A nucleic acid sequence construct for co-expressing TRIM11 and ApoE2 and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a nucleic acid sequence construct for co-expressing TRIM11 and ApoE2 and its application. The present invention utilizes a self-processing unit to connect the TRIM11 gene and the ApoE2 gene, wherein TRIM11 prevents the misfolding and aggregation of tau protein by promoting SUMOylation and proteasomal degradation of tau protein, and can also act as a disaggregating enzyme to dissolve existing tau aggregates; apolipoprotein ApoE2 is a secreted protein with a signal peptide that can bind to Aβ and promote Aβ clearance in a lipid state and ApoE subtype-dependent manner. The recombinant adeno-associated virus vector provided by the present invention achieves simultaneous downregulation of Tau protein and β-like protein by efficiently co-expressing TRIM11 and ApoE2. It can be used for the research and development of drugs for the treatment of dementia.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a nucleic acid sequence construct for co-expressing TRIM11 and ApoE2 and an application thereof. Background Art

[0002] Dementia is a syndrome that can be caused by a variety of diseases that destroy nerve cells and damage the brain over time, often leading to a deterioration in cognitive function (the ability to process thoughts) and a persistent decline in memory, thinking, behavior and social skills. These changes can affect a person's ability to care for themselves. Currently, more than 55 million people worldwide have dementia, more than 60% of whom live in low- and middle-income countries. There are nearly 10 million new cases each year. Dementia is caused by a variety of diseases and injuries that affect the brain. Alzheimer's disease (AD) is the most common form of dementia, which may account for 60-70% of cases, and the risk of developing the disease increases with age.

[0003] Alzheimer's disease is a neurodegenerative disorder with nearly 70% of its risk factors being genetic; other risk factors include head trauma, depression, and hypertension. Symptoms of AD include memory impairment, aphasia, apraxia, agnosia, and impaired visual-spatial abilities. Furthermore, patients experience impairment in abstract thinking and calculation, often accompanied by personality and behavioral changes.

[0004] The true cause of Alzheimer's disease remains unknown. Numerous hypotheses attempt to explain the pathogenesis of AD, including the Aβ cascade hypothesis, the cholinergic hypothesis, the abnormal Tau protein phosphorylation hypothesis, the neuroinflammation hypothesis, and the metal ion disorder hypothesis. Characteristic neuropathological changes in the AD brain include amyloid plaques formed by Aβ protein deposition, neurofibrillary tangles (intracellular aggregates composed of hyperphosphorylated tau protein), synaptic loss and atrophy, selective depletion of neurotransmitter systems (such as acetylcholine), and Lewy bodies (in rare cases). These conditions lead to impaired communication between neurons and even neuronal death, ultimately leading to AD. The β-amyloid protein (Aβ) deposition hypothesis and the abnormally phosphorylated Tau protein hypothesis are the most classic pathologies and hypotheses.

[0005] There is currently no treatment that can prevent or reverse the course of the disease. Only a few methods may temporarily relieve or improve symptoms. Current drug development ideas mainly focus on hot areas such as clearing β-amyloid protein (Aβ), regulating Tau protein, and acetylcholinesterase inhibitors (AChE).

[0006] Recent research on the tripartite motif (TRIM) protein family has revealed that several TRIM molecules possess a potent ability to prevent tau aggregation. They found that the expression of TRIM11 is significantly downregulated in Alzheimer's disease brains. Furthermore, there is a strong negative correlation between its expression and tau aggregation: in brains with high TRIM11 expression, tau aggregation levels are lower, and vice versa. Further mechanistic studies have revealed that TRIM11 can degrade both defective and excess normal tau proteins through the proteasome, thereby linking tau to the ubiquitin-proteasome pathway. TRIM11 also acts as a molecular chaperone to prevent tau misfolding and aggregation, and as a tau depolymerase to break down already formed tau aggregates. TRIM11 prevents both spontaneous and induced aggregation of tau proteins within cells, keeping tau proteins soluble. Therefore, TRIM11 is an important neuroprotective factor.

[0007] The protein encoded by the ApoE gene is called apolipoprotein E. It has multiple functions in the body, one of which is involved in lipid metabolism and cholesterol transport. There are three common ApoE alleles (E2, E3, and E4). These differ by alterations in two sites within the ApoE gene, which translate to cysteine ​​or arginine at amino acids 112 and 158. If both sites are Cys, the ApoE encoded on a chromosome is ApoE2. If Cys is at position 112 and Arg is at position 158, the apolipoprotein is ApoE3. If both sites are Arg, the apolipoprotein is ApoE4. These three alleles can combine to form six genotypes: ApoE2 / 2, 2 / 3, 2 / 4, 3 / 3, 3 / 4, and 4 / 4, encoding three ApoE protein isoforms: ApoE2, ApoE3, and ApoE4. AD is closely associated with polymorphisms in this gene. Using the ApoE3 homozygote as a baseline for AD incidence, those with ApoE4 showed a higher incidence than those with ApoE3 homozygotes. The incidence rates of heterozygotes containing E4 / E2 and E4 / E3 were 2.6 and 3.6 times higher than those with ApoE3 homozygotes, respectively. The incidence rate of pure E4 / E4 skyrocketed to 15.5 times. E2 can reduce the risk of AD.

[0008] Gene therapy is an emerging therapeutic approach that applies modern molecular biology techniques and precision medicine theories and practices. It is one of the most promising biomedical fields with promising future development prospects. AAV-based gene therapy has been actively promoted as a very promising alternative, and recent clinical trials have successfully demonstrated its practicality. LX1001 is an investigational AAV-based gene therapy developed by LEXEO Therapeutics. It is designed to deliver a transgene expressing the protective ApoE2 gene to the central nervous system (CNS) of Alzheimer's disease patients with two ApoE4 alleles to prevent or slow disease progression. In February 2022, positive data from a Phase 1 / 2 clinical trial of LX1001 in patients with homozygous ApoE4 were announced. Clinical data showed that in two patients who were followed for more than 12 months after a single treatment, Tau protein levels and phosphorylated Tau protein levels in the cerebrospinal fluid were significantly reduced compared to baseline, with no serious safety or tolerability issues observed. Recently, scientists used AAV to deliver TRIM11 into the brains of various tauopathy mouse models. They found that intracranial delivery of TRIM11 effectively prevented tau pathology and neuroinflammation. Furthermore, TRIM11 also effectively prevented cognitive and motor decline. Summary of the Invention

[0009] Based on this, the present invention provides a recombinant adeno-associated virus vector that co-expresses TRIM11 and ApoE2, achieving co-expression of TRIM11 and ApoE2 proteins while simultaneously downregulating Tau and β-like proteins. This can be used in the development of drugs for the treatment of Alzheimer's disease.

[0010] The technical solution adopted in the present invention is as follows:

[0011] A nucleic acid sequence construct is characterized in that it includes a composite sequence, wherein the composite sequence is composed of a nucleotide sequence encoding triple helical structure protein 11 (TRIM11) and human apolipoprotein E2 (ApoE2), and a self-processing unit sequence (APU) connecting the nucleotide sequences encoding the two proteins.

[0012] Preferably, the amino acid sequence of the triple helical structure protein 11 is shown in SEQ ID NO.1; the amino acid sequence of the human apolipoprotein E2 is shown in SEQ ID NO.3; the self-processing unit sequence includes but is not limited to an internal ribosome entry site sequence (IRES), a sequence encoding a 2A peptide or a 2A peptide-like sequence, an Intein, a type B bacterial Intein-like domain (BIL), a Furin sequence and its derivatives.

[0013] Preferably, the self-processing unit sequence 2A peptide or 2A-like peptide in the composite sequence includes but is not limited to F2A derived from hand, foot and mouth disease virus, E2A derived from equine rhinitis virus, P2A derived from porcine teschovirus and T2A derived from tricholoma clarkii virus.

[0014] Preferably, the self-processing unit sequence in the composite sequence is P2A derived from porcine teschovirus.

[0015] Preferably, the nucleotide sequence of the composite sequence has a nucleic acid having a nucleotide sequence as shown in any one of SEQ ID NOs. 7 to 11.

[0016] The composite sequence described in the present invention may also be a nucleic acid having one or more nucleotides substituted, deleted or added in any fragment of the sequence described in SEQ ID NO. 7 to 11; or a nucleic acid having at least 85% homology with the sequence of the nucleic acid and encoding TRIM11 (SEQ ID NO. 1) and ApoE2 (SEQ ID NO. 3) proteins; or a nucleic acid that is partially complementary or fully complementary to any of the above items.

[0017] The present invention also provides a eukaryotic expression vector comprising a nucleic acid having a nucleotide sequence as shown in any one of SEQ ID NOs. 7 to 11.

[0018] Preferably, the vector is one of viral, non-viral, and naked DNA.

[0019] Preferably, the non-viral vector is selected from one of nanocarriers and polymer carriers, such as cationic polymer carriers, liposome carriers and nanoparticle carriers, and the nucleotide sequence construct is encapsulated and delivered to target cells and tissues for expression; the viral vector is selected from at least one of a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0020] Preferably, the serotype of the adeno-associated viral vector includes but is not limited to AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, PHP.eB and AAV-retro.

[0021] The present invention also provides a use of the nucleic acid sequence construct or the eukaryotic expression vector in the preparation of a drug for treating dementia.

[0022] The nucleotide sequence constructs of the present invention can be administered via in vivo or in vitro routes to achieve expression of the target gene in target cells and tissues. The target gene can be introduced into the human body's own or allogeneic cells (or xenogeneic cells) in vitro, amplified in vitro, and then reintroduced into the body. Alternatively, the nucleotide sequence can be assembled into a specific eukaryotic expression vector and directly introduced into the body. Such vectors can be viral or non-viral, or even naked DNA. Routes of administration for such viral, non-viral vectors, or naked DNA include, but are not limited to, systemic intravenous administration, intracranial, or intrathecal injection.

[0023] The nucleotide sequence construct provided by the present invention connects the sequence encoding TRIM11 protein and the sequence encoding apolipoprotein ApoE through a self-processing unit (taking P2A as an example) to construct a recombinant adeno-associated viral vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt). The main construction idea is: connecting the TRIM11 gene and the ApoE2 gene through P2A, wherein TRIM11 prevents the misfolding and aggregation of tau protein by promoting SUMOylation and proteasome degradation of tau protein, and can also act as a disaggregating enzyme to dissolve existing tau aggregates. Apolipoprotein ApoE2 is a secretory protein with a signal peptide that can bind to Aβ and promote the clearance of Aβ in a lipid state and ApoE subtype-dependent manner. The sequence diagram of the target gene in the constructed recombinant adeno-associated viral vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) is shown in FIG. Figure 1 The amino acid sequence of TRIM11 is shown in SEQ ID NO.1, and the nucleotide sequence encoding TRIM11 is shown in SEQ ID NO.2; the amino acid sequence of ApoE2 is shown in SEQ ID NO.3, and the nucleotide sequence encoding ApoE2 is shown in SEQ ID NO.4; the amino acid sequence of P2A is shown in SEQ ID NO.5, and the nucleotide sequence encoding P2A is shown in SEQ ID NO.6.

[0024] The sequences of SEQ ID NOs. 7 to 11 in the present invention were optimized by selecting five different online codon optimization platforms: GenSmart, IDT, GeneArt, VectorBuilder, and Tsingke. Our TRIM11-P2A-ApoE2 gene was then transfected into 293 cells. It was found that the expression levels of the sequences optimized by IDT, GeneArt, and VectorBuilder (VB) were significantly improved, among which the optimization effect by Tsingke was the best. The expression levels of the optimized TRIM11 were 1.91 times that of the wild type, and ApoE2 were 2.57 times that of the wild type.

[0025] Studies have found that Aβ promotes the hyperphosphorylation and accumulation of Tau protein by activating JNK kinase. On the other hand, Tau protein can also inhibit the degradation of Aβ protein. Therefore, Aβ and Tau toxicity can interact to form a snowball cycle, which continuously aggravates the progression of AD. This mechanism suggests that previous drug development targeting Aβ or Tau alone may not effectively inhibit the progression of AD, and that combining Aβ and Tau as a combined target may be the most effective way to slow down AD. Therefore, the present invention pioneered the construction of AAV expression vectors for TRIM11 and ApoE2, and optimized the codons to obtain gene fragments that efficiently co-express TRIM11 and ApoE2 in cells. This led to the construction of a recombinant adeno-associated virus vector that efficiently co-expresses TRIM11 and ApoE2, as well as a drug for the treatment of Alzheimer's disease. Experiments showed that AAV viruses carrying TRIM11 and ApoE2 can directly infect SH-SY5Y neuroblastoma cells. Overexpression of AAV vectors carrying the target genes in target cells can simultaneously downregulate Tau protein and β-amyloid protein.

[0026] The present invention connects the target genes TRIM11 and ApoE2 by an auto-processing unit (APU), constructs a nucleotide sequence construct, verifies the expression of the target genes TRIM11 and ApoE2 by Western blot and ELISA, and co-transfects Tau. It is found that the expression of Tau is negatively correlated with the vector, and the expression of tau is downregulated with the expression of TRIM11, and shows a dose-dependent effect. The codons of the target gene are then optimized by different codon optimization platforms, and the gene fragments with high expression efficiency are obtained by Western blot screening. AAV vectors are constructed with the optimized sequence, and the AAV recombinant virus is packaged and purified. After infecting neuroblastoma SH-SY5Y, the expression of the target gene in the host cell is verified. At the same time, after the sequence-optimized vector is co-transfected with Tau, it is found that the optimized vector can efficiently inhibit the formation of Tau.

[0027] Compared with the existing technology, the present invention has the following advantages: the nucleotide sequence construct provided by the present invention can achieve efficient co-expression of TRIM11 and ApoE2, providing an important basis for the research of drugs for treating Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A diagram of the vector structure constructed for the present invention;

[0029] Figure 2The figure shows the Western blot expression results of target genes TRIM11 and ApoE2 after transient transfection of the expression vector;

[0030] Figure 3 This is the standard curve of ApoE ELISA test;

[0031] Figure 4 Real-Time PCR standard curve for AAV virus titer detection;

[0032] Figure 5 The figure shows the expression of TRIM11 and ApoE2 in human neuroblastoma cells SH-SY5Y detected by Western blot after recombinant AAV virus was infected;

[0033] Figure 6 This is the Western blot detection result of overexpression of TRIM11 and ApoE2 wild-type vector, which induced Tau protein degradation;

[0034] Figure 7 This is the Western blot detection result of protein expression after transient transfection using vectors optimized by different optimization platforms;

[0035] Figure 8 This is a diagram of Western blot analysis using ImageJ;

[0036] Figure 9 The results of Western blot analysis of target gene expression in neuroblastoma SH-SY5Y cells were obtained by infecting the recombinant adeno-associated virus (AAV) before and after sequence optimization on the VectorBuilder platform.

[0037] Figure 10 This is the Western blot detection result of Tau protein degradation induced by overexpression of the vector optimized by the VectorBuilder platform;

[0038] Figure 11 This is the Western blot detection result of the target gene expression after the sequence-optimized vector was co-expressed with BACE1 and APP;

[0039] Figure 12 This is the standard curve of Aβ40 ELISA detection after co-expression of sequence-optimized vector with BACE1 and APP;

[0040] Figure 13 This is an analysis chart of ELISA results. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] The PEI was purchased from Polyscience, catalog number 23966-100; the RIPA cell lysate was purchased from Sangon Biotechnology, catalog number C500007-0100; the TRIM11 and ApoE primary antibodies were purchased from Boster Biotechnology, catalog numbers BM4412 and A08971-2, respectively; the FLAG primary antibody was purchased from Proteintech, catalog number 66008-4-Ig; the HA primary antibody was purchased from Affinity, catalog number T0008; the GFP antibody was purchased from Proteintech, catalog number 66002-1; the secondary antibody HRP-conjugate daffinipure Goat anti Rabbit IgG (H+L) was purchased from Proteintech, catalog number SA0001-2; the Goat Abto Ms IgG (HRP) was purchased from Abcam, catalog number ab205719; the ApoE ELISA kit was purchased from Elabscience, catalog number E-EL-H0470; the 10kDa MWCO ultrafiltration tubes were purchased from Millipore, catalog number UFC901008.

[0043] Example 1 Construction of AAV viral vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt)

[0044] The construction process of the AAV viral vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) is as follows:

[0045] The wild-type TRIM11 and ApoE2 ORFs were connected through P2A, and the AgeI restriction site and 3×FLAG tag were fused to the N-terminus of TRIM11 in sequence. The HA tag and EcoRI restriction site were fused to the C-terminus of ApoE2 in sequence. Nanjing Qingke Biotechnology was commissioned to synthesize the AgeI-3×FLAG-TRIM11-P2A-ApoE2-HA-EcoRI full gene. The synthesized full gene fragment and pAAV vector backbone were double-digested with AgeI and EcoRI, respectively, and detected by agarose gel electrophoresis. The target gene fragment and linearized vector backbone fragment were then recovered by gel tapping. The recovered target gene and linearized vector backbone fragment were ligated overnight with T4 DNA ligase, and the ligation products were transformed into Stbl3 Escherichia coli competent cells. LB plates were applied, and single clones were selected. The colonies identified as recombinant plasmids by PCR were sent to Nanjing Qingke Biotechnology for sequencing. The recombinant plasmids with correct sequencing results were the constructed recombinant vectors.

[0046] TRIM11 prevents tau protein misfolding and aggregation by promoting SUMOylation and proteasomal degradation, while also acting as a disaggregase to dissolve existing tau aggregates. Apolipoprotein E2 is a secreted protein with a signal peptide that can bind to Aβ and promote Aβ clearance in a lipid state- and ApoE isoform-dependent manner.

[0047] The sequence diagram of the target gene in the recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) constructed by the present invention is as follows: Figure 1 The amino acid sequence of TRIM11 is shown in SEQ ID NO.1, and the nucleotide sequence encoding TRIM11 is shown in SEQ ID NO.2; the amino acid sequence of ApoE2 is shown in SEQ ID NO.3, and the nucleotide sequence encoding ApoE2 is shown in SEQ ID NO.4; the amino acid sequence of P2A is shown in SEQ ID NO.5, and the nucleotide sequence encoding P2A is shown in SEQ ID NO.6.

[0048] Experimental Example 1 Identification of protein expression

[0049] 1. Experimental process:

[0050] (1) HEK293 cells in the logarithmic growth phase with a cell viability of >98% were selected, diluted with DMEM + 10% FBS complete medium and plated on 6-well plates. The recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) was transfected into the cells using PEI. The recombinant adeno-associated virus vector pAAV-EGFP was transfected into the cells as a control. The transfection ratio of DNA:PEI was 1:3 (μg:μL). After transfection, the cells were placed in a 37°C, 5% CO2 incubator for static culture.

[0051] (2) After 48 h, cells and culture supernatant (Supernatant, SP) were collected, and untransfected HEK293 cells and their supernatant were used as blank controls;

[0052] (3) Cells were lysed with RIPA lysis buffer, centrifuged at 12,000 rpm for 15 minutes, the precipitate was discarded, and the supernatant was used for Western blot to detect the expression of TRIM11, FLAG, ApoE, HA, GFP, and β-actin;

[0053] (4) ApoE ELISA (Elabscience, E-EL-H0470) was used to detect the ApoE2 content in the cell culture supernatant.

[0054] 2. Experimental results: The experimental results are as follows Figure 2 、 Figure 3 shown. Figure 2 In the figure, 1 is a pAAV-EGFP vector, 2 is a pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA vector of the present invention; Figure 2 It can be seen that endogenous TRIM11 and ApoE2 are expressed in HEK293 cells. 48 hours after transfection with the recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) of the present invention, it was found that the amounts of TRIM11 and ApoE2 in the transfected group cells were significantly increased compared with HEK293 cells not transfected with the plasmid. The Western blot results of FLAG and HA further demonstrated that the recombinant adeno-associated virus vector pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) mediated the expression of the target gene TRIM11. At the same time, the expression of ApoE2 was also detected in the cell lysate (WCL) and cell culture supernatant, while it was not detected in the control group transfected with the pAAV-EGFP vector, indicating that ApoE2 was successfully expressed and secreted extracellularly after transfection. This indicates that both the target genes TRIM11 and ApoE2 are expressed.

[0055] Depend on Figure 3 It can be seen that the ApoE content in the cell culture supernatant was detected using an ApoE ELISA kit, and the data were analyzed using the online ELISA data analysis tool GainData developed by Arigo. The standard curve is as follows Figure 3 shown.

[0056] ELISA results showed that 48 hours after transfection, the concentration of ApoE2 in the cell supernatant was 932.29 ng / mL, while in the control group transfected with pAAV-EGFP, the concentration of ApoE2 was the background level of 0.451 ng / mL, indicating that ApoE2 was successfully expressed and secreted outside the cells after transfection.

[0057] Thus, Western blot can simultaneously detect TRIM11 and ApoE2 in cell lysates, indicating that the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA(wt) vector can co-express TRIM11 and ApoE2. ELISA further verified the secretory expression of ApoE2, indicating that this vector can co-express TRIM11 and ApoE2, and ApoE2 can be secreted extracellularly.

[0058] Experimental Example 2 Construction of Type II Recombinant Adeno-Associated Virus Vector and Protein Detection

[0059] 1. Experimental procedure: HEK293T cells in the logarithmic growth phase were passaged into 10 15 cm cell culture dishes. When the degree of confluence reached 80%, the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) expression vector (i.e., the wild-type vector constructed by the present invention), the capsid protein plasmid AAV2, and the auxiliary packaging plasmid pHelper were co-transfected into the cells at a molar ratio of 1:1:1 under PEI mediation. The mass ratio of total plasmid DNA (i.e., the total DNA of the vector of the present invention and AAV2) to PEI was 1:3. The cells were collected 72 h after transfection, resuspended in RIPA cell lysis buffer, and lysed by repeated freezing and thawing in liquid nitrogen and a 37°C water bath three times. The supernatant was collected by centrifugation at 12000 rpm for 20 min and filtered with a 0.45 μm filter to obtain the filtered AAV virus liquid. The AAV virus liquid was purified by iodixanol density gradient centrifugation as follows: 5 mL of 60% iodixanol, 5 mL of 40% iodixanol, 6 mL of 25% iodixanol and 8 mL of 15% iodixanol were added to the Beckman quick-seal tube in sequence. Finally, the filtered AAV virus liquid was carefully added on top of the iodixanol. The centrifuge tube was heat-sealed and centrifuged at 35,000 g for 90 minutes. The virus liquid layer containing AAV was removed from the side of the centrifuge tube with a syringe. The AAV virus layer was located at the junction of 60% and 40% iodixanol. The virus liquid was then filtered with a 0.45 μm filter and the AAV virus was concentrated and replaced with a Millipore 100 kDa MWCO ultrafiltration tube (Cat. No. UFC9100).

[0060] The virus titer was detected by Taqman qPCR. The primers used for AAV virus titer determination were: F: 5'-GGAACCCCTAGTGATGGAGTT-3', R: 5'-CGGCCTCAGTGAGCGA-3', and probe: 5'-FAM-CACTCCCTCTCTGCGCGCTCG-BBQ-3'. Plasmid pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) was serially diluted (2×10 9 , 2×10 8 , 2×10 7 , 2×10 6 , 2×10 5 qPCR was performed simultaneously to prepare a standard curve. The qPCR enzyme was Taq Pro U+Multiple Probe qPCR Mix, purchased from Novozymes, catalog number QN213-01. The qPCR system was prepared according to the system and conditions recommended in the instruction manual. The instrument used was the QuantStudio1 real-time fluorescence quantitative PCR system from Thermo Fisher Scientific.

[0061] Human neuroblastoma SH-SY5Y cells were infected with recombinant adeno-associated virus (AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt)) at an MOI of 10,000. Recombinant adeno-associated virus (AAV-EGFP) was used as a control. Four days later, cells and culture supernatant were collected and lysed with RIPA buffer. The expression of TRIM11 and ApoE2 in cells was detected by Western blot, and the secretory expression of ApoE in the cell supernatant was detected by ApoE ELISA.

[0062] 2. Experimental results: The experimental results are as follows Figure 4-Figure 5 As shown, Figure 4 The real-time PCR standard curve was constructed after the gradient dilution of the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) plasmid. The adeno-associated virus AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) (250-fold dilution) amplified at the same time had a Ct value of 21.391. The virus titer calculated by the standard curve equation was 1.16E+12 vg / mL.

[0063] Figure 5 In the figure, 1 is AAV-EGFP, 2 is AAV-3×FLAG-TRIM11-P2A-ApoE2-HA; Figure 5 Western blot results showed that endogenous TRIM11 and ApoE2 were expressed in human neuroblastoma cells SH-SY5Y. Four days after SH-SY5Y cells were infected with type 2 recombinant adeno-associated virus AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt), the expression of TRIM11 and ApoE2 in the cells was significantly increased. ApoE ELISA results detected that the concentration of ApoE2 in the supernatant was 281.75 ng / mL, while the concentration of ApoE2 in the supernatant of the control group cells infected with AAV-EGFP was the background expression of 1.508 ng / mL, indicating that ApoE2 was successfully expressed and secreted extracellularly after rAAV infection of human neuroblastoma cells SH-SY5Y.

[0064] Experimental Example 3: Detection of Tau protein levels

[0065] 1. Experimental Procedure: Tau P301L is a common variant of tau protein in familial tauopathy. Animal models that overexpress the tau mutant P301L to simulate tau protein pathology are widely used in Alzheimer's disease research.

[0066] Different amounts of plasmid pAAV-TRIM11-P2A-ApoE2 and pRK5-EGFP-Tau-P301L vector were co-transfected into HEK293 cells. After 48 hours, the cells were collected and lysed with RIPA cell lysis buffer. The expression of the target protein in the cells was detected by Western blot.

[0067] 2. Experimental results: Specific experimental results are as follows Figure 6 As shown, Figure 6 In the table, band 1 corresponds to the first column, i.e. pRK5-EGFP-Tau-P301L 1μg, pAAV-EGFP 4μg, and the following bands 2-5 correspond to the 2-5 columns of the table respectively. The pAAV vector is used as an empty vector to fill the total DNA transfected in each well to keep it consistent. Figure 6 The results showed that after transfection with different amounts of the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) vector, both TRIM11 and ApoE2 proteins were expressed, and the expression was positively correlated with the amount of transfected plasmid. Tau P301L aggregates were significantly degraded in a dose-dependent manner, indicating that our target protein can downregulate intracellular Tau protein levels.

[0068] Experimental Example 4 Sequence Optimization

[0069] 1. Experimental process: 5 different online codon optimization platforms were selected: GenSmart, IDT, GeneArt, VectorBuilder and Tsingke to optimize the TRIM11-P2A-ApoE2 gene of the present invention. During optimization, a 3×FLAG tag was added to the front end of TRIM11 and an HA tag was added to the back of ApoE2. The nucleotide sequence optimized by VectorBuilder is shown as SEQ ID NO.7, the nucleotide sequence optimized by GenSmart is shown as SEQ ID NO.8, the nucleotide sequence optimized by IDT is shown as SEQ ID NO.9, the nucleotide sequence optimized by GeneArt is shown as SEQ ID NO.10, and the nucleotide sequence optimized by Tsingke is shown as SEQ ID NO.11. The optimized sequences were then transfected into HEK293 cells together with other gene sequences. After 48 hours, the cells were collected and lysed, and Western blot detection was performed according to the method of Experimental Example 1. Due to the expression of endogenous genes, in order to remove background, FLAG was used to reflect the expression of TRIM11 and HA was used to reflect the expression of ApoE2 during the experiment.

[0070] 2. Experimental results: The test results are as follows Figure 7 As shown, Figure 7In the figure, 1 is the original sequence of 3×FLAG-TRIM11-P2A-ApoE2-HA (wt) of the present invention; 2 is the vector optimized using IDT software; 3 is the vector optimized using GeneArt software; 4 is the vector optimized using Tsingke software; 5 is the vector optimized using VectorBuilder software; 6 is the vector optimized using GenSmart software. Western blot results were analyzed using ImageJ. Figure 8 As shown, the expression levels of the sequences optimized by IDT, GeneArt and VectorBuilder (VB) were significantly improved, among which VectorBuilder optimization had the best effect. The expression levels of TRIM11 after optimization were 1.91 times that of the wild type, and ApoE2 was 2.57 times that of the wild type.

[0071] Comparison of protein expression in Experimental Example 5

[0072] 1. Experimental samples: sequence-optimized AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (VB), wild-type AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt);

[0073] 2. Experimental method: AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (VB) was packaged and purified, and the wild-type AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt) (refer to the method described in Experimental Example 2) was used to infect neuroblastoma SH-SY5Y cells at an MOI of 10,000. The AAV-EGFP group was infected with the same MOI as a control. Five days after infection, the cells were collected and lysed, and the detection was performed according to the method of Experimental Example 2.

[0074] 3. Experimental results: Western blot results are as follows Figure 9 As shown, Figure 9 Among them, 1 is AAV-EGFP, 2 is AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (wt), and 3 is AAV-3×FLAG-TRIM11-P2A-ApoE2-HA (VB). It can be seen that in the AAV group after target sequence optimization, the expression levels of TRIM11 and ApoE2 are significantly higher than those in the wild group, indicating that after the sequence-optimized AAV infects the target cells, the expression of the target gene is significantly increased.

[0075] Experimental Example 6: Detection of tau protein content

[0076] 1. Experimental Procedure: Tau P301L is a common familial tau mutation. Overexpression of tau P301L is commonly used as an animal model for Alzheimer's disease. 293T cells were co-transfected with varying amounts of TRIM11-P2A-ApoE2(VB) and tau P301L. After 48 hours, the cells were harvested and lysed, and assays were performed according to the protocol in Experimental Example 3.

[0077] 2. Experimental results: Western blot results are as follows Figure 10 As shown, it can be seen that overexpression of the target genes TRIM11 and ApoE2 promoted the obvious degradation of tau P301L aggregates, and the degradation showed a dose-dependent effect.

[0078] Test Example 7 Aβ protein detection

[0079] 1. Experimental Procedure: β-Amyloid protein (Aβ) is a polypeptide containing 39-43 amino acids derived from the amyloid precursor protein (APP). APP is a transmembrane glycoprotein that undergoes two degradation pathways in vivo: non-amyloid and amyloid pathways. In the amyloid pathway, APP is sequentially cleaved by BACE1 (beta-site APP cleaving enzyme-1, an aspartic protease also known as β-secretase) and γ-secretase, producing a series of Aβ fragments of varying lengths, primarily Aβ40 and Aβ42. These fragments readily aggregate to form insoluble particles, which are the primary components of senile plaques. HEK293T cells were co-transfected with APP, BACE1, and the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA(VB) plasmid. A control group was co-transfected with APP, BACE1, and pAAV-EGFP. After 48 hours, cells were harvested and lysed for Western blot and ELISA analysis. A standard curve was constructed using the ELISA method described in Experimental Example 1.

[0080] 2. Experimental results: The experimental results are as follows Figure 11-13 As shown, Figure 11 and Figure 13 In the figure, 1 is pAAV-EGFP, and 2 is pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA. Figure 11 Western blot was used to verify the expression of target genes. The results showed that after transfection with the pAAV-3×FLAG-TRIM11-P2A-ApoE2-HA(VB) plasmid, the target genes APP, BACE1, TRIM11, and ApoE2 were all expressed. The ELISA standard curve was as follows: Figure 12 As shown, pAAV-EGFP was used as the control group, and the ELISA results were analyzed as shown in Figure 13 As shown, overexpression of the target genes TRIM11 and ApoE2 can significantly inhibit the production of Aβ40.

Claims

1. A nucleic acid sequence construct, characterized in that: The invention comprises a composite sequence, which is composed of a nucleotide sequence encoding a triple helical structure protein 11 and a human apolipoprotein E2, and a self-processing unit sequence connecting the nucleotide sequences encoding the two proteins; the nucleotide sequence of the composite sequence is one of SEQ ID NOs. 7 to 11.

2. A eukaryotic expression vector, characterized in that: It comprises one of the nucleotide sequences shown in SEQ ID NOs. 7 to 11.

3. The eukaryotic expression vector according to claim 2, wherein The vector is one of viral, non-viral and naked DNA.

4. The eukaryotic expression vector according to claim 3, wherein The non-viral vector is selected from one of a nano-vector and a polymer vector, and the viral vector is selected from at least one of a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

5. The eukaryotic expression vector according to claim 4, wherein The serotypes of the adeno-associated virus vector include AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, AAV-PHP.eB and AAV-retro.

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