Codon-optimized ldlr gene and use thereof

Abstract

The present invention provides a nucleic acid molecule, a transgenic expression cassette, a recombinant adeno-associated virus, and a use thereof. The present invention assembles a transgenic expression cassette by means of combining a nucleic acid molecule encoding a human low-density lipoprotein receptor with an expression control elements conducive to liver cell expression, which is then used to prepare a recombinant adeno-associated virus (rAAV). The resulting rAAV can effectively reduce LDL-C and TC levels in subjects at low doses over the long term, showing promising use prospects in the treatment of familial hypercholesterolemia.

Description

Codon-optimized LDLR gene and its application Technical Field

[0001] The present invention belongs to the technical field of gene therapy, and specifically relates to a nucleic acid molecule, a transgenic expression cassette, a recombinant adeno-associated virus and applications thereof, as well as a medicine. Background Art

[0002] Cholesterol is an essential cellular component, and maintaining cholesterol homeostasis is crucial for normal physiological function. Elevated plasma cholesterol levels are associated with various pathological conditions, most notably coronary artery disease, in which high cholesterol levels lead to foam cell formation and arterial plaque accumulation, potentially leading to heart attack or stroke. Regulation of cellular cholesterol metabolism and plasma cholesterol levels depends on the uptake of LDL into specific cells, mediated by the low-density lipoprotein receptor (LDLR). LDL is the primary carrier of cholesterol in the blood, accounting for over 60% of total plasma cholesterol. LDL is taken up by the liver and extrahepatic tissues through receptor-mediated endocytosis triggered by the apoB-100-LDLR interaction. Internalized LDL particles are transported to lysosomes, where they are degraded into free cholesterol and amino acids. In humans, the liver is the most important organ for LDL-C (low-density lipoprotein cholesterol) catabolism and LDL-C receptor activity.

[0003] Hypercholesterolemia is one of the most important independent risk factors for atherosclerosis and cardiovascular disease, interacting with numerous genetic and environmental factors. Familial hypercholesterolemia (FH), a primary form of hypercholesterolemia, is primarily caused by genetic mutations in the low-density lipoprotein (LDL) receptor, leading to decreased or ineffective function. This slows plasma LDL clearance and increases cholesterol levels, leading to premature cardiovascular and cerebrovascular disease and often accompanied by skin and tendon xanthomas.

[0004] FH is an autosomal dominant genetic disease. The prevalence of LDLR heterozygous mutations is estimated to be 1 / 200-1 / 500, and the prevalence of homozygous mutations is approximately 1 / 100,000. FH is one of the most serious common inherited metabolic diseases. Despite its high prevalence, FH remains underdiagnosed and undertreated. It has been reported that more than 50% of patients over 60 years old with myocardial infarction have heterozygous mutations in the LDLR gene (Wiegman A, Gidding SS, Watts GF, et al. Familial hypercholesterolaemia in children and adolescents: gaining decades of life by optimizing detection and treatment [J]. Eur Heart J. 2015, 36(36): 2425-2437.). Most patients with homozygous mutations do not receive effective treatment and die before the age of 30. The LDL-C level of heterozygous patients is more than twice the normal level (approximately 5 mmol / L), while that of homozygous patients is more than 4 times (>13 mmol / L) (Nohara A, Tada H, Ogura M, et al. Homozygous Familial Hypercholesterolemia[J]. J Atheroscler Thromb. 2021, 28(7): 665-678.), (Defesche JC, Gidding SS, Harada-Shiba M, et al. Familial hypercholesterolaemia[J]. Nat Rev Dis Primers. 2017.). If LDL-C is above 5mmol / L, there is a high risk of developing severe familial hypercholesterolemia. The ideal LDL-C level is controlled at <2.5mmol / L. Reducing LDL-C by more than 50% in FH patients is the clinical treatment goal (Wierzbicki AS, Humphries SE, Minhas R. Familial hypercholesterolaemia: Summary of NICE guidance[J]. BMJ Clinical Research, 2008.).

[0005] Currently, the main marketed lipid-lowering drugs for FH patients include statins and PCSK9 inhibitors, such as inclisiran, evolocumab (REPATHA), and alirocumab (PRALUENT). Statins act by upregulating LDLR expression, resulting in significantly better therapeutic outcomes for adolescents and children with FH than for adults, and achieving treatment targets in most adolescents and children with HeFH. However, even at the highest dose, LDL-C plasma levels in most adults only decrease by 10-25%, with less than 30% achieving adult treatment targets. In patients with homozygous familial hypercholesterolemia (HoFH), the average reduction in LDL-C levels ranges from 14-31%, falling short of achieving treatment targets. PCSK9 inhibitors, by inhibiting LDLR degradation, have shown promising results for heterozygous hypercholesterolemia (HeFH), but are less effective for HoFH.

[0006] HoFH patients with LDRL mutations have no LDLR expression or express LDLR mutants with no / low activity, so they respond poorly to drugs that increase LDLR expression through drugs such as statins and PCSK9 inhibitors. This type of HoFH patients who are insensitive to major lipid-lowering drugs (statins and PCSK9 antibody drugs) can only undergo some non-drug intervention measures such as lipoprotein plasma exchange, liver transplantation and gene technology. These measures put great physiological and economic pressure on patients, and new treatment methods are urgently needed. Regenxbio, a US company, has developed an AAV-based liver-directed gene therapy RGX-501, which has obtained FDA orphan drug status and has conducted clinical studies to evaluate its efficacy in HoFH patients (clinical number NCT02651675). However, this clinical trial revealed that the low-dose group (2.5x10 12 GC / kg) did not achieve clinically meaningful changes in LDL-C levels, and some cases required adjunctive cortisol administration due to elevated transaminases.

[0007] In addition, no other clinical studies using optimized LDLR sequences for the treatment of FH have been reported. It is necessary to develop a safer and more effective LDLR gene therapy. Summary of the Invention

[0008] To address the aforementioned issues in the prior art, the present invention provides a nucleic acid molecule, a transgenic expression cassette, a recombinant adeno-associated virus, and their applications. The present invention uses an AAV vector to deliver a codon-optimized nucleic acid sequence encoding the human low-density lipoprotein receptor (hLDLR) protein, whose expression is controlled by a liver-specific expression control element, to hepatocytes. Animal studies in mice have shown that the recombinant adeno-associated virus of the present invention can significantly reduce LDL-C and TC levels in mice over a long period of time at relatively low doses.

[0009] In a first aspect, the present invention provides a nucleic acid molecule encoding human low-density lipoprotein receptor.

[0010] The nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NOs: 2-18.

[0011] In some preferred embodiments, the nucleotide sequence is shown as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 16. In some more preferred embodiments, the nucleotide sequence is shown as SEQ ID NO: 3 or SEQ ID NO: 11. Even more preferably, the nucleotide sequence is shown as SEQ ID NO: 11.

[0012] In the present invention, the nucleic acid sequence encoding human low-density lipoprotein receptor is codon-optimized, and compared with the nucleic acid sequence encoding wild-type human low-density lipoprotein receptor without codon optimization, the expression level of human low-density lipoprotein receptor is higher.

[0013] In a second aspect, the present invention provides a transgenic expression cassette.

[0014] The transgene expression cassette comprises the nucleic acid molecule of claim 1.

[0015] In some embodiments, the transgenic expression cassette further comprises a promoter, an intron, and a terminator, wherein the 3' end of the intron is operably linked to the 5' end of the nucleic acid molecule, the 3' end of the promoter is operably linked to the 5' end of the intron, and the 3' end of the nucleic acid molecule is operably linked to the 5' end of the terminator.

[0016] In some embodiments, the promoter is selected from the group consisting of SerpinA1 gene promoter, SerpinG1 gene promoter, ALB gene promoter, SerpinA1 gene promoter, HLP gene promoter, or LP gene promoter. In some preferred embodiments, the promoter is the SerpinA1 gene promoter. In some more preferred embodiments, the SerpinA1 gene promoter is the human α1 antitrypsin promoter, whose nucleotide sequence is shown in SEQ ID NO: 21; and / or,

[0017] The intron is a truncated SerpinA1 intron. In some preferred embodiments, the nucleotide sequence of the truncated SerpinA1 intron is shown in SEQ ID NO: 22; and / or,

[0018] The terminator is selected from BGH polyA, hGH polyA or GH polyA. In some preferred embodiments, the terminator is BGH polyA. In some more preferred embodiments, the BGH polyA is a bovine growth hormone polyadenylation signal, the nucleotide sequence of which is shown in SEQ ID NO: 23.

[0019] In some embodiments, the transgenic expression cassette further comprises a 5'ITR, a 3'ITR and an enhancer, wherein the 5'ITR is located at the 5' end of the transgenic expression cassette, the 3'ITR is located at the 3' end of the transgenic expression cassette, the 3' end of the enhancer is operably connected to the 5' end of the promoter, the nucleotide sequence of the 5'ITR is shown in SEQ ID NO: 19, and the nucleotide sequence of the 3'ITR is shown in SEQ ID NO: 24; in some preferred embodiments, the enhancer is an ApoE HCR enhancer; in some more preferred embodiments, the ApoE HCR enhancer is the hepatocyte control region of human apolipoprotein E, and its nucleotide sequence is shown in SEQ ID NO: 20.

[0020] In some embodiments, the transgenic expression cassette further comprises a Kozak sequence located between the intron and the nucleic acid molecule described above, and the nucleotide sequence of the Kozak sequence is gccacc.

[0021] In the present invention, the liver-specific expression control element is selected and assembled with the nucleic acid sequence encoding the human low-density lipoprotein receptor, and the obtained expression cassette can express the human low-density lipoprotein receptor protein with high efficiency.

[0022] Furthermore, the nucleotide sequence of the transgenic expression cassette is shown in SEQ ID NO: 32.

[0023] In a third aspect, the present invention provides a recombinant adeno-associated virus.

[0024] The recombinant adeno-associated virus comprises an AAV capsid, and the nucleic acid molecule described above or the transgenic expression cassette described above; in some preferred embodiments, the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or rhAAV10; in some more preferred embodiments, the AAV is AAV8.

[0025] In the present invention, by selecting an AAV capsid with liver tropism and constructing it together with a transgene expression cassette into a recombinant adeno-associated virus for gene delivery, the expression cassette containing the human low-density lipoprotein receptor can be efficiently delivered to liver cells.

[0026] In a fourth aspect, the present invention provides a composition.

[0027] The composition comprises the nucleic acid molecule described above, the transgenic expression cassette described above, or the recombinant adeno-associated virus described above.

[0028] In some preferred embodiments, the composition is a pharmaceutical composition. In some more preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0029] In a fifth aspect, the present invention provides use of the nucleic acid molecule described above, the transgenic expression cassette described above, the recombinant adeno-associated virus described above, or the composition described above in the preparation of a drug for treating hypercholesterolemia.

[0030] In some preferred embodiments, the hypercholesterolemia is familial hypercholesterolemia.

[0031] In some more preferred embodiments, the familial hypercholesterolemia includes homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia.

[0032] In the present invention, the pharmaceutical composition can be used to transduce cells in vitro or transduce animals (such as mice or humans) in vivo, thereby treating familial hypercholesterolemia.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] In the present invention, by codon-optimizing the nucleic acid sequence encoding the human low-density lipoprotein receptor, the expression level of the human low-density lipoprotein receptor is increased in vitro and in vivo, as well as the lipid-lowering activity. Furthermore, by combining a nucleic acid molecule encoding the human low-density lipoprotein receptor with an expression control element that facilitates expression in liver cells, a transgenic expression cassette is generated and used to prepare a recombinant adeno-associated virus (rAAV). The resulting rAAV can reduce LDL-C and TC levels in subjects over a long period of time at a relatively low dose, and has good application prospects in the treatment of familial hypercholesterolemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG1 is a schematic diagram of the structure of the hLDLR expression cassette constructed in Example 1 of the present invention;

[0036] FIG2 shows the expression detection results of the codon-optimized hLDLR opt in HepG2 cells in Example 2 of the present invention; wherein (A) is the Western-Blot detection result; (B) is the quantitative result of grayscale analysis;

[0037] FIG3 shows the lipid uptake results of the codon-optimized hLDLR opt in Huh7 cells in Example 3 of the present invention;

[0038] Figure 4 shows the expression of AAV8 recombinant virus carrying the codon-optimized hLDLR gene in high-fat feeding LDLR in Example 4 of the present invention. - / - Results of the detection of LDL-C content and hLDLR concentration in mouse serum; (A) shows the results of LDL-C content in mouse serum at week 1, week 2, and week 3; (B) shows the results of LDL-C content in mouse serum at week 3;

[0039] Figure 5 shows that the AAV8 recombinant virus carrying the hLDLR gene in Example 5 of the present invention reduces the LDLR in high-fat diet - / - Dose-response results of LDL-C and TC in mouse serum; (A) shows the Western-blot analysis of hLDLR protein expression in mouse liver 5 weeks after administration; (B) shows the weekly levels of LDL-C and TC in mouse serum within 5 weeks after administration;

[0040] Figure 6 shows that the NGGT-006 recombinant virus in Example 6 of the present invention continuously reduces LDLR in high-fat diet - / - Results of LDL-C and TC levels in mouse serum; (A) is the LDL-C content test result; (B) is the TC content test result;

[0041] Figure 7 shows the NGGT-006 recombinant virus in Example 7 of the present invention in high-fat fed LDLR - / -Results of sustained and effective reductions in blood LDL-C and TC levels and plaque deposition in a mouse model; (A) shows the results of LDL-C and TC levels; (B) shows the size and distribution of atherosclerotic plaques after Oil Red O staining; (C) shows the area of ​​aortic deposition;

[0042] Figure 8 shows the NGGT-006 recombinant virus and the TBG.PI.hLDLRco.RGB AAV8 recombinant virus in Example 8 of the present invention in the presence of high-fat fed LDLR - / - Comparative analysis results of the effects of lowering LDL-C and TC in mouse models, where (A) is the LDL-C content detection result; (B) is the TC content detection result;

[0043] Figure 9 shows that the NGGT-006 recombinant virus in Example 9 of the present invention continuously and stably reduces LDLR in high-fat diet - / - Results of LDL-C and TC levels in golden hamsters;

[0044] Figure 10 shows that the NGGT-006 recombinant virus in Example 10 of the present invention continuously and stably reduces the normal feeding LDLR + / - Results of LDL-C and TC levels in golden hamsters. DETAILED DESCRIPTION

[0045] The technical solutions of the present invention will be described clearly and completely below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0046] For experimental methods in the examples where specific conditions are not specified, generally conventional conditions and conditions described in the manual or conditions recommended by the manufacturer were followed. The general equipment, materials, reagents, etc. used were all commercially available unless otherwise specified.

[0047] The experimental animals used in the present invention are as follows:

[0048] LDLR - / - Mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd., strain number: C000114. The serum cholesterol (TC) content of normal mice is about 2.07-2.58 mmol / L, of which low-density lipoprotein cholesterol (LDL-C) accounts for about 20% of the total cholesterol (TC) content. - / -Serum cholesterol (TC) levels in mice range from approximately 5.17 to 10.34 mmol / L. When fed a high-fat diet, serum cholesterol (TC) levels rise significantly (approximately 50 mmol / L). This mouse model is currently a recognized animal model for studying lipid metabolism and LDLR gene therapy.

[0049] LDLR - / - Golden hamsters and LDLR + / - Golden hamsters were donated by the Institute of Cardiovascular Diseases, Peking University. The serum cholesterol (TC) content of normal golden hamsters is about 3.46-3.82 mmol / L, of which low-density lipoprotein cholesterol (LDL-C) accounts for about 22% of the total cholesterol (TC) content. - / - Golden hamsters and LDLR + / - Serum cholesterol (TC) levels in golden hamsters range from 13.86 to 27.77 mmol / L and 11.5 to 15.22 mmol / L, respectively. After four weeks of feeding a high-fat, high-cholesterol diet, serum cholesterol (TC) levels increased significantly (approximately 60 mmol / L). Compared to mice, golden hamsters express cholesterol ester transfer protein (CETP), making them more susceptible to arteriosclerosis (AS), with the coronary arteries being the most susceptible sites. This makes them more similar to humans in lipid metabolism and AS pathology, making them an ideal model for studying lipid metabolism.

[0050] In the present invention, the mouse tail vein injection method is as follows:

[0051] Place the mouse in a small animal cage and irradiate it with an infrared heating lamp until the blood vessels in the mouse's tail dilate. Remove the mouse from the cage and place it on a mouse and rat tail injection vein imaging device, exposing the tail. Wipe it repeatedly with an alcohol cotton ball to fully dilate the tail vein. Use a disposable 1mL sterile syringe (B. Braun, #1.0ml U-40 type 7) to administer the drug. Slowly push the injection into the mouse through the tail vein. The injection time must be greater than 10s, and the injection speed should be slow and uniform. After the injection is completed, gently pull out the needle and use a clean dry cotton ball to compress the injection site to stop bleeding.

[0052] The injection method for the external jugular vein of golden hamsters is as follows:

[0053] The hamster's neck skin was depilated with an animal razor and anesthetized with isoflurane gas. Under a dissecting microscope, the hamster's clavicle was located. Above the clavicle, just to the right of the trachea, the skin was incised with surgical scissors. Forceps were used to separate the skin, subcutaneous muscle, fat, and other tissues to avoid bleeding. Under a dissecting microscope, the hamster's external jugular vein was located and the surrounding tissue was separated as much as possible. Administration was performed using a disposable sterile 1 mL syringe (B. Braun, #1.0 mL U-40 Type 7). After withdrawing the appropriate viral sample, the syringe needle was gently bent using forceps. Gently lift the upper end of the vein with forceps, keeping it straight. The viral sample was then inserted into the vein using the syringe described above and slowly injected. After the injection was complete, the syringe needle was left in the vein. A piece of autologous fat of appropriate size was pre-cut and used to cover the injection site upon needle removal to promote hemostasis. After confirming that there is no bleeding at the injection site, the surgical wound is sutured using a surgical needle and suture thread to suture the muscle layer and skin layer respectively. The surgical site is disinfected with iodine and appropriate postoperative care is provided.

[0054] Mouse / golden hamster serum is obtained as follows:

[0055] The day before collection, mice / hamsters were fasted overnight but not water. The next day, mice / hamsters were anesthetized with isoflurane gas anesthesia, and 200 μl of blood was collected from the orbital venous plexus. The whole blood was kept at room temperature for at least 1 hour and incubated at 5000 rpm / min for 15 minutes to obtain serum. Serum was stored at -20°C for one week and at -80°C for long-term storage.

[0056] The method for Oil Red staining of mouse aortic arch is as follows:

[0057] Place the mouse in a small animal anesthesia machine for anesthesia and euthanasia. Fix under a dissecting microscope, disinfect the mouse's abdomen with alcohol, use surgical instruments to cut the mouse open, expose the abdominal cavity and chest cavity, and find the complete blood vessels to be stripped, including the aortic arch. Perform cardiac perfusion on the mouse with 1×PBS. Stop the perfusion when the outflowing perfusion fluid is no longer bloody. Use ophthalmic scissors and ophthalmic forceps to completely strip out the mouse's blood vessels and try to remove the fat around the blood vessels. Cut the stripped blood vessels in the middle so that they can be spread flat on a glass slide, including the vascular branches near the aortic arch, which also need to be cut one by one. Place the cut complete blood vessels in a 1×PBS solution.

[0058] Before use, thoroughly mix 6 parts of saturated Oil Red O dye solution with 4 parts of distilled water, then filter with qualitative filter paper to obtain Oil Red O working solution. Keep the working solution away from light. Remove the blood vessels from 1×PBS and spread them flat on a glass slide with the blood vessel plaque side facing up. Use the prepared Oil Red O working solution to stain the blood vessels spread on the slide for about 7 minutes, keeping them away from light throughout the process. Rinse the stained blood vessels with 75% alcohol solution until the part without plaques is colorless, and place them in 4% paraformaldehyde solution for fixation. Spread the fixed stained blood vessels flat on a glass slide for scanning and photographing. During the photographing process, drip 1×PBS on the slide in time to keep the blood vessels moist.

[0059] The method for measuring serum LDL-C and TC is as follows:

[0060] The total cholesterol (TC / TCH) assay kit (Cat. No. A111-1-1, Nanjing Jiancheng Bioengineering Institute) was used to measure the serum TC concentration. The low-density lipoprotein cholesterol (LDL-C) assay kit (Cat. No. A113-1-1, Nanjing Jiancheng Bioengineering Institute) was used to measure the serum LDL-C concentration.

[0061] Example 1 Construction of adeno-associated virus vector and virus production, purification and quantification

[0062] 1.1 Codon optimization of the nucleic acid sequence encoding human low-density lipoprotein receptor

[0063] The nucleic acid sequence encoding the wild-type human low-density lipoprotein receptor (hLDLR WT) (GeneID: 3949) was found in the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, and its sequence is specifically shown in SEQ ID NO: 1. The NCBI accession number of hLDLR WT is NP_000518.1.

[0064] The nucleic acid sequence encoding the wild-type human low-density lipoprotein receptor (WT hLDLR) was codon-optimized to obtain codon-optimized encoding nucleic acid sequences, denoted as hLDLR opt1-17, whose nucleotide sequences are shown in SEQ ID NOs: 2-18, respectively.

[0065] 1.2 Construction of hLDLR expression cassette

[0066] The structure of the hLDLR expression cassette is shown in Figure 1, wherein the hLDLR expression cassette comprises, from the 5' end to the 3' end, the 5' ITR, the ApoE HCR enhancer, the SerpinA1 promoter, the truncated SerpinA1 intron, the Kozak sequence, the target gene (hLDLR WT or codon-optimized hLDLR opt1-17), the BGH polyA, and the 3' ITR. In the hLDLR expression cassette:

[0067] The nucleotide sequence of 5'ITR is shown in SEQ ID NO: 19;

[0068] The ApoE HCR enhancer is the hepatocyte control region of human apolipoprotein E, and its nucleotide sequence is shown in SEQ ID NO: 20;

[0069] SerpinA1 promoter is human α1 antitrypsin promoter, and its nucleotide sequence is shown in SEQ ID NO: 21;

[0070] The SerpinA1 intron is a truncated α1 antitrypsin intron, 261 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 22;

[0071] The Kozak sequence was inserted before the hLDLR gene sequence, and its nucleotide sequence was: gccacc;

[0072] BGH polyA is the bovine growth hormone polyadenylation signal, the nucleotide sequence of which is shown in SEQ ID NO: 23;

[0073] The nucleotide sequence of 3'ITR is shown in SEQ ID NO:24;

[0074] Exemplarily, an expression cassette containing wild-type human low-density lipoprotein receptor (hLDLR WT) comprises, from the 5' end to the 3' end, 5' ITR, ApoE HCR enhancer, SerpinA1 promoter, truncated SerpinA1 intron, Kozak sequence, target gene (hLDLR WT), BGH polyA and 3' ITR; its nucleotide sequence is shown in SEQ ID NO: 25.

[0075] The expression cassette containing the codon-optimized human low-density lipoprotein receptor (hLDLR opt1-17) comprises, from the 5' end to the 3' end, a 5' ITR, an ApoE HCR enhancer, a SerpinA1 promoter, a truncated SerpinA1 intron, a Kozak sequence, the target gene (codon-optimized hLDLR opt1-17), a BGH polyA, and a 3' ITR; the nucleotide sequence of the hLDLR opt1-17 expression cassette is obtained by replacing the nucleotide sequence of hLDLR WT with one of the nucleotide sequences of hLDLR opt1-17 based on the nucleotide sequence of the hLDLR-WT expression cassette (taking the hLDLR opt1 expression cassette as an example, it is obtained by replacing the nucleotide sequence of hLDLR WT with the nucleotide sequence of hLDLR opt1 based on the nucleotide sequence of the hLDLR-WT expression cassette). Since the nucleotide sequences of hLDLR opt1-17 have been described above, the nucleotide sequences of the hLDLR-opt1-17 expression cassettes are clear.

[0076] 1.3 Production and purification of adeno-associated virus

[0077] The expression cassette in step 1.2 was constructed into a shuttle plasmid (the plasmid containing hLDLR WT was designated as pAAV8-ATT-LDLR-WT, and its nucleotide sequence is shown in SEQ ID NO: 26; the plasmid containing codon-optimized hLDLR opt1 to 17 was designated as pAAV8-ATT-LDLR-opt1 to 17). pAAV8-ATT-LDLR-opt1 to 17 were obtained by replacing the nucleotide sequence of hLDLR WT with one of the nucleotide sequences of hLDLR opt1 to 17 on the basis of pAAV8-ATT-LDLR-WT (taking pAAV8-ATT-LDLR-opt1 as an example, it was obtained by replacing the nucleotide sequence of hLDLR WT with the nucleotide sequence of hLDLR opt1 on the basis of the nucleotide sequence of pAAV8-ATT-LDLR-WT). Since the nucleotide sequences of hLDLR opt1 to 17 have been described above, the nucleotide sequences of pAAV8-ATT-LDLR-opt1 to 17 are clear. A three-plasmid system (shuttle plasmid pAAV8-ATT-LDLR-WT or pAAV8-ATT-LDLR-opt1~17, pRepCap plasmid carrying AAV vector rep and cap genes (its nucleotide sequence is shown in SEQ ID NO:27, and synthesized by Jin Weizhi) and helper plasmid pHelper (its nucleotide sequence is shown in SEQ ID NO:28, and synthesized by Jin Weizhi) were used to co-transfect HEK293 cells with PEI as the transfection reagent to recombinantly package the AAV viral vector; the cells were harvested 48 hours after transfection, and the harvested fluid was purified to obtain recombinant AAV viral vectors of a certain purity, which were named AAV8-ATT-LDLR-WT and AAV8-ATT-LDLR-opt1~17, respectively.

[0078] The purification method is as follows:

[0079] First, the harvested fluid was pretreated: HEK293 cells were fully lysed to release the AAV viral vector in the cells, and nuclease was added to digest the free nucleic acid. After digestion, the supernatant was removed by centrifugation to remove cell debris, and the filtrate was filtered through a 0.22 μm filter membrane for affinity chromatography.

[0080] Affinity chromatography utilizes the specific adsorption of ligands and proteins to capture the AAV viral vector in the harvested fluid and remove most process-related impurities, achieving both concentration and impurity removal. The collected eluate is mixed, neutralized with neutralization buffer, and stored in a sterile reservoir bottle as the sample for anion chromatography.

[0081] Anion chromatography uses the difference in isoelectric points of different components to separate solid and empty AAV viruses, while continuing to remove residual impurities. The eluate is collected in a new sterile storage bottle and then replaced with a buffer solution that is stable for the preparation by ultrafiltration concentration. At the same time, the virus titer is concentrated to about 1×10 13 vg / mL, and finally sterilize, filter, and aliquot for later use.

[0082] 1.4 Quantification of AAV viral vector titer

[0083] The primer probe was designed based on the consensus sequence BGH PolyA of the AAV viral vector, and its sequence is shown below:

[0084] F primer sequence: 5′-tgccttccttgaccctgg-3′ (SEQ ID NO: 29);

[0085] R primer sequence: 5′-actcagacaatgcgatgcaa-3′ (SEQ ID NO: 30);

[0086] Probe sequence: 5′-cactcccactgtcctttcctaata-3′ (SEQ ID NO: 31);

[0087] Q-PCR was then performed to quantify the AAV viral vector.

[0088] Among them, in the process of genome titer detection, the standard curve must be established first, and the positive standard plasmid is diluted to 2×10 7 , 2×10 6 , 2×10 5 , 2×10 4 , 2×10 3 , 2×10 2 Copies / μl, as a standard curve template, the standard curve needs to control its linearity and amplification efficiency, generally requiring R 2 >0.99, with an amplification efficiency between 90% and 110%. Purified rAAV viral vector (rAAV) samples were then diluted and tested with Q-PCR to ensure the sample Ct value was within the standard curve range. The rAAV sample genome titer was calculated based on the sample Ct value substituted into the standard curve to quantify the product content.

[0089] Example 2 In vitro expression detection of codon-optimized hLDLR opt

[0090] In this example, the in vitro expression level of codon-optimized hLDLR opt was evaluated in the HepG2 hepatocyte cell line (purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number: TCHu72).

[0091] The shuttle plasmids pAAV8-ATT-LDLR-WT and pAAV8-ATT-LDLR-opt1-17 in step 1.3 of Example 1 were transfected into HepG2 cells using liposome transfection to transiently transfect and express the target gene. Western blotting analysis was then performed to determine the expression level of the target protein in the HepG2 cell lysate. The results are shown in Figure 2.

[0092] Wherein, the method of liposome transfection plasmid is as follows:

[0093] Human hepatoma cell line HepG2 was digested and 1×10 5 Cells were seeded into 24-well plates. Lipo3K / DNA transfection complex was added for plasmid transfection. Lipofectamine 3000 and P3000 (Thermo, L3000015) transfection reagents were premixed with the plasmids and added to HepG2 cells in a 24-well plate at a concentration of 240 ng / well of pAAV8-ATT-LDLR-WT or pAAV8-ATT-LDLR-opt1-17 shuttle plasmid, 360 nL / well of 3000, and 360 nL / well of Lipo 3000 (Thermo, L3000015). The cells were then cultured in a CO2 incubator for 48 h.

[0094] The Western blot analysis method is as follows:

[0095] Cultured cells were seeded in a 24-well plate and lysed for 5 minutes in RIPA lysis buffer (Beyotime, P0013B). The cells were then incubated on ice for 30 minutes. The protein lysate was harvested and incubated on ice for 30 minutes. The supernatant was then collected by centrifugation. After adding protein loading buffer to the protein sample, the cells were denatured at 95°C for 5 minutes. Twenty μL of protein was loaded onto a SDS-PAGE electrophoresis plate and transferred to a PVDF membrane. The membrane was incubated with anti-LDLR (abcam, ab52818), anti-Flag (abcam, ab49763), and anti-GAPDH (TransGen, HC301) antibodies. Images were analyzed using a ChemiDoc Touch Imaging System (Bio-Rad) and grayscale analysis was performed using Image J software.

[0096] As can be seen from Figure 2, the expression level of a portion of the codon-optimized hLDLR opt gene is significantly higher than that of the wild-type hLDLR WT gene. Therefore, in the present invention, through careful design and extensive experiments, the inventors obtained the nucleotide sequences of hLDLR opt2 (as shown in SEQ ID NO: 3), hLDLR opt3 (as shown in SEQ ID NO: 4), hLDLR opt4 (as shown in SEQ ID NO: 5), hLDLR opt10 (as shown in SEQ ID NO: 11), hLDLR opt11 (as shown in SEQ ID NO: 12), and hLDLR opt15 (as shown in SEQ ID NO: 16) with high expression levels.

[0097] Example 3: Testing of codon-optimized hLDLR opt for lipid uptake in hepatocytes

[0098] The recombinant viruses AAV8-hLDLR-WT, opt2, opt3, opt4, opt10, and opt15 prepared in Example 1 were infected at different doses of 2E+5, 4E+5, and 8E+5 MOI, respectively, into human hepatoma cells huh7 (purchased from the cell bank of the Committee for Typical Culture Collection of the Chinese Academy of Sciences, catalog number: TCHu182) (1E+5 cells / well) seeded in a 24-well plate one day in advance, and 300 μl of a working solution containing 10 μg / ml of Dil-LDL (YEASEN, 20614ES76) was added. The fluorescence signal intensity was tested using the PE channel in FACS (excitation light 549 nm, emission light 565 nm). The results are shown in Figure 3.

[0099] Figure 3 shows that under the three different infection doses, hepatocyte lipid uptake capacity increased with increasing infection dose, indicating that the codon-optimized genes significantly enhanced hepatocyte lipid uptake compared to the wild-type gene. Furthermore, although opt10 and opt2 were expressed at lower levels than opt3 in transfected cells, they were more effective in enhancing lipid uptake. The recombinant virus AAV8-hLDLR-opt10 exhibited the highest lipid uptake capacity, followed by AAV8-hLDLR-opt2.

[0100] Example 4 AAV8 recombinant virus carrying the codon-optimized hLDLR gene was used to express LDLR in high-fat diet - / - Study on the effect of lowering LDL-C levels in mouse models

[0101] LDLR at 6 weeks of age - / -Male mice were fed a high-fat and high-cholesterol diet (2% fat + 0.15% cholesterol, Rudi Biotechnology, D12079B) for 8 weeks, and the LDL-C content in the mouse serum was tested. When the LDL-C content was 10 mM, the mice were divided into 6 groups, with 5 mice in each group. Each group of mice was injected with a single tail vein injection of AAV8-hLDLR-WT, opt2, opt3, opt4, opt10, and opt15 recombinant viruses (prepared in Example 1) at a dose of 8E+10 vg / kg. The LDL-C content in the mouse serum was detected in the 1st, 2nd, and 3rd weeks, and the hLDLR concentration in the mouse serum was detected in the 3rd week. The results are shown in Figure 4.

[0102] As can be seen from Figures 4A and 4B, after drug treatment, the LDL-C content in the mouse serum gradually decreased, and the recombinant viruses AAV8-hLDLR-opt2 and AAV8-hLDLR-opt10 statistically significantly reduced the LDL-C in the mice.

[0103] Based on the results of Figures 4A and 4B , the recombinant virus AAV8-hLDLR-opt10, which has a better lipid-lowering effect and a higher concentration of hLDLR in mouse serum, was named NGGT-006.

[0104] As can be seen from Examples 1-4, the present invention first performs codon optimization on the nucleic acid sequence encoding the human low-density lipoprotein receptor (hLDLR) to obtain several codon-optimized coding nucleotide sequences (hLDLR opt1-17), and assembles them in combination with expression control elements that are beneficial to liver cell expression to prepare a shuttle plasmid, which is transfected into a liver cell host cell via liposomes. The protein expression levels of the codon-optimized coding nucleic acid sequences are compared and screened by protein immunoblotting (WB) to obtain sequences with significantly improved expression levels (hLDLR opt2, hLDLR opt3, hLDLR opt4, hLDLR opt10, hLDLR opt11, and hLDLR opt15 nucleotide sequences). The sequences with higher expression levels are further compared in terms of in vitro lipid uptake activity, thereby obtaining sequences with higher lipid uptake capacity, hLDLR-opt2 and hLDLR-opt10. Finally, a three-plasmid system is used to package the candidate optimized gene into a recombinant virus of the AAV8 serotype in HEK293 cells, and the recombinant virus is expressed in high-fat fed LDLR mice. - / - The lipid-lowering activity of the candidate sequences was compared in a mouse model, and further comparison revealed sequences (hLDLR-opt2 and hLDLR-opt10) with better LDL-C lowering effects at the same viral dose.

[0105] For example, the recombinant virus NGGT-006 is used for long-term lipid-lowering studies in subsequent animal experiments.

[0106] Example 5 AAV8 recombinant virus carrying hLDLR gene reduces LDLR- / -Dose response of LDL-C and TC in mouse models

[0107] LDLR at 6 weeks of age - / - Male mice were fed a high-fat and high-cholesterol diet (2% fat + 0.15% cholesterol, Rudi Biotechnology, D12079B) for 8 weeks, and the LDL-C content in the mouse serum was tested. When the LDL-C content was 10 mM, the mice were randomly divided into three groups, 5 mice in each group, and given a single tail vein injection of AAV8-hLDLR-WT virus (prepared in Example 1) at different doses of 8E+10 vg / kg, 4E+11 vg / kg, and 2E+12 vg / kg in a gradient manner. The LDL-C and TC levels in the mouse serum were detected at the 1st, 2nd, 3rd, 4th and 5th weeks, and the protein expression of hLDLR in the mouse liver was detected at the 5th week. The results are shown in Figure 5.

[0108] As can be seen from Figure 5A, with the increase of the dosage, the protein expression of hLDLR in the liver increased significantly; as can be seen from Figure 5B, with the increase of the dosage, the levels of LDL-C and TC in the serum also decreased accordingly. When the lowest dose of 8E+10vg / kg was given, the decrease in LDL-C and TC was about 50%, while when the high dose of 2E+12vg / kg was given, the decrease in LDL-C and TC could reach more than 90%. The results showed that AAV8 carrying the recombinant virus expressing the wild-type hLDLR gene can significantly reduce the levels of LDL-C and TC in the serum of FH mice, and there is a dose-dependent phenomenon.

[0109] Example 6 NGGT-006 recombinant virus continuously reduces LDLR in high-fat diet - / - LDL-C and TC levels in mouse models

[0110] LDLR in 6-week-old males of the same sex - / -Mice were fed a high-fat, high-cholesterol diet (2% fat + 0.15% cholesterol, Rudi Biotech, D12079B) for 7 weeks to establish a hypercholesterolemia model (FH). After the LDL-C content in the mouse serum was detected to 10 mM, they were randomly divided into two groups, 5 mice in each group, and given a single tail vein injection of AAV8-hLDLR-WT virus and NGGT-006 virus (prepared in Example 1) at a dose of 8E+10 vg / kg, respectively. The LDL-C and TC levels in the mouse serum were measured after injection and at 1, 2, 3, 4, 8, 16, and 19 weeks. The results are shown in Figure 6.

[0111] As can be seen in Figure 6A, both the AAV8-delivered wild-type hLDLR gene and NGGT-006 were able to significantly reduce LDL-C by more than 40% starting from week 1, and this LDL-C-lowering effect was sustained for more than 12 weeks. However, at this dosage and in the high-fat-fed FH model, the AAV8-delivered wild-type hLDLR gene increased LDL-C levels to above 10 mM at week 16, while NGGT-006 maintained LDL-C levels below 10 mM until week 19. As can be seen in Figure 6B, both the AAV8-delivered wild-type hLDLR gene and NGGT-006 were able to significantly reduce TC by more than 45% starting from week 1. However, starting from week 4, TC levels in mice with the AAV8-delivered wild-type hLDLR gene increased to above 10 mM, while NGGT-006 maintained LDL-C levels around 10 mM until week 19. The results show that the optimized NGGT-006 designed in the present invention not only has a higher expression capacity than the wild-type hLDLR gene, but also has better pharmacokinetics in the body, and can maintain a therapeutically significant lipid-lowering effect over a longer time window, thus having the potential to significantly reduce the frequency of dosing and will have better application development value in clinical practice.

[0112] Example 7 NGGT-006 recombinant virus in high-fat fed LDLR - / - Sustained and effective reduction of LDL-C and TC levels in blood and reduced plaque deposition in mouse models

[0113] LDLR in 6-week-old males of the same sex - / -Mice were fed a high-fat, high-cholesterol diet (2% fat + 0.15% cholesterol, Rudi Biotech, D12079B) for 7 weeks to establish a hypercholesterolemia model (FH). After the LDL-C content in the mouse serum was detected to 10 mM, the mice were randomly divided into two groups, each with 5 mice. Each group was given a single tail vein injection of NGGT-006 virus (prepared in Example 1) at a dose of 4E+11 vg / kg and an equal volume of solvent control. The LDL-C and TC levels in the mouse serum were measured after injection and at 1, 2, 5, 9 and 12 weeks. At the end of the 12-week experiment, the mice were sacrificed and the whole aorta was isolated. The size and distribution of atherosclerotic plaques were observed by Oil Red O staining. The results are shown in Figure 7.

[0114] As can be seen from Figure 7A, compared with the vehicle control group, the drug-treated group can significantly reduce the LDL-C and TC levels in the mouse serum, and can maintain the LDL-C and TC levels at a low level for at least 12 weeks; as can be seen from Figures 7B and 7C, Oil Red O staining showed that FH mice given a dose of 4E+11 vg / kg of NGGT-006 and continuously fed with high fat and high cholesterol for 19 weeks (7 weeks of high fat feeding before grouping + 12 weeks of observation period after drug administration) had only mild atherosclerosis, while the vehicle control group mice had severe atherosclerosis in the entire aorta and aortic sinus after 19 weeks of feeding a high-fat and high-cholesterol diet, and the area of ​​aortic deposition in the drug-treated group was significantly lower than that in the vehicle group. The results showed that the drug-treated group can significantly reduce the LDL-C and TC levels in the mouse serum. At the same time, 3 months after administration, the area of ​​atherosclerotic plaques in the drug-treated group mice was reduced by approximately 80% compared with the control group.

[0115] Example 8 NGGT-006 and TBG.PI.hLDLRco.RGB recombinant viruses in high-fat fed LDLR - / - Comparative analysis of the effects of lowering blood LDL-C and TC in mouse models

[0116] In Example 7, NGGT-006 was 11 vg / kg dose significantly reduced the LDL-C and TC levels in the mouse serum, and at the same time reduced the area of ​​atherosclerotic plaques in the mice, and had a significant lipid-lowering effect in the FH mouse model.

[0117] At the same time, according to existing public reports, the sequence TBG.PI.hLDLRco.RGB (disclosed in U.S. Patent US20190002917A1) in ongoing clinical research (NCT02651675) was used as a control group to compare it with the NGGT-006 disclosed in this article in terms of LDLR - / - Differences in lipid-lowering effects in FH mouse models.

[0118] LDLR in 6-week-old males of the same sex - / -Mice were fed a high-fat, high-cholesterol diet (2% fat + 0.15% cholesterol, Rudi Biotech, D12079B) for 7 weeks to establish a hypercholesterolemia model (FH). After the LDL-C content in the mouse serum was detected to 10 mM, they were randomly divided into two groups, each with 5 mice. Each group was given a single tail vein injection of NGGT-006 (prepared in Example 1) and TBG.PI.hLDLRco.RGB AAV8 recombinant virus at a dose of 8E+10 vg / kg, respectively. The LDL-C and TC levels in the mouse serum were measured at 1, 2, 3, 4, and 8 weeks after injection.

[0119] As can be seen from Figure 8, at a dose of 8E10vg / kg, NGGT-006 can significantly reduce the levels of LDL-C and TC in the blood, while the use of the same dose of TBG.PI.hLDLRco.RGB AAV8 cannot reduce the levels of LDL-C and TC in the blood. According to US20190002917A1, TBG.PI.hLDLRco.RGB did not observe any effect of reducing TC content when the amount of administered virus was 1.5E+11GC / kg (same vg / kg), and it was only at 5.0E+11GC / kg that the effect of significantly reducing TC content was observed. It can be seen that the NGGT-006 disclosed herein can achieve the effect of reducing TC content and LDL-C content at a significantly lower dose (reduced by about 6 times). The reduced effective dosage also means that the NGGT-006 disclosed herein has a higher safety potential.

[0120] Example 9 NGGT-006 recombinant virus continuously and stably reduces LDLR in high-fat diet - / - LDL-C and TC levels in golden hamsters

[0121] LDLR at 6 weeks of age - / - Golden hamsters were fed a high-fat, high-cholesterol diet (15% fat + 0.5% cholesterol, Rudi Biotechnology, D06050505) to establish a hypercholesterolemia model (FH). After the LDL-C level in the hamster serum was measured to 10 mM, the hamsters were randomly divided into a male experimental group, a male control group, a female experimental group, and a female control group, with 5 hamsters in each group. The experimental group received a single external jugular vein injection of 1.5E+13 vg / kg of NGGT-006 virus (prepared in Example 1), while the control group received an equal volume of vehicle. The LDL-C and TC levels in the hamster serum were measured at different time points after injection. The results are shown in Figure 9.

[0122] As shown in Figure 9, NGGT-0006 has an effect on the LDLR of females and males. - / -Homozygotes all showed significant reductions in serum LDL-C and TC levels, and this lipid-lowering effect lasted for at least 4 weeks, suggesting that gene therapy based on the optimized sequence disclosed herein can achieve long-term, sustained, and stable reductions in LDL-C and TC with just a single administration.

[0123] Example 10 NGGT-006 recombinant virus effectively reduces natural dietary LDLR + / - LDL-C and TC levels in golden hamsters

[0124] In order to investigate the lipid-lowering effect of NGGT-006 in LDLR heterozygous genotype, this example will conduct a natural diet (SPF rat maintenance feed, purchased from Pizhou Xiaohe Technology Development Co., Ltd.) of LDLR + / - Golden hamsters (30 weeks old, female) were randomly divided into an experimental group and a control group, with 5 hamsters in each group. At week 0, 1.5E+13 vg / kg of NGGT-006 virus (prepared in Example 1) and an equal volume of vehicle were administered via a single external jugular vein injection. Serum LDL-C and TC levels were measured at various time points after injection. The results are shown in Figure 10.

[0125] As can be seen from the figure, under normal dietary conditions, the serum LDL-C and TC levels of heterozygous golden hamsters were slightly elevated (control group); in contrast, one week after receiving NGGT-006 administration, the serum LDL-C and TC levels were significantly reduced (to about 50%), and this reduction effect can be maintained for a long time (at least 20 weeks). The above results show that the lipid-lowering effect of NGGT-006 is effective not only for homozygous genotypes lacking endogenous LDLR expression, but also for LDLRs with a certain amount of endogenous LDLR gene expression. + / - In heterozygous genotypes, the blood lipid lowering effect can also be effectively achieved, suggesting that gene therapy based on the optimized sequence disclosed in this article is clinically applicable to LDLR - / - Homozygous population and LDLR + / - Potential of heterozygous populations.

[0126] The above embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field should understand that any technician familiar with this technical field can still modify the technical solutions recorded in the above embodiments within the technical scope disclosed by the present invention, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered by the protection scope of the present invention.

Claims

1. A nucleic acid molecule encoding a human low density lipoprotein receptor, characterized in that, Its nucleotide sequence is as shown in any one of SEQ ID NO: 2-18; preferably, the nucleotide sequence is as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO:

16.

2. A transgenic expression cassette, characterized in that, It contains the nucleic acid molecule described in claim 1.

3. The transgenic expression cassette according to claim 2, wherein The transgenic expression cassette further contains a promoter, an intron and a terminator. The 3' end of the intron is operably linked to the 5' end of the nucleic acid molecule, the 3' end of the promoter is operably linked to the 5' end of the intron, and the 3' end of the nucleic acid molecule is operably linked to the 5' end of the terminator.

4. The transgenic expression cassette according to claim 3, wherein The promoter is selected from the SerpinA1 gene promoter, the SerpinG1 gene promoter, the ALB gene promoter, the SerpinA1 gene promoter, the HLP gene promoter or the LP gene promoter. Preferably, the promoter is the SerpinA1 gene promoter. More preferably, the nucleotide sequence of the promoter is as shown in SEQ ID NO: 21; and / or, The intron is a truncated SerpinA1 intron. Preferably, the nucleotide sequence of the truncated SerpinA1 intron is as shown in SEQ ID NO: 22; and / or, The terminator is selected from BGH polyA, hGH polyA or GH polyA. Preferably, the terminator is BGH polyA. More preferably, the nucleotide sequence of BGH polyA is as shown in SEQ ID NO:

23.

5. The transgenic expression cassette according to any one of claims 3-4, characterized in that, The transgenic expression cassette further includes a 5' ITR, a 3' ITR and an enhancer. The 5' ITR is located at the 5' end of the transgenic expression cassette, the 3' ITR is located at the 3' end of the transgenic expression cassette, and the 3' end of the enhancer is operably linked to the 5' end of the promoter.

6. The transgenic expression cassette according to claim 5, characterized in that, The enhancer is the ApoE HCR enhancer. Preferably, the nucleotide sequence of the ApoE HCR enhancer is as shown in SEQ ID NO: 20; Furthermore, the nucleotide sequence of the transgenic expression cassette is as shown in SEQ ID NO:

32.

7. A recombinant adeno-associated virus, characterized in that, It includes an AAV capsid and the nucleic acid molecule described in claim 1 or the transgenic expression cassette described in any one of claims 2-6; preferably, the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or rhAAV10; more preferably, the AAV is AAV8.

8. A composition, characterized in that, It includes the nucleic acid molecule described in claim 1, the transgenic expression cassette described in any one of claims 2-6 or the recombinant adeno-associated virus described in claim 7. Preferably, the composition is a pharmaceutical composition. More preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

9. Use of the nucleic acid molecule according to claim 1, the transgenic expression cassette according to any one of claims 2-6, the recombinant adeno-associated virus according to claim 7 or the composition according to claim 8 in the preparation of a medicament for treating hypercholesterolemia, preferably, the hypercholesterolemia is familial hypercholesterolemia.

10. The application according to claim 9, wherein, The familial hypercholesterolemia includes homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia.

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