library as a template for PCR amplification and introduce homology arms. The amplification primers are:

[0117] KZ-F1:tcactaggggttcctAACGTTGAGTTCGCAGTT(SEQ ID NO:21)

[0118] KZ-R1: ggctgaatatcgcgaAGAGTAGACTCCTTCTGTGTT (SEQ ID NO: 22).

[0119] After amplification, the fragments were recovered from the gel.

[0120] 2. Construction of pAAV-MGP-Gluc backbone plasmid: The backbone plasmid was constructed basically in the manner described in CN111088264A. In brief, based on the AAV vector pAAV2neo vector, the MGP promoter (human matrix glial protein MGP gene promoter, SEQ ID NO: 16) was connected to the pAAV2neo vector from which the CMV promoter had been cut off, and the AAV plasmid vector pAAV-MGP containing the MGP promoter was screened and identified. Using the pCMV-Gluc2 plasmid (NEB, USA) as a template, a fragment containing the Gluc coding sequence (secretory luciferase coding sequence derived from Gaussia) was amplified by PCR, and the fragment was connected to the pAAV-MGP plasmid, and the AAV backbone plasmid vector pAAV-MGP-Gluc (containing the Gluc gene coding region) was screened and identified. Figure 2A ).

[0121] 3. Linearize pAAV-MGP-Gluc backbone plasmid: Digest pAAV-MGP-Gluc with Xho I endonuclease to serve as the backbone for library insertion. Ligate the PCR amplification product obtained in step 1 (hereinafter referred to as "RE") with the linearized backbone pAAV-MGP-Gluc to obtain a plasmid containing the RE-MGP-Gluc construct (e.g. Figure 2B The AAV plasmid vector pAAV-RE-MGP-Gluc (shown in Figure 2) was transformed and plated, and monoclonal colonies were picked to form the final screening library. As a control, the enhancer portion of the eMGP promoter reported by Jun-Kai Tan et al. (2023, doi: 10.18240 / ijo.2023.08.03) (enhancer, sequence see SEQ ID NO: 17) was ligated into the linearized backbone pAAV-MGP-Gluc to obtain the AAV plasmid vector pAAV-eMGP-Gluc containing the eMGP-Gluc construct.

[0122] Example 2 Construction of AAV-RE-MGP-Gluc viral vector and in vitro activity study

[0123] Reference (Xiao X, et al. Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus. J Virol. 1998; 72(3): 2224-2232), the Rep / Cap plasmid expressing AAV Cap protein and Rep protein, the helper plasmid, and the AAV vector plasmid pAAV-RE-MGP-Gluc were mixed at a molar ratio of 1:1:1 by a three-plasmid packaging method and co-transfected into HEK293 cells using the calcium phosphate method. After 72 hours of transfection, the cells and culture supernatant were collected, and the recombinant AAV virus was isolated and purified to obtain a recombinant AAV virus product. The genome titer of the prepared rAAV virus was determined by dot blot hybridization to obtain the recombinant virus AAV-RE-MGP-Gluc. In the same manner, the control recombinant virus AAV-eMGP-Gluc was obtained.

[0124] Human trabecular meshwork cells (HTMC) were derived from ScienCellResearch Laboratories (Carlsbad, CA, USA; for details, see the website http: / / www.sciencellonline.com) and cultured according to the literature (Tan J, et al. Invest Ophthalmol Vis Sci. 2018; 59(12): 4937-4944.). The prepared recombinant AAV virus was used to infect human trabecular meshwork cells (HTMC) at an MOI (multiplicity of infection) of 5000, and the Gluc activity in the cell supernatant was detected after 72 hours. Specifically, 20 μL of cell culture supernatant was taken and reacted with 1:200 diluted coelenterazine (Gluc luciferase substrate, Solebol IC2920) in a GLOMAX (Promega) device to measure the results. The results are shown in Figure 3 Results showed that compared to the MGP promoter, the following promoters resulted in higher Gluc activity: RE055-MGP, RE071-MGP, RE082-MGP, RE083-MGP, and RE089-MGP. Sequencing yielded the DNA sequences of enhancer fragments RE055, RE071, RE082, RE083, and RE089, as shown in SEQ ID Nos. 1 to 5, respectively; the DNA sequences of promoters RE055-MGP, RE071-MGP, RE082-MGP, RE083-MGP, and RE089-MGP, as shown in SEQ ID Nos. 6 to 10, respectively.

[0125] Example 3 Construction of AAV-RE-eMGP-Gluc viral vector and in vitro activity study

[0126] In order to test whether the RE enhancer fragments screened have the ability to synergistically enhance expression with eMGP, RE055, RE071, RE082, RE083, and RE089 were embedded between the enhancer of eMGP and the MGP promoter to produce the following: Figure 4 The construct RE-eMGP-Gluc drives the expression of Gluc. The DNA sequences of promoters RE055-eMGP, RE071-eMGP, RE082-eMGP, RE083-eMGP and RE089-eMGP in each construct are shown in SEQ ID Nos. 11 to 15, respectively.

[0127] Referring to the method shown in Example 1, an AAV plasmid vector pAAV-RE-eMGP-Gluc containing the RE-eMGP-Gluc construct was constructed.

[0128] Referring to Example 2, a three-plasmid packaging method was used to prepare recombinant viruses AAV-RE055-eMGP-Gluc, AAV-RE071-eMGP-Gluc, AAV-RE082-eMGP-Gluc, AAV-RE083-eMGP-Gluc, and AAV-RE089-eMGP-Gluc. The prepared recombinant AAV viruses were used to infect HTMC cells at an MOI of 5000, and Gluc activity in the cell supernatant was detected after 72 hours. The results are shown in FIG. Figure 5 As shown, the above RE055, RE071, RE082, RE083, and RE089 fragments can significantly enhance the expression capacity of the eMGP promoter.

[0129] Example 4 Construction of AAV-RE-eMGP / MGP-C3 viral vector and in vitro activity study

[0130] Based on the requirements of sequence length and viral packaging yield, RE055-eMGP, RE071-eMGP, RE089-eMGP, and RE089-MGP promoters were selected to construct viral vectors that can express C3 protein for testing the gene delivery expression ability in human trabecular meshwork cells.

[0131] The pAAV-RE-eMGP-C3 vector and the pAAV-RE-MGP-C3 vector were constructed according to essentially the same method as in CN111088264A. Figure 6 Schematic diagram of the RE-eMGP-C3 construct and the RE-MGP-C3 construct contained in the vector is shown. Figure 6 In the figure, "RE" represents the enhancer fragment of the present invention, RE055, RE071 or RE089; "MGP" represents the MGP promoter (SEQ ID NO.16); "Enhancer" represents the enhancer portion of the eMGP promoter (SEQ ID NO.17); "C3" represents the coding region sequence of the Clostridium botulinum C3 gene synthesized by optimizing human codon expression (SEQ ID NO.19), with the Kozak sequence "5'gccacc3'" added to the 5' end of the synthetic sequence and the stop codon "5'TGATAA3'" added to the 3' end.

[0132] Referring to Example 2, a three-plasmid packaging method was used to prepare recombinant viruses AAV-RE055-eMGP-C3, AAV-RE071-eMGP-C3, AAV-RE089-eMGP-C3, and AAV-RE089-MGP-C3. The packaged recombinant viruses were infecting HTMC cells with the same MOI. 48 hours after infection, total cell RNA was extracted and reverse transcribed to obtain cDNA according to the method reported by Jun-Kai Tan et al. (2023, doi: 10.18240 / ijo.2023.08.03); the copy number of the target gene C3 and the internal reference gene GAPDH in the sample was detected by real-time quantitative polymerase chain reaction analysis, and the relative content of the C3 copy number was corrected by the internal reference to determine the expression ability of different promoters.

[0133] The results are as follows Figure 7 The results showed that when driving C3 protein expression, the RE055-eMGP promoter resulted in the highest expression increase of 2.5-fold compared to the eMGP promoter (SEQ ID NO: 20). RE089-eMGP resulted in a 2.3-fold increase, and RE089-MGP resulted in a 2-fold increase. However, RE071-eMGP did not show a significant increase. This suggests that the sequence of the target gene may have a certain impact on promoter activity.

[0134] Sequence Listing

[0135] SEQ ID No. 1: DNA sequence of enhancer RE055

[0136] SEQ ID No. 2: DNA sequence of enhancer RE071

[0137] SEQ ID No. 3: DNA sequence of enhancer RE082

[0138] SEQ ID No. 4: DNA sequence of enhancer RE083

[0139] SEQ ID No. 5: DNA sequence of enhancer RE089

[0140] SEQ ID No. 6: DNA sequence of promoter RE055-MGP

[0141] SEQ ID No. 7: DNA sequence of promoter RE071-MGP

[0142] SEQ ID No. 8: DNA sequence of promoter RE082-MGP

[0143] SEQ ID No. 9: DNA sequence of promoter RE083-MGP

[0144]

[0145] SEQ ID No. 10: DNA sequence of promoter RE089-MGP

[0146] SEQ ID No. 11: DNA sequence of promoter RE055-eMGP

[0147] SEQ ID No. 12: DNA sequence of promoter RE071-eMGP

[0148]

[0149] SEQ ID No. 13: DNA sequence of promoter RE082-eMGP

[0150] SEQ ID No. 14: DNA sequence of promoter RE083-eMGP

[0151]

[0152] SEQ ID No. 15: DNA sequence of promoter RE089-eMGP

[0153] SEQ ID No. 16: DNA sequence of MGP promoter

[0154]

[0155] SEQ ID No. 17: Sequence of the enhancer fragment of eMGP

[0156] SEQ ID No. 18: Amino acid sequence of Clostridium botulinum C3 protein

[0157] SEQ ID No. 19: Optimized nucleotide sequence encoding Clostridium botulinum C3 protein

[0158] SEQ ID No. 20: DNA sequence of eMGP promoter

[0159]

[0160]

[0161] SEQ ID No. 21: Amplification primer KZ-F1

[0162]

[0163] SEQ ID No. 22: Amplification primer KZ-R1

[0164]

[0165] SEQ ID No.23: Promoter of human chitinase 3-like protein 1 (chitinase3-like1) gene .

Claims

1. An enhancer polynucleotide comprising a nucleotide sequence selected from SEQ ID NO: 1-5, or a nucleotide sequence having at least 95% identity thereto.

2. A promoter polynucleotide capable of enhancing transcription, wherein the polynucleotide comprises a promoter sequence operably linked to the enhancer polynucleotide of claim 1.

3. The promoter polynucleotide of claim 2, comprising operably linked: a. an enhancer sequence, wherein the enhancer sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, and b. Promoter sequence expressed in trabecular meshwork cells.

4. The promoter polynucleotide of any one of claims 2-3, wherein the promoter polynucleotide further comprises an operably linked second enhancer sequence.

5. The promoter polynucleotide of any one of claims 2 to 4, wherein the promoter polynucleotide comprises operably linked: (a) an enhancer sequence selected from the group consisting of SEQ ID NOs: 1-5; and (b) the promoter sequence shown in SEQ ID NO: 16, and Optionally, (c) a second enhancer sequence shown in SEQ ID NO:

17.

6. The promoter polynucleotide of any one of claims 2-5, wherein the promoter polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 6-15, or a nucleotide sequence that is at least about 90% identical thereto, e.g., about 95%, 96%, 97%, 98%, 99% or more identical thereto.

7. A gene expression cassette comprising the enhancer according to claim 1 or the promoter polynucleotide according to any one of claims 2 to 6 operably linked to a gene of interest.

8. The gene expression cassette of claim 7, wherein the gene of interest comprises a nucleotide sequence encoding exoenzyme C3 transferase.

9. The gene expression cassette of claim 8, wherein the gene of interest comprises a nucleotide sequence encoding an exoenzyme C3 transferase as set forth in SEQ ID NO: 18, or at least about 90% identical to SEQ ID NO: 18; or comprises a nucleotide sequence of SEQ ID NO: 19, or a nucleotide sequence having about 90% identity thereto (e.g., about 95%, 96%, 97%, 98%, 99% or more identity) and encoding an exoenzyme C3 transferase.

10. A nucleic acid expression vector, wherein the nucleic acid expression vector comprises the enhancer polynucleotide according to claim 1, the promoter polynucleotide according to any one of claims 2 to 6, or the gene expression cassette according to any one of claims 7 to 9.

11. A recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises an enhancer polynucleotide according to claim 1, a promoter polynucleotide according to any one of claims 2-6, a gene expression cassette according to any one of claims 7-9, or a nucleic acid expression vector according to claim 10.

12. The rAAV vector of claim 11, wherein the rAAV vector is a recombinant AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11 or AAV12 vector, preferably a recombinant AAV2 vector.

13. The rAAV vector of claim 11 or 12, wherein the rAAV vector is a ssAAV or scAAV vector.

14. A pharmaceutical composition comprising the nucleic acid expression vector according to claim 10 or the recombinant adeno-associated virus vector according to any one of claims 11 to 13.

15. Use of the nucleic acid expression vector according to claim 10 or the recombinant adeno-associated virus vector according to any one of claims 11 to 13 for alleviating or treating glaucoma, or in the preparation of a medicament for alleviating or treating glaucoma.

16. The use according to claim 15, wherein the glaucoma is primary open-angle glaucoma or primary angle-closure glaucoma.

17. The use according to claim 15 or 16, wherein the nucleic acid expression vector or the recombinant adeno-associated virus vector is administered by intracameral injection.

18. Use of the enhancer polynucleotide of claim 1 or the promoter polynucleotide of any one of claims 2 to 6 for enhancing the transcription of a target gene in trabecular meshwork cells.

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