Vectors comprising elac2 gene and use thereof in diseases related to nerve injury

Abstract

The present application relates to a vector containing an Elac2 gene and its application in diseases related to nerve injury. Specifically, the present application provides an adeno-associated virus vector containing an Elac2 gene, which can be constructed by a three-plasmid system: an AAV-CAG-ELAC2 vector plasmid, a pAAV2-RC packaging plasmid, and a pAAV-Helper. The present application uses Elac2 and the protein encoded thereby for nerve protection and nerve regeneration, provides a gene therapy means for nerve regeneration, and opens up a new way for the treatment of related diseases.

Description

Technical Field

[0001] This invention relates to the fields of neuroscience and gene therapy, and in particular to genes related to neuroprotection and nerve regeneration and their applications. Background Technology

[0002] Neurological damage and neurodegenerative diseases (such as Alzheimer's and Parkinson's) are generally global medical challenges. Neuronal damage and death lead to permanent functional impairment because the regenerative capacity of neurons in the central nervous system is extremely limited. Currently, research on neuroprotection and neuroregeneration mainly focuses on neurotrophic factors, cell therapy, and gene therapy. However, existing technologies still have many limitations in promoting neuroregeneration and neuroprotection; for example, neurotrophic factors have difficulty penetrating the blood-brain barrier, and cell therapy faces problems such as immune rejection.

[0003] Recent studies have found that the regulation of certain genes can significantly promote nerve regeneration. For example, knockdown of the lipin1 gene can activate the mTOR and STAT3 signaling pathways, thereby promoting nerve regeneration after spinal cord injury. Furthermore, the Nfe2l3 gene has also been shown to promote neuroprotection and long-distance axon regeneration after injury in vivo. Viral vectors (such as adeno-associated virus AAV) are widely used in gene therapy to deliver neurotrophic factors or other genes that promote nerve regeneration to damaged nerve cells. For example, delivering specific genes via AAV vectors can activate signaling pathways within nerve cells, promoting axon regeneration.

[0004] Current treatments (such as neurotrophic factor delivery and cell therapy) can promote nerve regeneration to some extent, but their effects are limited and it is difficult to achieve efficient nerve regeneration and functional recovery. There is a lack of effective targets: Although some genes and signaling pathways have been found to be related to nerve regeneration, the regulatory mechanisms of these targets are complex, and their effects vary in different nerve injury models.

[0005] Therefore, there is a need to develop new targets and treatments that promote nerve regeneration and functional recovery. Summary of the Invention

[0006] Technical Purpose

[0007] The technical objective of this invention is to develop a novel therapeutic target for neurological injury-related diseases.

[0008] Another technical objective of this invention is to develop new products for neurodegenerative diseases targeting this novel therapeutic target.

[0009] Another technical objective of this invention is to develop new treatment methods for neurodegenerative diseases targeting this novel therapeutic target.

[0010] Invention Summary

[0011] On the one hand, the present invention provides a vector containing the Elac2 gene, wherein the vector is a viral vector or a non-viral vector.

[0012] In specific embodiments, the viral vector includes: adeno-associated virus vector, lentiviral vector, or herpes simplex virus vector.

[0013] In a specific embodiment, the non-viral carrier is a liposome or a polymer nanoparticle.

[0014] In a specific embodiment, the vector is an adeno-associated virus vector, which is constructed by containing the following three-plasmid system:

[0015] The AAV-CAG-ELAC2 vector plasmid, pAAV2-RC packaging plasmid, and pAAV-Helper, wherein the sequence of the AAV-CAG-ELAC2 vector plasmid is SEQ ID No:1.

[0016] In a specific embodiment, the sequence of the pAAV2-RC packaging plasmid is SEQ ID No:2, and the sequence of the pAAV-Helper is SEQ ID No:3.

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising a vector containing the Elac2 gene or a protein encoding the Elac2 gene as described above, and optionally, pharmaceutically acceptable excipients.

[0018] In another aspect, the present invention provides a host cell containing the vector containing the Elac2 gene described above.

[0019] In a specific embodiment, the host cell is an AAV-293 cell.

[0020] In another aspect, the present invention also provides the use of the vector containing the Elac2 gene, the protein encoded by the Elac2 gene, the pharmaceutical composition described above, and / or the host cell in the preparation of a medicament for the prevention or treatment of neurological injury-related diseases in a subject.

[0021] In another aspect, the present invention provides the use of the Elac2 gene or its encoded protein as a biomarker in the diagnosis of neurological injury-related diseases.

[0022] In another aspect, the present invention provides a kit for detecting neurological injury-related diseases, the kit comprising reagents for detecting the expression level of the Elac2 gene.

[0023] In a specific implementation, the nerve injury-related diseases are selected from spinal cord injury, brain injury, and neurodegenerative diseases.

[0024] In a specific implementation, the neurodegenerative disease is selected from glaucoma, Alzheimer's disease, and Parkinson's disease.

[0025] In another aspect, the present invention provides a method for treating neurological injury-related diseases, comprising administering to a subject in need the aforementioned vector containing the Elac2 gene, the protein encoded by the Elac2 gene, or a pharmaceutical composition.

[0026] In another aspect, the present invention provides a combined treatment method for neurological injury-related diseases, the method comprising:

[0027] Administer to subjects in need the above-described vector containing the Elac2 gene, the protein encoded by the Elac2 gene, or a pharmaceutical composition, as well as one or more therapies selected from neurotrophic factor delivery, cell therapy, and drug therapy.

[0028] In a specific implementation, the neurological injury-related diseases are selected from spinal cord injury, brain injury, neurodegenerative diseases, and optic nerve injury.

[0029] In a specific implementation, the neurodegenerative disease is selected from glaucoma, Alzheimer's disease, and Parkinson's disease.

[0030] In another aspect, the present invention provides the use of reagents for overexpressing the Elac2 gene in the preparation of medicaments for the prevention or treatment of neurological injury-related diseases in subjects.

[0031] In a specific implementation, the neurological injury-related diseases are selected from spinal cord injury, brain injury, neurodegenerative diseases, and optic nerve injury.

[0032] In a specific implementation, the neurodegenerative disease is selected from glaucoma, Alzheimer's disease, and Parkinson's disease.

[0033] Beneficial effects

[0034] The inventors of this application unexpectedly discovered that Elac2 overexpression significantly enhances neuronal survival and axonal regeneration. By enhancing the expression of the Elac2 gene, neuronal axonal regeneration capacity can be significantly improved, thereby increasing the efficiency of nerve regeneration. This effect was verified in a model of optic nerve injury. The discovery of Elac2 and its encoded protein provides a new target for neuroprotection and nerve regeneration. This discovery not only enriches the gene therapy methods for nerve regeneration but also opens up new avenues for the treatment of related diseases. Attached Figure Description

[0035] Figure 1AAV vector map of Elac2 overexpression.

[0036] Figure 2 Effects of Elac2 overexpression on axonal growth in adult mouse DRG cells in vitro. Left image: Immunofluorescence staining image; Right image: Statistical data based on the left image.

[0037] Figure 3 The effect of Elac2 overexpression via AAV vector injection into the vitreous cavity of mouse eyes on optic nerve regeneration under an optic nerve compression model. Left image: Images taken (green represents labeled regenerating axons); Right image: Statistical data based on the left image.

[0038] Figure 4 In a optic nerve compression model, overexpression of Elac2 via AAV vector injection into the vitreous cavity of mouse eyes promoted the survival of retinal ganglion cells (RGCs). Left image: Immunofluorescence staining image (red signals represent optic ganglion cells); Right image: Statistical data based on the left image.

[0039] in, Figure 2 In the statistical data, each black dot represents a cell. Figure 3-4 In the statistical data, each black dot represents a mouse, and the data analysis method used is Student's t test (two-tailed). *P<0.05. In the figure, EGFP represents enhanced green fluorescent protein, and GFP represents green fluorescent protein. Detailed Implementation

[0040] The present application will be described in detail below through specific embodiments to enable those skilled in the art to better understand the technical content of the present application. However, as will be understood by those skilled in the art, the scope of protection of the present application is not limited to these embodiments, but also includes various equivalent variations made by those skilled in the art based thereon.

[0041] Example 1:

[0042] 1.1 Construction of AAV-CAG-ELAC2 plasmid vector: For nerve cells, the AAV2 serotype was selected (also applicable to AAV2 / 9, AAV-PHP.B and other serotypes), which showed high transduction efficiency in the central nervous system.

[0043] The Elac2 gene was inserted into the expression cassette of the AAV vector, which included a promoter, the Elac2 gene, and a terminator. The ITR sequences at both ends of the expression cassette were preserved. The sequence of the constructed AAV-CAG-ELAC2 plasmid is shown in SEQ ID No:1.

[0044] cDNA of the Elac2 gene (NCBI Gene ID: 60528, ID: 68626) was extracted. The pAAV vector (#37825, Addgene) and the amplified Elac2 gene cDNA were digested with restriction enzymes. The digested vector was purified by ethanol precipitation. The digested vector was subjected to 1% agarose gel electrophoresis for 6 hours at 50V to remove undigested vector bands, and then the digested vector bands were extracted using a gel extraction kit. The digested Elac2 gene fragment and pAAV vector were mixed, and the volume was adjusted to 10 μL with 1X DNA dilution buffer. Then, 10 μL of 2× DNA ligation buffer and 1 μL (1U) T4 DNA ligase were added, and the mixture was incubated at room temperature for 30 minutes. 2 μL of the ligation reaction solution was used to transform *E. coli* cells (Sangon Biotech, B528413-0100). The next day, three colonies were selected from each plate for sequencing, and the correct plasmid was amplified.

[0045] 1.2 AAV virus packaging and purification

[0046] (1) Plasmid transfection: AAV-293 (Procell, #CL-0019) cells were transfected with polyethylenimine (PEI) (1 mg / mL). A three-plasmid system was used for cell transfection, and AAV-293 cells were cultured in 10cm culture dishes. Add 4 μg of AAV-CAG-ELAC2 plasmid (SEQ ID No:1), 6 μg of pAAV2-RC (SEQ ID No:2) (Addgene, #104963) packaging plasmid, and 8 μg of pAAV-Helper (SEQ ID No:3) (Addgene, #112867) helper plasmid to 1 mL of serum-free DMEM medium. Add 100 μL of PEI to 1 mL of serum-free DMEM. Mix the plasmid and PEI and incubate at room temperature for 30 min. Mix the DMEM containing the plasmid and PEI and shake well. Add the mixture to AAV-293 cells (density of about 80%) in a 10 cm culture dish and change the medium after 24 h.

[0047] (2) Virus collection: The virus was extracted 72 hours after transfection. Since the AAV2 virus is not secretory, the culture medium was discarded. The cells were blown off with 1 mL of sterile PBS and collected in a 1.5 mL centrifuge tube.

[0048] (3) Cell lysis: The cell suspension was frozen at -80℃. After complete freezing, the cells were placed in a 37℃ water bath and repeatedly frozen and thawed three times. Then, the cells were centrifuged at 8000 rpm and 4℃ for 10 min to remove cell debris, collect the supernatant, filter through a 0.22 μm filter membrane to remove impurities, and collect the virus suspension.

[0049] (4) Virus density gradient centrifugation: 15%, 25%, 40%, and 60% of iodixanol were added to the ultracentrifuge tube in sequence, and finally the virus suspension was added; the ultracentrifuge was centrifuged at 360,000g and 4℃ for 90 min; after centrifugation, the 40% layer of virus suspension was collected (this step of extracting the crude virus extract is not performed).

[0050] (5) Concentrate the virus, add an equal volume of AAVS1 (2 M NaCl (Sigma-Aldrich, S9888), 20% PEG8000 (Sigma-Aldrich, 89510)) solution to the virus suspension, mix well, precipitate overnight at 4°C, centrifuge at 12000 rpm at 4°C for 30 min, resuspend the virus precipitate in sterile PBS, and aliquot the virus into 1.5 mL EP tubes (10 μL per tube).

[0051] Example 2:

[0052] In vitro experiments: The effects of overexpressing the Elac2 gene and the control group GFP on neuronal survival and axonal growth were observed in cultured neurons in vitro.

[0053] Specific experimental procedure: First, DRGs (dorsal root ganglion cells) were dissected from 6–8 week old female ICR mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. SCXK (Zhejiang) 2024-0004, housing conditions: barrier facility). The cells were digested at 37 °C for 70 min with 1 mg / ml type I collagenase (Thermo Fisher Scientific) and 5 mg / ml dispersin II (Thermo Fisher Scientific), followed by washing three times with HBSS. The digested DRGs were placed in MEM medium containing 10% fetal bovine serum and 1× penicillin / streptomycin (Thermo Fisher Scientific) and mechanically dissociated into single cells by pipetting. The cell suspension was filtered through a 100 μm cell sieve and centrifuged at 1000 rpm for 8.5 min. The collected cell pellet was used for in vitro electroporation.

[0054] External electroporation

[0055] Cell preparation: Prepare a single-cell suspension according to the above instructions.

[0056] Mixing transfection reagents with cells: Mix the cell suspension with the AAV-CAG-ELAC2 plasmid from Example 1 and the control plasmid pAAV vector pAAV-CAG-GFP (#37825, Addgene) until homogeneous.

[0057] Setting up the electroporation program: Select the appropriate electroporation program and parameters based on cell type and transfection purpose. The Lonza electroporator offers a variety of preset programs, and users can also customize settings as needed.

[0058] Perform electroporation: Add the mixed cell-transfection reagent solution to the electroporation cuvette, place the cuvette in the electroporator, and start the electroporation program.

[0059] Cell culture and detection: After electroporation, the cells are transferred to a culture plate containing appropriate culture medium for culture.

[0060] Immediately after electroporation, cells were mixed with an appropriate amount of preheated medium (as above) and seeded onto glass coverslips pre-coated with a mixture of 100 μg / ml poly-D-lysine (Sigma-Aldrich) and 10 μg / ml laminin (Thermo Fisher Scientific). After complete cell attachment (approximately 6 hours), the medium was completely replaced to remove the electroporation buffer and dead cells. Cells were then cultured in MEM containing 5% fetal bovine serum, 1×GlutaMAX-I (Thermo Fisher Scientific), 1× penicillin / streptomycin, and antimitotic reagents (20 μM 5-fluoro-2'-deoxyuridine and 20 μM uridine, both purchased from Sigma-Aldrich). No exogenous growth factors were added during culture, and the cells were cultured for 2 days. Immunofluorescence staining was used to detect axonal length.

[0061] Experimental results are as follows Figure 2 As shown in the figure, axonal growth is significant when ELAC2 is overexpressed.

[0062] Example 3:

[0063] Animal model experiments: In a mouse model of nerve injury, the Elac2 gene was activated through gene therapy to evaluate its effect on nerve regeneration and functional recovery.

[0064] Experimental procedure: Mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. SCXK (Zhejiang) 2024-0004, housing conditions: barrier facility) were anesthetized by intraperitoneal injection of a mixture containing ketamine (100 mg / kg) and toluenethiazide (10 mg / kg). The titer was greater than 1×10⁻⁶ per μL. 13In Example 1, AAV2 virus obtained at a concentration of vg / mL was injected into the vitreous cavity of 6- to 8-week-old wild-type mice via a glass microtube connected to a Picospritzer II (Parker Inc.) (pressure: 15 psi; duration: 6 ms). Two weeks after the intravitreal injection, the optic nerve in the right orbit was exposed, and a model of optic nerve compression injury was successfully established by clamping the nerve approximately 1 mm posterior to the optic disc for 2 seconds using Dumont #5 forceps.

[0065] To label retinal ganglion cell (RGC) axons in the optic nerve using anterograde labeling, 1.5 μL of CTB (Cholera Toxin Subunit B) conjugated with Alexa-594 (2 μg / μL, Invitrogen) (2 days prior to sacrifice) was injected intravitreally into the vitreous cavity of the animals. After sacrifice, the animals were fixed by perfusion of 20 mL of 0.1 M phosphate-buffered saline (PBS), followed by perfusion of 40 mL of 4% paraformaldehyde (PFA) at a rate of 5 mL / min. The retina and optic nerve segments were dissected and post-fixed overnight in 4% PFA (4°C). The fixed optic nerve was cleared before imaging and quantification. Additionally, optic ganglion cells were labeled and counted by immunofluorescence staining. Experimental results are as follows: Figure 3 and 4 As shown in the image.

[0066] from Figure 3 It can be seen that overexpression of ELAC2 in vivo promotes the regeneration of the central nervous system (optic nerve).

[0067] from Figure 4 It can be seen that overexpression of ELAC2 in vivo is beneficial to the survival of optic ganglion cells.

Claims

1. Use of an adeno-associated virus vector containing the Elac2 gene in the preparation of a medicament for treating optic nerve injury in a subject.

2. The use according to claim 1, wherein, The adeno-associated virus vector is constructed by a three-plasmid system containing the following: AAV-CAG-ELAC2 vector plasmid, pAAV2-RC packaging plasmid, and pAAV-Helper, wherein the sequence of the AAV-CAG-ELAC2 vector plasmid is SEQ ID No:

1.

3. The use according to claim 2, wherein, The sequence of the pAAV2-RC packaging plasmid is SEQ ID No:2, and the sequence of the pAAV-Helper is SEQ ID No:

3.

4. Use of the pharmaceutical composition in the preparation of a medicament for treating optic nerve injury in a subject, wherein, The pharmaceutical composition comprises: an adeno-associated virus vector containing the Elac2 gene, and pharmaceutically acceptable excipients.

5. The use according to claim 4, wherein, The adeno-associated virus vector is constructed by a three-plasmid system containing the following: AAV-CAG-ELAC2 vector plasmid, pAAV2-RC packaging plasmid, and pAAV-Helper, wherein the sequence of the AAV-CAG-ELAC2 vector plasmid is SEQ ID No:

1.

6. The use according to claim 5, wherein, The sequence of the pAAV2-RC packaging plasmid is SEQ ID No:2, and the sequence of the pAAV-Helper is SEQ ID No:3.

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