Nucleic acid molecules encoding human nuclear factor e2-related factor 2, expression vectors and uses thereof

Abstract

The present application relates to the field of biological medicine, in particular to nucleic acid molecule encoding human nuclear factor E2 related factor 2, expression vector and application thereof. The present application discloses a nucleotide sequence encoding human nuclear factor E2 related factor 2 (Nrf2) protein, which comprises the nucleic acid encoding human nuclear factor E2 related factor 2 (Nrf2) protein. Further disclosed is a recombinant expression vector comprising the above nucleic acid. The nucleic acid encoding human nuclear factor E2 related factor 2 (Nrf2) protein has faster expression time and higher expression level, and thus can better treat optic nerve related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to nucleic acid molecules encoding human nuclear factor E2-related factor 2, expression vectors, and their applications. Background Technology

[0002] The optic nerve originates from the ganglion cell layer of the retina and is part of the central nervous system. Visual information received by the retina is transmitted to the brain via the optic nerve. Many diseases are related to optic nerve damage, including glaucoma, ischemic optic neuropathy, Leber's hereditary optic neuropathy (LHON), and autosomal dominant optic atrophy (DOA). Damage to ganglion cells and their axons is irreversible and is a major cause of vision loss and even blindness. Currently, there are no effective drugs for optic nerve damage on the market. Research on drugs for optic nerve protection mainly focuses on neurotrophic factors, such as BDNF and CNTF, whose mechanism of action is to promote neuronal growth and repair damaged optic nerves, but the therapeutic effects are not ideal.

[0003] Research has found that the pathogenesis of many optic nerve injuries is due to the energy dependence and sensitivity of retinal ganglion cells and their axons to free radicals, leading to damage and even apoptosis, which in turn causes blindness. Therefore, optic nerve protection can be achieved by scavenging free radicals in diseased cells and enhancing the antioxidant capacity of cells, thereby preventing or delaying apoptosis of ganglion cells (RGCs).

[0004] Oxidative stress is caused by the excessive production of reactive oxygen species (ROS) and electrophiles by cells. Excessive ROS can induce free radical chain reactions, damaging cellular macromolecules such as proteins, lipids, and DNA, leading to cell death and tissue damage. At the same time, ROS also participate in other pathological processes such as inflammation and fibrosis. The body has formed a complex oxidative stress response system. When exposed to electrophilic agents or reactive oxygen species, it can induce a series of protective proteins to alleviate the damage to cells.

[0005] Oxidative stress, particularly mitochondrial oxidative stress, can lead to a vicious cycle that directly damages cells. The retina's constant exposure to reactive oxygen species (ROS) causes oxidative stress, inflammation, and neuronal degeneration, which are also associated with retinal diseases. Increasing evidence suggests that oxidative stress plays a crucial role in the accumulation of retinal diseases.

[0006] Nuclear factor erythroid 2-associated factor 2 (Nrf2) is a redox-sensitive transcription factor observed in many tissues, particularly those exposed to the external environment (skin, lungs, and gastrointestinal tract) and tissues involved in detoxification (liver and kidneys). At rest, Nrf2 is captured in the cytosol by the adaptor protein Keap1 and rapidly degraded via the proteasome pathway. Under oxidative stress, Nrf2 can enter the nucleus, bind to the antioxidant response element ARE, and initiate the transcription and expression of genes encoding numerous antioxidant enzymes and phase II detoxification enzymes. Nrf2 targets more than 250 genes, including NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), glutamate-cysteine ​​ligase, glutathione S-transferase, glutathione peroxidase, catalase, superoxide dismutase, and thioredoxin UDP-glucuronyltransferase, among others. Keap1-Nrf2 signaling activates the expression of these downstream genes, thereby responding to various stimuli (such as active toxins, pro-inflammatory factors, apoptosis, and carcinogenesis), rescuing tissue cells, and reducing damage. Activation of Nrf2 function is one of the key defense mechanisms against oxidative stress in many species. The well-known Nrf2 activator methylbardoxolone and its derivatives have been the subject of numerous clinical trials, including those aimed at treating chronic kidney disease, pulmonary hypertension, and mitochondrial myopathy. Recent studies have shown that Nrf2 activation can protect the retina from retinal diseases. In particular, this finding is supported by the discovery that Nrf2 knockout mice exhibit age-related retinal degeneration.

[0007] Nrf2 contains seven highly conserved structural and functional domains, Neh1–Neh6 (Nrf2-ECH homology). The Neh1 region contains a bZIP structure. When Nrf2 translocates to the nucleus, bZIP forms a heterodimer with small Maf proteins in the nucleus, enabling Nrf2 to recognize and bind to AREs, thereby initiating the transcription of downstream related genes. The Neh2 domain binds to Keap1 protein (Kelch-like ECH-associated protein-1) and is then ubiquitinated and degraded in the cytoplasm. Neh3, located at the C-terminus, can bind to CHD6 (a chromo-ATPase / helicase DNA-binding protein), promoting the regulation of related gene transcription by antioxidant response elements (AREs). Neh4 and Neh5 are domains involved in initiating downstream gene transcription. When Nrf2 translocates into the nucleus, it binds to ARE in the form of Nrf2-Maf, but transcription cannot be initiated at this stage. Neh4, Neh5, and CREB are required to activate transcription. The Neh6 region is rich in serine residues and is a regulatory region for Nrf2 degradation that is not KEAP1-dependent. Neh7 binds to the RXRa nuclear receptor factor, inhibiting Nrf2 transcriptional activity. We deleted the domains that inhibit Nrf2 activity and those responsible for Nrf2 degradation, while retaining the functional domains that activate downstream genes, and constructed them into a double-stranded AAV vector. The scAAV-Nrf2 mutant (miniNrf2) showed higher efficiency and stronger activation of the transcription and expression of downstream antioxidant and anti-inflammatory genes compared to single-stranded AAV, and could be used to treat dry-AMD and other chronic ophthalmic diseases related to antioxidants and anti-inflammation.

[0008] Adeno-associated virus (AAV) belongs to the genus *Dependent Virus* of the family Parvoviridae. It is a single-stranded DNA-deficient virus with a genomic DNA of less than 5 kb, no envelope, and a naked icosahedral particle shape. AAV can infect humans and some other primates. AAV vectors are genetically engineered vectors that can be used for artificial transgenic expression, utilizing certain characteristics of naturally occurring AAV. They have no potential pathogenicity and exhibit good infectivity in skeletal muscle, retina, hepatocytes, cardiac smooth muscle cells, neurons, pancreatic B cells, and synovial cells, with extremely high in vivo infection efficiency. At least a dozen serotypes of AAV have been identified, primarily differing in their capsid protein (Cap), leading to varying infection efficiencies in different tissues and cells.

[0009] Recombinant adeno-associated virus (rAAV) is a widely used tool for gene transduction and gene therapy, capable of transducing exogenous genes into dividing and terminally differentiated cells and maintaining relatively long-term gene expression. Due to its safety, low immunogenicity, and non-pathogenicity, rAAV is favored and has been widely used as an ideal vector for in vivo research and gene therapy. Adeno-associated virus is a single-stranded DNA virus (ssAAV), and its single-stranded genome must first be converted into a transcriptionally active double-stranded form before expression can begin. This process limits AAV vector-mediated gene transduction and directly affects viral infection efficiency. Summary of the Invention

[0010] In view of this, the present invention provides a nucleotide sequence encoding the human nuclear factor E2-associated factor 2 (Nrf2) protein, which can be used to effectively treat ophthalmic diseases.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0012] This invention provides a nucleic acid molecule having:

[0013] (I) A nucleotide sequence as shown in any of SEQ ID No. 1, 2 or 3; or

[0014] (II) A complementary nucleotide sequence to any of the nucleotide sequences shown in SEQ ID No. 1, 2 or 3; or

[0015] (III) A nucleotide sequence that has at least 95% sequence identity with the nucleotide sequence described in (I) or (II).

[0016] In some specific embodiments of the present invention, the nucleic acid molecule has a nucleotide sequence that is at least 98% sequence identical to the nucleotide sequence of (I) or (II).

[0017] The present invention also provides polypeptides or proteins encoded by the aforementioned nucleic acid molecules.

[0018] The present invention also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0019] In some specific embodiments of the present invention, the expression vector is a viral vector, including a double-stranded adeno-associated virus vector.

[0020] In some specific embodiments of the present invention, the serotype of the viral vector is selected from AAV2, AAV5, AAV7, or AAV8 or a combination thereof.

[0021] In some specific embodiments of the present invention, the double-stranded adeno-associated virus vector further includes a promoter.

[0022] In some specific embodiments of the present invention, the promoter includes one or more of SYN, CMV, or CAG.

[0023] Based on the above research, the present invention also provides the application of the nucleic acid molecule, the polypeptide or protein, and the expression vector in the preparation of medicaments for the prevention and / or treatment of optic nerve injury-related diseases.

[0024] In some specific embodiments of the present invention, the optic nerve injury-related diseases include, but are not limited to, autosomal dominant optic atrophy (DOA), Leber's hereditary optic neuropathy (LHON), ischemic optic neuropathy, or glaucoma.

[0025] In some specific embodiments of the present invention, the optic nerve injury-related disease is acute optic nerve injury or chronic optic nerve injury.

[0026] In some specific embodiments of the present invention, the activities of the nucleic acid molecule, the polypeptide or protein, and the expression vector include, but are not limited to, any of the following:

[0027] (1) Inducing stable high expression of the Nrf2 gene in retinal ganglion cells; and / or

[0028] (2) Long-term treatment of optic nerve injury-related diseases; and / or

[0029] (3) Activate cellular anti-inflammatory or antioxidant responses; and / or

[0030] (4) Increase the survival rate of ganglion cells; and / or

[0031] (5) Preventing or delaying ganglion cell apoptosis; and / or

[0032] (6) Eliminate free radicals in diseased cells.

[0033] More importantly, the present invention also provides a medicament comprising the nucleic acid molecule, the polypeptide or protein, the expression vector, and pharmaceutically acceptable excipients.

[0034] This invention also provides a drug combination comprising the nucleic acid molecule, the polypeptide or protein, the expression vector, and any other active ingredients. Drug combinations prepared by those skilled in the art based on the drugs provided by this invention and combined with any other active ingredients are all within the scope of protection of this invention.

[0035] The present invention also provides a pharmaceutical formulation comprising the nucleic acid molecule, the polypeptide or protein, the expression vector, and a pharmaceutically acceptable carrier or excipient.

[0036] In some specific embodiments of the present invention, the pharmaceutical preparation includes a liquid preparation.

[0037] In addition, the present invention also provides a method for drug delivery, wherein the drug, the drug combination, or the drug formulation is applied to the eye via intravitreal administration.

[0038] The present invention also provides a treatment method for optic nerve-related diseases, including the administration of the drug, the drug combination, or the drug formulation.

[0039] In some specific embodiments of the present invention, the optic nerve injury-related diseases include, but are not limited to, autosomal dominant optic atrophy (DOA), Leber's hereditary optic neuropathy (LHON), ischemic optic neuropathy, or glaucoma.

[0040] In some specific embodiments of the present invention, the optic nerve injury-related diseases include acute optic nerve injury or chronic optic nerve injury.

[0041] In some specific embodiments of the present invention, the activities of the drug, the drug combination, and the drug formulation include, but are not limited to, any of the following:

[0042] (1) Inducing stable high expression of the Nrf2 gene in retinal ganglion cells; and / or

[0043] (2) Long-term treatment of optic nerve injury-related diseases; and / or

[0044] (3) Activate cellular anti-inflammatory or antioxidant responses; and / or

[0045] (4) Increase the survival rate of ganglion cells; and / or

[0046] (5) Preventing or delaying ganglion cell apoptosis; and / or

[0047] (6) Eliminate free radicals in diseased cells.

[0048] Adeno-associated viruses (AAVs) are single-stranded DNA viruses (ssAAVs). They must first complete the conversion of their single-stranded genome into a transcriptionally active double-stranded form before expression can begin. This process limits AAV vector-mediated gene transduction and directly affects viral infection efficiency. Self-complementary double-stranded DNA AAVs (scAAVs) overcome this limitation, eliminating the need for a single-strand to double-strand conversion process. In other words, double-stranded AAVs can be directly expressed after entering cells, with faster expression times and higher expression levels.

[0049] This invention discloses an expression vector for human nuclear factor E2-related factor 2 (Nrf2) protein and its applications. Specifically, this invention discloses a nucleotide sequence encoding Nrf2 protein, which contains the nucleic acid encoding Nrf2 protein. A recombinant expression vector is further disclosed, which contains the aforementioned nucleic acid. The nucleic acid encoding Nrf2 protein described in this invention expresses the protein more quickly and at a higher level, thus providing better treatment for optic nerve-related diseases. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0051] Figure 1 The image shows the plasmid electrophoresis pattern. In the electrophoresis pattern, numbers 1-5 represent pscAAV-mini Nrf2-1, numbers 6-10 represent pscAAV-mini Nrf2-2, and numbers 11-15 represent pscAAV-mini Nrf2-3.

[0052] Figure 2 The image shows plasmid restriction enzyme digestion. In the electrophoresis diagram, lanes 1, 2, and 3 are pscAAV-mini Nrf2-1 after restriction enzyme digestion; lanes 4, 5, and 6 are pscAAV-mini Nrf2-2 after restriction enzyme digestion; and lanes 7, 8, and 9 are pscAAV-mini Nrf2-3 after restriction enzyme digestion. From top to bottom, the images show the undigested circular plasmid, the digested linear plasmid, and the target band after digestion.

[0053] Figure 3(a) shows the characteristic spectrum of psAAV-CMV-miniNrf2-1 plasmid; Figure 3(b) shows the characteristic spectrum of psAAV-CMV-miniNrf2-2 plasmid; Figure 3(c) shows the characteristic spectrum of psAAV-CMV-miniNrf2-3 plasmid.

[0054] Plasmid map and start and end sites of each element: including promoter, gene sequence, and viral packaging ITR sequence;

[0055] Figure 4 The diagram shows the protein expression detection after transfection of HEK293 cells with the constructed vector. Lanes 1-5 represent the protein expression detection (40X) after transfection of HEK293 cells with blank cells, pscAAV-GFP, pscAAV-mini Nrf2-1, pscAAV-mini Nrf2-2, and pscAAV-mini Nrf2-3 plasmids, respectively.

[0056] Figure 5 Subcellular localization map;

[0057] Figure 6a This indicates that plasmid transfection regulates the expression level of the downstream gene HO-1. Figure 6b The plasmid transfection regulates the expression of the downstream gene NQO1;

[0058] Figure 7 The expression of mini-Nrf21-3 in mouse retinal RGC cells is shown; among them, Figure 7 a shows the expression of mini-Nrf2-1 in mouse retinal RGC cells; Figure 7 b shows the expression of mini-Nrf2-2 in mouse retinal RGC cells; Figure 7 c shows the expression of mini-Nrf2-3 in mouse retinal RGC cells;

[0059] Figure 8(a) shows the activation of transcription of the downstream target gene NQO1 in mice after viral infection; Figure 8(b) shows the activation of transcription of the downstream target gene HO-1 in mice after viral infection. Detailed Implementation

[0060] This invention discloses a nucleic acid molecule encoding human nuclear factor E2-related factor 2, its expression vector, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0061] In view of the shortcomings of the prior art, the first aspect of the present invention provides a nucleotide sequence encoding the human nuclear factor E2-related factor 2 (Nrf2) protein, which can be used to effectively treat ophthalmic diseases.

[0062] Preferably, the nucleotide sequence encoding the Nrf2 protein is one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0063] More preferably, the sequence is selected from sequences that have ≥95% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0064] More preferably, the sequence is selected from sequences that have ≥98% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0065] More preferably, the sequence is selected from sequences that have ≥99% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0066] In this invention:

[0067] Sequence 1. mini Nrf2-1 (as shown in SEQ ID No. 1)

[0068]

[0069] Sequence 2. mini Nrf2-2 (as shown in SEQ ID No. 2)

[0070]

[0071]

[0072] In a second aspect, the present invention provides an adeno-associated virus vector encoding the human nuclear factor E2-associated factor 2 (Nrf2) protein.

[0073] Specifically, the present invention provides an adeno-associated virus vector capable of long-term treatment of optic nerve injury-related diseases, wherein the adeno-associated virus vector includes a sequence of promoter expressing Nrf2 protein.

[0074] Preferably, the promoter is one of CAG, SYN, or CMV.

[0075] More preferably, the promoter is selected from CMV.

[0076] More preferably, the plasmid map of the vector is shown in Figure 3.

[0077] In a third aspect, the present invention provides the use of any of the aforementioned nucleotides, adeno-associated virus vectors, and plasmids in the treatment of acute or chronic optic nerve-related diseases; preferably, the diseases are DOA, LHON, ischemic optic neuropathy, and glaucoma, etc.

[0078] In a fourth aspect, the present invention provides a pharmaceutical formulation comprising the aforementioned viral vector and a pharmaceutically acceptable carrier or excipient; preferably, the pharmaceutical formulation is a liquid formulation.

[0079] A fifth aspect of the invention provides a method for delivering the above-described pharmaceutical preparation, wherein the pharmaceutical preparation is injected intraocularly. More preferably, the intraocular injection is an intravitreal injection.

[0080] More preferably, the disease is an eye disease.

[0081] More preferably, the eye disease is an optic nerve injury-related disease, such as acute optic nerve injury or chronic optic nerve injury.

[0082] More preferably, the optic nerve injury-related disease is autosomal dominant optic atrophy (DOA), Leber's hereditary optic neuropathy (LHON), ischemic optic neuropathy, or glaucoma.

[0083] The aforementioned vector or drug formulation can induce stable high expression of the Nrf2 gene in retinal ganglion cells.

[0084] The aforementioned carrier or drug formulation can provide long-term treatment for diseases related to optic nerve damage.

[0085] The carrier or drug formulation described herein can activate cellular anti-inflammatory or antioxidant responses.

[0086] The aforementioned carrier or drug formulation can increase the survival rate of ganglion cells.

[0087] The aforementioned carrier or drug formulation can prevent or delay apoptosis of ganglion cells.

[0088] The carrier or drug preparation can eliminate free radicals in diseased cells.

[0089] This invention provides an expression vector for human nuclear factor E2-related factor 2 (Nrf2) protein and its application. Specifically, this invention discloses a nucleotide sequence encoding Nrf2 protein, which contains the nucleic acid encoding Nrf2 protein. A recombinant expression vector is further disclosed, which contains the aforementioned nucleic acid. The nucleic acid encoding Nrf2 protein described in this invention expresses the protein more quickly and at a higher level, thus providing better treatment for optic nerve-related diseases.

[0090] The nucleic acid molecule encoding human nuclear factor E2-related factor 2, the expression vector, and their applications provided by this invention all use commercially available raw materials and reagents.

[0091] The present invention will be further illustrated below with reference to the embodiments:

[0092] Example 1. Vector Construction

[0093] Using the existing vector pAAV-nrf2 as a template, a first round of PCR was performed using primeSTAR GXL premix high-fidelity amplification enzyme to amplify the intermediate target fragment. After PCR, the PCR products were subjected to agarose gel electrophoresis, and the target fragment was recovered according to the size of the target product. Using the first round of PCR products as a template, a second round of nested PCR was performed to amplify the target fragment. The PCR products were subjected to agarose gel electrophoresis, and the target fragment was recovered. The target product was digested with the target vector, and the digested target band was recovered by electrophoresis. The target fragment and vector were ligated using T4 ligase and incubated overnight at 37°C. The ligation product was transformed into stbl3 competent cells, plated, cultured overnight, and single colonies were picked and cultured by shaking for plasmid extraction. The extracted plasmids were identified by enzyme digestion, and the correctly digested plasmids were sent to a sequencing company for sequencing identification.

[0094] Table 1: PCR primers

[0095]

[0096] PCR amplification of miniNrf2-1 reaction system

[0097]

[0098] PCR computer operation procedure:

[0099]

[0100] PCR amplification of miniNrf2-2 reaction system

[0101]

[0102]

[0103] PCR computer operation procedure:

[0104]

[0105] PCR amplification of miniNrf2-3 reaction system

[0106]

[0107]

[0108]

[0109] PCR computer operation procedure:

[0110]

[0111] Primers are shown in Table 1. Vector construction and electrophoresis are as follows: Figure 1 As shown, the constructed vector was identified by enzyme digestion. Figure 2 As shown in Figure 3, the vector spectrum is as follows.

[0112] Figure 1 The pscAAV-mini Nrf2-1 shows a value of approximately 5kD, the pscAAV-mini Nrf2-2 shows approximately 5kD, and the pscAAV-mini Nrf2-3 shows approximately 4.7kD, which matches the theoretical size. Figure 2 After enzyme digestion, the size met expectations. Combined with sequencing results, the vector construction was confirmed to be correct.

[0113] Example 2. Expression and functional detection of the vector in in vitro experiments.

[0114] 2.1 Vector Expression

[0115] The expression of the vector was detected by Western blot. Six-well plates were divided into eight groups, and each group was seeded with 5 × 10⁶ cells / well. 5 Cells were seeded in each well, and the cell count was 1 × 10⁻⁶ cells 24 hours later. 6Each well was transfected with 3 μg of plasmid. After 48 hours of culture, HEK293 cells from different experimental groups were collected, the culture medium was removed, and the cells were washed twice with PBS. 150 μL of lysis buffer was added to each well, and the cells were placed on ice for complete lysis. The cell lysis buffer was collected, centrifuged at 10,000-14,000 g at 4°C for 3-5 minutes, and the supernatant was collected. After determining the protein concentration by BCA method, the loading amount for the experimental and control groups was calculated based on a total protein of 50 μg. SDS-PAGE gel electrophoresis and Western blot were performed. After incubation with primary antibody (anti-flag 1:2000) and secondary antibody (anti-rabbit HRP 1:10000), chemiluminescence imaging was performed.

[0116] The results are as follows Figure 4 As shown, the protein sizes expressed by pscAAV-mini Nrf2-1, pscAAV-mini Nrf2-2, and pscAAV-mini Nrf2-3 are consistent with theoretical values, and all three vectors exhibit significant expression. This indicates that the three vectors were successfully constructed and can express the target proteins in large quantities.

[0117] 2.2 Cell localization

[0118] The nuclear localization of the vector was detected by immunofluorescence staining. HEK293 cells were digested and seeded into 6-well plates at a ratio of 4*10^5 cells / well. After overnight culture, 3 μg of plasmid / well was transfected with PEI, resulting in four groups: blank, pAAV-GFP, pAAV-dN2, pAAV-dN2N6, and pAAV-dN2N6N7. Cells were harvested 30 hours after transfection. The culture medium was discarded, and the cells were washed twice with PBS. 1 ml of 4% PFA fixative was added to each well, and the cells were fixed at room temperature for 10 min. The fixative was discarded, leaving a small amount of PBS in the 6-well plate, and the cells were temporarily stored at 4°C. Cell slides were transferred to 24-well plates, washed with PBS, and punched with 1% Triton for 15 min, then blocked at room temperature for 2 hours. Primary antibody (anti-flag 1:200) was added, and the cells were incubated overnight at 4°C. Secondary antibody was added, and the cells were incubated at room temperature for 1 hour. DAPI staining solution was added, and the slides were mounted and observed under a fluorescence microscope. The results are as follows. Figure 5 As shown,

[0119] The blank control group and the pAAV-CAG-GFP group did not express the target protein. However, cells transfected with pscAAV-mini Nrf2-1, pscAAV-mini Nrf2-2, and pscAAV-mini Nrf2-3 groups could specifically express the target protein. The target protein was clearly aggregated in the cell nucleus, indicating that the vector normally expressed the protein and that the target protein was located in the cell nucleus.

[0120] 2.3 qPCR detection of the function of each Nrf2 mutant

[0121] pAAV-Nrf2 mutant plasmid was transfected into HEK293 cells (1.5ug, 3ug). RNA was extracted from the cells 31 hours after transfection, and qPCR was used to detect the mRNA levels of Nrf2 downstream target genes NQO-1 and HO-1. Results are as follows: Figure 6a , Figure 6b As shown.

[0122] Figure 6a In this study, HO-1 is a downstream target gene of the Nrf2 gene. After transfection of cells with pscAAV-mini Nrf2-1, pscAAV-miniNrf2-2, and pscAAV-mini Nrf2-3, HO-1 was expressed to varying degrees, with the miniNrf2-2 vector showing the strongest downstream activation effect. Figure 6b The downstream NQO1 gene of Nrf2 is also expressed to varying degrees.

[0123] Based on the above experimental results, mini Nrf2-1, 2, and 3 can all express the target protein well and enter the cell nucleus to regulate the expression of downstream target genes, and their functions are stronger than those of the full-length Nrf2 vector.

[0124] Example 3. Virus Preparation

[0125] The three-plasmid system (pAAV2-, pHelper, pRC) was used to transfect HEK293 cells when the confluence was 85% (PEI transfection reagent was used for transfection). A total of 30 micrograms of plasmids (the three plasmids in a 10:10:10 ratio) were transfected into 10 cm culture dishes. The medium was not changed on the day of transfection, but was changed to 5% FBS DMEM the next day. Cells were harvested after 72 hours. They were placed in cryovials and subjected to three freeze-thaw cycles at -70°C and 37°C, each lasting at least 10 minutes. After each thaw, the cells were vigorously vortexed for 2 minutes. Then, the cells were centrifuged at 14,000 rpm for 5 minutes, and the supernatant was collected. The virus was purified by gradient centrifugation with cesium chloride solution.

[0126] The physical titer of the virus was detected using quantitative real-time PCR. SYBR II (Takara), target fragment primers (20 μM), and the target plasmid used to package the virus were used for accurate quantification. The virus was then detected quantitatively using real-time PCR. The PCR reaction conditions in an eight-tube Bio-Rad were: pre-denaturation: 95℃ for 10 minutes; cycling: 95℃ for 15 seconds, 60℃ for 1 minute. The final determined genomic titer of the viral vector described in this application was 1 × 10⁻⁶. 13 vg / mL.

[0127] Example 4: In vivo retinal expression and functional detection of scAAV-mini Nrf1-1, -2, and -3

[0128] 1. Detection of expression in mouse retina

[0129] Mice were administered 1E10 vg / eye via intravitreal injection. Four weeks later, mice were euthanized by spinal dislocation, and eyeballs were fixed with 4% PFA. After OCT embedding, frozen sections were prepared. The sections were punched in 1% Triton X-100 for 15 min, blocked for 30 min, and subjected to antigen retrieval. Primary antibody (anti-flag 1:200) was incubated overnight at 4°C, followed by secondary antibody (anti-rabbit Alexa594 1:1000) incubated at room temperature for 2 hours. The expression and distribution of mini Nrf protein on the retina were observed. Figure 7 The results showed that the mini Nrf21-3 protein was mainly expressed in retinal RGC cells.

[0130] 2. Activation of downstream target genes in mice

[0131] Mice were administered 1E10 vg / eye via intravitreal injection. Four weeks after administration, the mice were sacrificed by spinal dislocation, and the eyeballs and retinas were removed. Total RNA was extracted using the TRIZOL method, and cDNA was reverse transcribed. The expression of NQO1 and HO-1 genes downstream of the NRF2 transcription factor was detected using qPCR.

[0132] As shown in Figure 8, mini Nrf2-1, -2, and -3 can all activate the expression of NQO1 (as shown in Figure 8a) and HO-1 (as shown in Figure 8b) downstream target genes of Nrf2 in mice, demonstrating that these vectors are also functional in vivo.

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Wuhan NewFos Biotechnology Co., Ltd. <120> Nucleic acid molecules, expression vectors and applications encoding human nuclear factor E2-related factor 2 <130> MP21016163 <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1503 <212> DNA <213> Artificial Sequence <400> 1 ggatccgcca ccatggatgc tttgtacttt gatgactgca tgcagctttt ggcgcagaca 60 ttcccgtttg tagatgacaa tgaggtttct tcggctacgt ttcagtcact tgttcctgat 120 attcccggtc acatcgagag cccagtcttc attgctacta atcaggctca gtcacctgaa 180 acttctgttg ctcaggtagc ccctgttgat ttagacggta tgcaacagga cattgagcaa 240 gtttgggagg agctattatc cattcctgag ttacagtgtc ttaatattga aaatgacaag 300 ctggttgaga ctaccatggt tccaagtcca gaagccaaac tgacagaagt tgacaattat 360 catttttact catctatacc ctcaatggaa aaagaagtag gtaactgtag tccacatttt 420 cttaatgctt ttgaggattc cttcagcagc atcctctcca cagaagaccc caaccagttg 480 acagtgaact cattaaattc agatgccaca gtcaacacag attttggtga tgaattttat 540 tctgctttca tagctgagcc cagtatcagc aacagcatgc cctcacctgc tactttaagc 600 cattcactct ctgaacttct aaatgggccc attgatgttt ctgatctatc actttgcaaa 660 gctttcaacc aaaaccaccc tgaaagcaca gcagaattca atgattctga ctccggcatt 720 780. tcactaaaca caagtcccag tgtggcatca ccagaacact cagtggaatc ttccagctat ggagacacac tacttggcct cagtgattct gagtggaag agctagatag tgcccctgga 840 agtgtcaaac agaatggtcc taaaacacca gtacattctt ctggggatat ggtacaaccc ttgtcaccat ctcaggggca gagcactcac gtgcatgatg cccaatgtga gaacacacca gagaagaat tgcctgtaag tcctggtcat cggaaaaccc cattcacaaa agacaaacat tcaagccgct tggaggctca tctcacaaga gatgaactta gggcaaaagc tctccatatc ccattccctg tagaaaaaat cattaacctc cctgttgttg acttcaacga aatgatgtcc aaagagcagt tcaatgaagc tcaacttgca ttaattcggg atatacgtag gaggggtag aataaagtgg ctgctcagaa ttgcagaaaa agaaaactgg aaaatatagt agaactagag caagatttag atcatttgaa agatgaaaaa gaaaattgc tcaaagaaaa aggagaaaat gacaaaagcc ttcacctact gaaaaaacaa ctcagcacct father agttttcagc atgctacgtg atgaagatgg aaaaccttat tctcctagtg aatactccct gcagcaaaca agagatggca atgttttcct tgttcccaaa agtaagaagc cagatgttaa gaaaaacctc 1500 gag 1503 <210> 2 <211> 1356 <212> DNA <213> Artificial Sequence <400> 2 ggatccgcca ccatggccca gcacatccag tcagaaacca gtggatctgc caactactcc 60 caggttgccc acattcccaa atcagatgct ttgtactttg atgactgcat gcagcttttg 120 gcgcagacat tcccgtttgt agatgacaat gaggtttctt cggctacgtt tcagtcactt 180 gttcctgata ttcccggtca catcgagagc ccagtcttca ttgctactaa tcaggctcag 240 tcacctgaaa cttctgttgc tcaggtagcc cctgttgatt tagacggtat gcaacaggac 300 attgagcaag tttgggagga gctattatcc attcctgagt tacagtgtct taatattgaa 360 aatgacaagc tggttgagac taccatggtt ccaagtccag aagccaaact gacagaagtt 420 gacaattatc atttttactc atctataccc tcaatggaaa aagaagtagg taactgtagt 480 ccacattttc ttaatgcttt tgaggattcc ttcagcagca tcctctccac agaagacccc 540 aaccagttga cagtgaactc attaaattca gatgccacag tcaacacaga ttttggtgat 600 gaattttat ctgctttcat agctgagccc agtatcagca acagcatgcc ctcacctgct 660 actttaagcc attcactctc tgaacttcta aatgggccca aacagaatgg tcctaaaaca 720 ccagtacatt cttctgggga tatggtacaa cccttgtcac catctcaggg gcagagcact 780 cacgtgcatg atgcccaatg tgagaacaca ccagagaaag aattgcctgt aagtcctggt 840 catcggaaaa ccccattcac aaaagacaaa cattcaagcc gcttggaggc tcatctcaca 900 agagatgaac ttagggcaaa agctctccat atcccattcc ctgtagaaaa aatcattaac 960 ctccctgttg ttgacttcaa cgaaatgatg tccaaagagc agttcaatga agctcaactt 1020 gcattaattc gggatatacg taggaggggt aagaataaag tggctgctca gaattgcaga 1080 aaaagaaaac tggaaaatat agtagaacta gagcaagatt tagatcattt gaaagatgaa 1140 aaagaaaaat tgctcaaaga aaaaggagaa aatgacaaaa gccttcacct actgaaaaaa 1200 caactcagca ccttatatct cgaagttttc agcatgctac gtgatgaaga tggaaaacct 1260 tattctccta gtgaatactc cctgcagcaa acaagagatg gcaatgtttt ccttgttccc 1320 aaaagtaaga agccagatgt taagaaaaac ctcgag 1356 <210> 3 <211> 1032 <212> DNA <213> Artificial Sequence <400> 3 ggatccgcca ccatggccca gcacatccag tcagaaacca gtggatctgc caactactcc 60 caggttgccc acattcccaa atcagatgct ttgtactttg atgactgcat gcagcttttg 120 gcgcagacat tcccgtttgt agatgacaat gaggtttctt cggctacgtt tcagtcactt 180 gttcctgata ttcccggtca catcgagagc ccagtcttca ttgctactaa tcaggctcag 240 tcacctgaaa cttctgttgc tcaggtagcc cctgttgatt tagacggtat gcaacaggac 300 attgagcaag tttgggagga gctattatcc attcctgagt tacagtgtct taatattgaa 360 aatgacaagc tggttaaaca gaatggtcct aaaacaccag tacattcttc tggggatatg 420 gtacaaccct tgtcaccatc tcaggggcag agcactcacg tgcatgatgc ccaatgtgag 480 aacacaccag agaaagaatt gcctgtaagt cctggtcatc ggaaaacccc attcacaaaa 540 gacaaacatt caagccgctt ggaggctcat ctcacaagag atgaacttag ggcaaaagct 600 ctccatatcc cattccctgt agaaaaaatc attaacctcc ctgttgttga cttcaacgaa 660 atgatgtcca aagagcagtt caatgaagct caacttgcat taattcggga tatacgtagg 720 aggggtaaga ataaagtggc tgctcagaat tgcagaaaaa gaaaactgga aaatatagta 780 gaactagagc aagatttaga tcatttgaaa gatgaaaaag aaaaattgct caaagaaaaa 840 ggagaaaatg acaaaagcct tcacctactg aaaaaacaac tcagcacctt atatctcgaa 900 gttttcagca tgctacgtga tgaagatgga aaaccttatt ctcctagtga atactccctg 960 cagcaaacaa gagatggcaa tgttttcctt gttcccaaaa gtaagaagcc agatgttaag 1020 aaaaacctcg ag 1032 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <400> 4 agctggatcc gccaccatgg atgctttgta ctttgatgac tgcatg 46 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <400> 5 atcgctcgag gtttttctta acatctggct tcttactttt gg 42 <210> 6 <211> 38 <212> DNA <213> Artificial Sequence <400> 6 agctggatcc gccaccatgg cccagcacat ccagtcag 38 <210> 7 <211> 49 <212> DNA <213> Artificial Sequence <400> 7 ctggtgtttt aggaccattc tgtttgggcc catttagaag ttcagagag 49 <210> 8 <211> 49 <212> DNA <213> Artificial Sequence <400> 8 ctctctgaac ttctaaatgg gcccaaacag aatggtccta aaacaccag 49 <210> 9 <211> 54 <212> DNA <213> Artificial Sequence <400> 9 ctggtgtttt aggaccattc tgtttaacca gcttgtcatt ttcaatatta agac 54 <210> 10 <211> 54 <212> DNA <213> Artificial Sequence <400> 10 gtcttaatat tgaaaatgac aagctggtta aacagaatgg tcctaaaaca ccag 54 <210> 11 <211> 501 <212> PRT <213> Artificial Sequence <400> 11 Gly Ser Ala Thr Met Asp Ala Leu Tyr Phe Asp Asp Cys Met Gln Leu 1 5 10 15 Leu Ala Gln Thr Phe Pro Phe Val Asp Asp Asn Glu Val Ser Ser Ala 20 25 30 Thr Phe Gln Ser Leu Val Pro Asp Ile Pro Gly His Ile Glu Ser Pro 35 40 45 Val Phe Ile Ala Thr Asn Gln Ala Gln Ser Pro Glu Thr Ser Val Ala 50 55 60 Gln Val Ala Pro Val Asp Leu Asp Gly Met Gln Gln Asp Ile Glu Gln 65 70 75 80 Val Trp Glu Glu Leu Leu Ser Ile Pro Glu Leu Gln Cys Leu Asn Ile 85 90 95 Glu Asn Asp Lys Leu Val Glu Thr Thr Met Val Pro Ser Pro Glu Ala 100 105 110 Lys Leu Thr Glu Val Asp Asn Tyr His Phe Tyr Ser Ser Ile Pro Ser 115 120 125 Met Glu Lys Glu Val Gly Asn Cys Ser Pro His Phe Leu Asn Ala Phe 130 135 140 Glu Asp Ser Phe Ser Ser Ile Leu Ser Thr Glu Asp Pro Asn Gln Leu 145 150 155 160 Thr Val Asn Ser Leu Asn Ser Asp Ala Thr Val Asn Thr Asp Phe Gly 165 170 175 Asp Glu Phe Tyr Ser Ala Phe Ile Ala Glu Pro Ser Ile Ser Asn Ser 180 185 190 Met Pro Ser Pro Ala Thr Leu Ser His Ser Leu Ser Glu Leu Leu Asn 195 200 205 Gly Pro Ile Asp Val Ser Asp Leu Ser Leu Cys Lys Ala Phe Asn Gln 210 215 220 Asn His Pro Glu Ser Thr Ala Glu Phe Asn Asp Ser Asp Ser Gly Ile 225 230 235 240 Ser Leu Asn Thr Ser Pro Ser Val Ala Ser Pro Glu His Ser Val Glu 245 250 255 Ser Ser Ser Tyr Gly Asp Thr Leu Leu Gly Leu Ser Asp Ser Glu Val 260 265 270 Glu Glu Leu Asp Ser Ala Pro Gly Ser Val Lys Gln Asn Gly Pro Lys 275 280 285 Thr Pro Val His Ser Ser Gly Asp Met Val Gln Pro Leu Ser Pro Ser 290 295 300 Gln Gly Gln Ser Thr His Val His Asp Ala Gln Cys Glu Asn Thr Pro 305 310 315 320 Glu Lys Glu Leu Pro Val Ser Pro Gly His Arg Lys Thr Pro Phe Thr 325 330 335 Lys Asp Lys His Ser Ser Arg Leu Glu Ala His Leu Thr Arg Asp Glu 340 345 350 Leu Arg Ala Lys Ala Leu His Ile Pro Phe Pro Val Glu Lys Ile Ile 355 360 365 Asn Leu Pro Val Val Asp Phe Asn Glu Met Met Ser Lys Glu Gln Phe 370 375 380 Asn Glu Ala Gln Leu Ala Leu Ile Arg Asp Ile Arg Arg Arg Gly Lys 385 390 395 400 Asn Lys Val Ala Ala Gln Asn Cys Arg Lys Arg Lys Leu Glu Asn Ile 405 410 415 Val Glu Leu Glu Gln Asp Leu Asp His Leu Lys Asp Glu Lys Glu Lys 420 425 430 Leu Leu Lys Glu Lys Gly Glu Asn Asp Lys Ser Leu His Leu Leu Lys 435 440 445 Lys Gln Leu Ser Thr Leu Tyr Leu Glu Val Phe Ser Met Leu Arg Asp 450 455 460 Glu Asp Gly Lys Pro Tyr Ser Pro Ser Glu Tyr Ser Leu Gln Gln Thr 465 470 475 480 Arg Asp Gly Asn Val Phe Leu Val Pro Lys Ser Lys Lys Pro Asp Val 485 490 495 Lys Lys Asn Leu Glu 500 <210> 12 <211> 452 <212> PRT <213> Artificial Sequence <400> 12 Gly Ser Ala Thr Met Ala Gln His Ile Gln Ser Glu Thr Ser Gly Ser 1 5 10 15 Ala Asn Tyr Ser Gln Val Ala His Ile Pro Lys Ser Asp Ala Leu Tyr 20 25 30 Phe Asp Asp Cys Met Gln Leu Leu Ala Gln Thr Phe Pro Phe Val Asp 35 40 45 Asp Asn Glu Val Ser Ser Ala Thr Phe Gln Ser Leu Val Pro Asp Ile 50 55 60 Pro Gly His Ile Glu Ser Pro Val Phe Ile Ala Thr Asn Gln Ala Gln 65 70 75 80 Ser Pro Glu Thr Ser Val Ala Gln Val Ala Pro Val Asp Leu Asp Gly 85 90 95 Met Gln Gln Asp Ile Glu Gln Val Trp Glu Glu Leu Leu Ser Ile Pro 100 105 110 Glu Leu Gln Cys Leu Asn Ile Glu Asn Asp Lys Leu Val Glu Thr Thr 115 120 125 Met Val Pro Ser Pro Glu Ala Lys Leu Thr Glu Val Asp Asn Tyr His 130 135 140 Phe Tyr Ser Ser Ile Pro Ser Met Glu Lys Glu Val Gly Asn Cys Ser 145 150 155 160 Pro His Phe Leu Asn Ala Phe Glu Asp Ser Phe Ser Ser Ile Leu Ser 165 170 175 Thr Glu Asp Pro Asn Gln Leu Thr Val Asn Ser Leu Asn Ser Asp Ala 180 185 190 Thr Val Asn Thr Asp Phe Gly Asp Glu Phe Tyr Ser Ala Phe Ile Ala 195 200 205 Glu Pro Ser Ile Ser Asn Ser Met Pro Ser Pro Ala Thr Leu Ser His 210 215 220 Ser Leu Ser Glu Leu Leu Asn Gly Pro Lys Gln Asn Gly Pro Lys Thr 225 230 235 240 Pro Val His Ser Ser Gly Asp Met Val Gln Pro Leu Ser Pro Ser Gln 245 250 255 Gly Gln Ser Thr His Val His Asp Ala Gln Cys Glu Asn Thr Pro Glu 260 265 270 Lys Glu Leu Pro Val Ser Pro Gly His Arg Lys Thr Pro Phe Thr Lys 275 280 285 Asp Lys His Ser Ser Arg Leu Glu Ala His Leu Thr Arg Asp Glu Leu 290 295 300 Arg Ala Lys Ala Leu His Ile Pro Phe Pro Val Glu Lys Ile Ile Asn 305 310 315 320 Leu Pro Val Val Asp Phe Asn Glu Met Met Ser Lys Glu Gln Phe Asn 325 330 335 Glu Ala Gln Leu Ala Leu Ile Arg Asp Ile Arg Arg Arg Gly Lys Asn 340 345 350 Lys Val Ala Ala Gln Asn Cys Arg Lys Arg Lys Leu Glu Asn Ile Val 355 360 365 Glu Leu Glu Gln Asp Leu Asp His Leu Lys Asp Glu Lys Glu Lys Leu 370 375 380 Leu Lys Glu Lys Gly Glu Asn Asp Lys Ser Leu His Leu Leu Lys Lys 385 390 395 400 Gln Leu Ser Thr Leu Tyr Leu Glu Val Phe Ser Met Leu Arg Asp Glu 405 410 415 Asp Gly Lys Pro Tyr Ser Pro Ser Glu Tyr Ser Leu Gln Gln Thr Arg 420 425 430 Asp Gly Asn Val Phe Leu Val Pro Lys Ser Lys Lys Pro Asp Val Lys 435 440 445 Lys Asn Leu Glu 450 <210> 13 <211> 344 <212> PRT <213> Artificial Sequence <400> 13 Gly Ser Ala Thr Met Ala Gln His Ile Gln Ser Glu Thr Ser Gly Ser 1 5 10 15 Ala Asn Tyr Ser Gln Val Ala His Ile Pro Lys Ser Asp Ala Leu Tyr 20 25 30 Phe Asp Asp Cys Met Gln Leu Leu Ala Gln Thr Phe Pro Phe Val Asp 35 40 45 Asp Asn Glu Val Ser Ser Ala Thr Phe Gln Ser Leu Val Pro Asp Ile 50 55 60 Pro Gly His Ile Glu Ser Pro Val Phe Ile Ala Thr Asn Gln Ala Gln 65 70 75 80 Ser Pro Glu Thr Ser Val Ala Gln Val Ala Pro Val Asp Leu Asp Gly 85 90 95 Met Gln Gln Asp Ile Glu Gln Val Trp Glu Glu Leu Leu Ser Ile Pro 100 105 110 Glu Leu Gln Cys Leu Asn Ile Glu Asn Asp Lys Leu Val Lys Gln Asn 115 120 125 Gly Pro Lys Thr Pro Val His Ser Ser Gly Asp Met Val Gln Pro Leu 130 135 140 Ser Pro Ser Gln Gly Gln Ser Thr His Val His Asp Ala Gln Cys Glu 145 150 155 160 Asn Thr Pro Glu Lys Glu Leu Pro Val Ser Pro Gly His Arg Lys Thr 165 170 175 Pro Phe Thr Lys Asp Lys His Ser Ser Arg Leu Glu Ala His Leu Thr 180 185 190 Arg Asp Glu Leu Arg Ala Lys Ala Leu His Ile Pro Phe Pro Val Glu 195 200 205 Lys Ile Ile Asn Leu Pro Val Val Asp Phe Asn Glu Met Met Ser Lys 210 215 220 Glu Gln Phe Asn Glu Ala Gln Leu Ala Leu Ile Arg Asp Ile Arg Arg 225 230 235 240 Arg Gly Lys Asn Lys Val Ala Ala Gln Asn Cys Arg Lys Arg Lys Leu 245 250 255 Glu Asn Ile Val Glu Leu Glu Gln Asp Leu Asp His Leu Lys Asp Glu 260 265 270 Lys Glu Lys Leu Leu Lys Glu Lys Gly Glu Asn Asp Lys Ser Leu His 275 280 285 Leu Leu Lys Lys Gln Leu Ser Thr Leu Tyr Leu Glu Val Phe Ser Met 290 295 300 Leu Arg Asp Glu Asp Gly Lys Pro Tyr Ser Pro Ser Glu Tyr Ser Leu 305 310 315 320 Gln Gln Thr Arg Asp Gly Asn Val Phe Leu Val Pro Light Ser Light Light 325 330 335 Pro Asp Val Lys Lys Asn Leu Glu 340

Claims

1. A nucleic acid molecule, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.

1.

2. The polypeptide or protein encoded by the nucleic acid molecule as described in claim 1, characterized in that, The amino acid sequence of the polypeptide or protein is shown in SEQ ID NO:

11.

3. An expression carrier, characterized in that, Includes the nucleic acid molecules as described in claim 1.

4. The expression vector as described in claim 3, characterized in that, The expression vector is a viral vector, and the viral vector is an adeno-associated virus vector.

5. The expression vector as described in claim 4, characterized in that, The serotype of the viral vector is selected from AAV2, AAV5, AAV7, or AAV8 or a combination thereof.

6. The expression vector as described in claim 4 or 5, characterized in that, The adeno-associated virus vector also includes a promoter.

7. The expression vector as described in claim 6, characterized in that, The promoter is selected from the SYN promoter, CMV promoter, or CAG promoter.

8. A drug, characterized in that, It includes the nucleic acid molecule as described in claim 1, the polypeptide or protein as described in claim 2, the expression vector as described in any one of claims 3 to 7, and pharmaceutically acceptable excipients.

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