Non-alcoholic fatty liver disease mouse model based on humanized PNPLA3 I148M transgene as well as construction method and application of non-alcoholic fatty liver disease mouse model

Abstract

The invention belongs to the technical field of animal model construction, and particularly relates to a non-alcoholic fatty liver disease mouse model based on humanized PNPLA3 I148M transgene as well as a construction method and application thereof, the construction method comprises the following steps: step i: designing CDS of a human gene and a key 3 'UTR element for patent medicine of a small nucleic acid drug in gene plasmid DNA of the constructed model; step ii, using Luc2 or iRFP to report gene expression in the gene plasmid DNA for constructing the model; and (iii) a disease model of the fatty liver disease induced by combining plasmid DNA transient transfer with high-fat diet. The mouse model constructed by the invention has the advantages that a specific exogenous gene expression signal of the liver is detected in real time through small animal living imaging, a human PNPLA3 protein is detected and overexpressed through serum ELISA, PNPLA3 I148M mRNA is detected and overexpressed through liver RT-qPCR, the phenotype of the non-alcoholic fatty liver disease of the liver is determined through 15-week-old pathology, and the whole process of modeling for 8 weeks does not need to be operated in a biosafety secondary laboratory.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a non-alcoholic fatty liver disease mouse model based on humanized PNPLA3I148M transgene, its construction method, and its application. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to a wide range of liver diseases that can progress from simple fatty liver (steatohepatitis) to non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis (irreversible late-stage scarring of the liver), liver failure, and even end-stage hepatocellular carcinoma (HCC). All stages of NAFLD share the common feature of fat accumulation and infiltration in hepatocytes. NASH is a more severe form of NAFLD, characterized by more than 5% hepatocyte steatosis combined with intralobular inflammation and ballooning degeneration of hepatocytes, with or without liver fibrosis.

[0003] NAFLD is the most common form of liver disease in Western countries, and about 10% of liver transplants performed in the United States are for cirrhosis associated with NAFLD[1]. In developing countries, including my country, while living standards have improved and dietary structures have changed, the prevalence of NAFLD, a metabolic disorder caused by multiple factors, is as high as 29.2% among adults in my country, an increase of 10% over the past 10 years. Currently, the global prevalence of NAFLD is about 25%[2], and it is estimated that by 2030, there will be 490 million NAFLD patients worldwide, and the global NAFLD drug market will grow to US$32.2 billion[3]. The FDA has not yet approved any new NAFLD drugs, and new drug development is a blue ocean market. However, constructing suitable animal models for evaluating the efficacy of new NAFLD drugs is the cornerstone of new drug development.

[0004] NAFLD is defined as excessive fat accumulation in the liver exceeding 5% due to causes other than alcohol intake. It has a strong genetic component. Genetic variations of the phospholipase domain protein 3 (PNPLA3) confer susceptibility to fatty liver disease. Researchers conducted genome-wide association studies on multiple ethnic groups and identified that the I148M point mutation of PNPLA3 is closely related to elevated liver fat levels and liver inflammation. This mutation is most common in Hispanics. Patients with homozygous PNPLA3 I148M have a liver fat content >2 times higher [4]. The overall incidence of NAFLD in people with the PNPLA3 I148M marker is as high as 30%-50%, and the carriers of this mutation account for 11% of patients with cirrhosis. In the Han Chinese population, as early as ten years ago, ultrasound confirmed that the PNPLA3 I148M genetic variation is associated with the degree of steatosis in NAFLD patients in the Chinese population [5].

[0005] The mechanism by which PNPLA3 I148M causes NAFLD is that PNPLA3 protein has hydrolytic enzyme activity on triglycerides (TG) and retinyl esters, promoting lipid droplet remodeling in hepatocytes and hepatic stellate cells. However, the enzyme activity of the PNPLA3 I148M mutant protein is reduced, and excessive TG accumulates in the liver, causing fatty liver and liver fibrosis, eventually progressing to liver failure and hepatocellular carcinoma [6].

[0006] Companies such as Arrowhead (Janssen), AstraZeneca (Ionis), Eli Lilly, Regeneron, and Alnylam have developed small nucleic acid drug pipelines targeting PNPLA3 for the treatment of NAFLD. Arrowhead, whose products are already in Phase I / II clinical trials, publicly announced its TRIM-based drug development in February 2023. TM The platform (TRIM) utilizes ligand-mediated delivery to achieve tissue-specific targeting. TMThe platform contains a highly efficient RNA trigger that can optimize the targeting ligand with high affinity, the structure that enhances pharmacokinetics, and the specific nucleic acid sequence for each candidate drug to achieve targeted drug delivery and maintain the best pharmacological activity and safety. The original siRNA drug ARO-PNPLA3[7] (formerly JNJ-75220795 developed by Janssen, a subsidiary of Johnson & Johnson) was developed in a Phase I / II clinical study (NCT04844450). 112 subjects with certain NAFLD genetic predispositions and who were identified as having hepatic steatosis at screening were injected subcutaneously. ARO-PNPLA3 inhibited hepatic steatosis by up to 40% in homozygous patients with the I148M mutation in a dose-dependent manner. PNPLA3 is a popular target for small nucleic acid drugs for the treatment of NAFLD.

[0007] Currently, the best humanized PNPLA3 I148M stable transgenic mouse model available in China [8] has the humanized PNPLA3 coding region (CDS) and 3'UTR non-coding region. This model fully considers that the drug sequences of most commercially available siRNA small nucleic acid drugs are 3'UTR. However, the limitation of this model is that it is not a liver tissue-specific model. The humanized PNPLA3I148M gene is expressed not only in the liver, but also non-specifically in organs such as the kidney, epididymal fat, and subcutaneous fat. Furthermore, the pathological features of fatty liver disease and the biochemical test values ​​of hyperlipidemia in this model have not been disclosed.

[0008] Arrowhead's ARO-PNPLA3, an original siRNA drug targeting PNPLA3 for the treatment of NAFLD, was used to construct a humanized PNPLA3-SEAP transient transformation mouse model using the secretory human placental alkaline phosphatase (SEAP) reporter gene. This model was then tested using Phospha-Light. TM SEAP reporter gene assay kit was used to detect mouse serum and evaluate the efficacy of ARO-PNPLA3 candidate drugs. However, the expression of the model containing human PNPLA3 CDS and 3'UTR uncoding regions was unstable. Finally, the human PNPLA3 cDNA was truncated[7]. Therefore, the transient transformation model could not express human PNPLA3 CDS and 3'UTR sequences at the same time.

[0009] Alnylam disclosed in its patent that the in vivo efficacy evaluation of its PNPLA3-targeting small nucleic acid drug in mice used an AAV8-hPNPLA3 mouse model, which is a transient transfection mouse model. The GLuc fluorescent reporter gene and the human PNPLA3 gene are packaged by adeno-associated virus AAV8, and the efficacy evaluation is carried out 14 days after modeling [9-10]. The human PNPLA3 gene is packaged by AAV8 virus through three plasmid co-transfection. The cell fluid is collected and then purified and concentrated by ultracentrifugation. According to the list of human infectious pathogens formulated by the National Health Commission

[11] , AAV infection animal experiments should be carried out in an animal biosafety level 2 laboratory (ABSL-2).

[0010] However, none of the above-mentioned humanized PNPLA3 I148M transgenic mouse models showed important NAFLD pathological features such as fatty liver disease and hyperlipidemia biochemical test values. The University of Gothenburg in Sweden and AstraZeneca / Ionis collaborated to knock in NAFLD mice with the PNPLA3I148M gene[6] to verify the efficacy of the small nucleic acid drug targeting PNPLA3 ASO in vivo. However, this was a mouse-derived rather than a humanized mouse model. PNPLA3 I148M gene knock-in NAFLD mice were introduced into the mouse genome using homologous recombination. Male mice were fed a high-fat diet and showed significant pathological features of hepatocyte steatosis and hepatocyte ballooning degeneration in the liver. Both the liver and serum showed high TG values. Female mice fed a high-sucrose diet and showed high TG values ​​in the serum. Although not a humanized model, it showed important NAFLD indicators.

[0011] To overcome the limitations of existing models, this invention discloses a method for constructing a mouse model of NAFLD based on humanized PNPLA3I148M transgenic genes. Seven-week-old mice are transiently transgenic and then fed a high-fat diet for eight weeks. This model enables real-time detection of liver-specific exogenous gene expression signals via in vivo imaging, detection of overexpression of humanized PNPLA3 protein via serum ELISA, detection of overexpression of PNPLA3 I148M mRNA via liver RT-qPCR, and pathological identification of a non-alcoholic fatty liver disease phenotype in the liver at 15 weeks of age, all without requiring ABSL-2 manipulation. In addition to the humanized PNPLA3 I148M CDS, this model also possesses the 3'UTR untranslated region sequence commonly used in marketed siRNA small nucleic acid drugs. Besides protein drugs, it is particularly suitable for efficacy evaluation in the development of new small nucleic acid drugs with stringent requirements for nucleotide sequences.

[0012] The references cited in this invention are as follows:

[0013] [1]McCullough AJ.The clinical features,diagnosis and natural historyof nonalcoholic fatty liver disease.Clin Liver Dis.2004Aug;8(3):521-533.

[0014] [2]Luukkonen PK,Porthan K,Ahlholm N,Rosqvist F,Dufour S,Zhang XM, TE, W,Orho-Melander M,Hodson L,Petersen KF,Shulman GI,Yki- H.The PNPLA3I 148M variant increases ketogenesis and decreaseshepatic de novo lipogenesis and mitochondrial function in humans.CellMetab.2023Nov 7;35(11):1887-1896.

[0015] [3]Tan DJH,Ng CH,Lin SY,Pan XH,Tay P,Lim WH,Teng M,Syn N,Lim G,YongJN,Quek J,Xiao J,Dan YY,Siddiqui MS,Sanyal AJ,Muthiah MD,Loomba R,HuangDQ.Clinical characteristics,surveillance,treatment allocation,and outcomes ofnon-alcoholic fatty liver disease-related hepatocellular carcinoma:asystematic review and meta-analysis.Lancet Oncol.2022Apr;23(4):521-530.

[0016] [4] Announcement No. US8785128B2, Genetic diagnosis of hepatic steatosis, The University of Texas system, July 22, 2014.

[0017] [5]Li Y,

[0018] [6]Lindén D,Ahnmark A,Pingitore P,Ciociola E,Ahlstedt I,AndréassonAC,Sasidharan K,Madeyski-Bengtson K,Zurek M,Mancina RM,Lindblom A,Bjursell M, G, M,Bohlooly-Y M,Haynes WG,Carlsson B,Graham M,Lee R,Murray S,Valenti L,Bhanot S, P, Romeo S. Pnpla3 silencing with antisense ol igonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148Mknock-in mice. Mol Metab. 2019Apr; 22:49-61.

[0019] [7] Publication No. CN115397436A, RNAi agents for inhibiting PNPLA3 expression, pharmaceutical compositions thereof and methods of use, Arrowhead, Inc., Publication Date: September 30, 2021.

[0020] [8] B6-hPNPLA3(I148M)mice(Strain NO.T054371) of the GemPharmatech (Nanjing, China) website. https: / / cn.gempharmatech.com / shop / productDetails / 62573.

[0021] [9] Announcement No. US11052103B2, Patatin-like phospholipase domain containing 3 (PNPLA3) iRNA compositions and methods of use thereof, Alnylam, July 6, 2021.

[0022]

[10] Announcement No. US10597661B2, Polynucleotide agents targeting patatin-like phospholipase domain containing 3 (PNPLA3) and methods of use thereof, Alnylam, March 24, 2020.

[0023]

[11] Catalogue of human-transmissible pathogens, National Health Commission of the People's Republic of China, 2023, No. 24 of 2023. Summary of the Invention

[0024] In view of the above situation, and to overcome the shortcomings of existing animal models, the present invention adopts the following technical solution:

[0025] A method for constructing a mouse model of non-alcoholic fatty liver disease based on humanized PNPLA3 I148M transgene, specifically including the following steps:

[0026] Step i: The CDS of the human gene and the 3'UTR element, which is key to the drug development of small nucleic acid drugs, are designed into the gene plasmid DNA for constructing the model; the CDS of the human gene can stably express human PNPLA3 I148M CDS in mouse liver for a long time.

[0027] Among the several marketed siRNA small nucleic acid drugs, the double-stranded siRNA sequences of the finished drugs are mostly located in the 3'UTR. Therefore, for humanized transgenic animal models used to evaluate the efficacy of small nucleic acid drug candidates, it is necessary to consider introducing the 3'UTR gene into the model. However, replacing the 3'UTR of mouse genomic DNA with human DNA through homologous recombination is difficult to achieve in stable transgenic models due to the excessively long sequence. This invention designs the CDS of the human gene and the key 3'UTR element for small nucleic acid drug development into the gene plasmid DNA used to construct the model, enabling the expression of the CDS and 3'UTR genes in humanized mouse models.

[0028] Step ii: Use Luc2 or iRFP to express the reporter gene in the gene plasmid DNA used to construct the model;

[0029] Preferably, the Luc2 reporter gene is used.

[0030] The Luciferase (Luc2) gene, which can detect bioluminescence in small animals by in vivo imaging after the application of a luciferase substrate, is currently the most commonly used reporter gene for in vivo detection. In addition to using Luc2, this invention also selected a chemiluminescent near-infrared fluorescent protein (iRFP), which does not require substrate application or can reduce costs, as a reporter gene, and expressed them separately in the gene plasmid DNA used to construct the model.

[0031] Step iii: Inducing a disease model of fatty liver disease by transtransferring plasmid DNA with a high-fat diet.

[0032] By transfecting human PNPLA3 I148M CDS and 3'UTR plasmid DNA containing a reporter gene into the mouse liver and then cleverly combining it with a high-fat diet, the pathological phenotype of fatty liver disease can be induced in mice in a short period of time. The humanized transgenic disease model disclosed in this invention can be operated entirely in a low biosafety level SPF laboratory, without being restricted by the ABSL-2 operation required for AAV modeling according to regulations.

[0033] In a preferred embodiment, the present invention adopts the following technical solution:

[0034] Step a. Design the CDS with the humanized PNPLA3 gene I148M point mutation into the gene plasmid DNA for constructing the model;

[0035] Step b. Incorporate the 3'UTR of the humanized PNPLA3 gene I148M point mutation into the gene plasmid DNA used to construct the model;

[0036] Preferably, restriction enzyme cleavage sites MluI and SacI are designed at the 5' and 3' ends of the humanized PNPLA3 I148M gene and designed in the gene plasmid DNA for constructing the model, enabling the humanized mouse model to express the CDS and 3'UTR genes.

[0037] Step c. Use Luc2 or iRFP reporter gene to express in the gene plasmid DNA of the constructed model;

[0038] Preferably, sequence P2A is designed between the reporter gene and the humanized gene. When the reporter gene-P2A-human gene DNA is transcribed into mRNA and translated into a multi-protein of reporter protein-P2A-human protein, the P2A protein will self-recognize and cleave, enabling the reporter protein and the human protein to be expressed separately.

[0039] Step d. Amplification, purification, and quality control of the gene plasmid DNA.

[0040] Preferably, the amplification step uses the heat shock method to transform plasmid DNA into Escherichia coli competent cell DH5α, coat it on an LB plate containing 30 μg / ml antibiotic kanamycin sulfate, pick positive clones and inoculate them into LB broth containing 30 μg / ml Kan, shake the bacteria at 37 °C in a shaker for small-scale culture, dilute and inoculate, and shake the bacteria at 37 °C in a shaker overnight for large-scale culture and amplification to obtain amplified bacterial liquid.

[0041] The purification step extracts and purifies plasmid DNA using a plasmid extraction kit. Preferably, the obtained plasmid DNA is pLC-Luc2-P2A-hPNPLA3 I148M-3UTR. Preferably, it is finally quantified to 1 mg / ml and stored at -20 °C.

[0042] Preferably, the quality control step is to measure the A260 / 280 value of the plasmid DNA using a micro-spectrophotometer to be between 1.8 and 2.0. After the DNA fragments obtained by sequencing are spliced, compare with the original design, and if they are consistent, it is judged that the quality control is qualified.

[0043] Step e. Transiently transfect the plasmid DNA and combine it with a disease model of fatty liver disease induced by a high-fat diet.

[0044] Preferably, the plasmid DNA transient transfection step includes designing the plasmid DNA to model at three concentrations of 2.5, 5, and 10 pmol / mouse, using DNA / RNA-free sterile saline as the solvent, preparing a 10% plasmid DNA solution according to the mouse weight, and using 7-week-old wild-type mice of C57BL / 6, BALB / c, or ICR, and transiently transfecting the plasmid DNA into the mice through the tail vein based on the principle of high-speed hydrodynamic.

[0045] Preferably, the plasmid DNA solution is administered via high-velocity hydrodynamic tail vein injection to the mice at 4-week intervals, using a 10% plasmid DNA solution, based on the mice's current body weight.

[0046] Preferably, plasmid DNA is transduced into mice via the tail vein in an average of about 6 seconds.

[0047] Preferably, the high-fat diet is a 45% high-fat feed diet.

[0048] Another aspect of the present invention provides a mouse model of non-alcoholic fatty liver disease based on humanized PNPLA3 I148M transgene.

[0049] In another aspect, the present invention provides an application of the mouse model described above in the preparation or screening of drugs for non-alcoholic fatty liver disease based on humanized PNPLA3 I148M transgene.

[0050] This invention provides a human PNPLA3 I148M transgenic mouse model that exhibits NAFLD pathological characteristics, enables real-time in vivo detection of liver-specific humanized gene expression in mice without sacrificing mice, can be operated in low biosafety level laboratories, and is suitable for evaluating the efficacy of small nucleic acid drugs. The beneficial effects of this invention are as follows: First, the model disclosed in this invention does not sacrifice mice, enabling real-time in vivo visual detection of the humanized PNPLA3 I148M gene, filling the gap in real-time in vivo imaging of the liver using models expressing humanized PNPLA3 I148M; Second, the mouse model disclosed in this invention exhibits the fatty liver pathological phenotype of NAFLD and a high TG value at 8 weeks of modeling (i.e., 15 weeks of model age), expressing pathological indicators earlier than existing stable or transient conversion models, shortening the cycle for humanized PNPLA3 I148M mice to become NAFLD pathological models, thus accelerating drug development; Third, the model disclosed in this invention contains the 3'UTR sequence of humanized PNPLA3 I148M, making it suitable for efficacy evaluation applications of small nucleic acid drugs with stringent requirements for nucleotide sequences; Fourth, the transient conversion model disclosed in this invention reduces biosafety risks compared to the AAV transient conversion model; Fifth, the model disclosed in this invention has a more economical modeling cost, reducing drug development costs; Sixth, the mouse model disclosed in this invention addresses the NAFLD-causing key gene PNPLA3. The I148M transgene is restricted to liver-specific expression in mice, avoiding the expression of the pathogenic PNPLA3 I148M gene in other organs. This solves the difficulty of non-specific expression in organs such as the kidney, epididymal fat, and subcutaneous fat in domestically available stable transgenic humanized PNPLA3 I148M mouse models, in addition to liver expression. Seventh, the mouse model constructed in this invention has the ability to detect liver-specific exogenous gene expression signals in real time using in vivo imaging, detect overexpression of human PNPLA3 protein using serum ELISA, detect overexpression of PNPLA3 I148M mRNA using liver RT-qPCR, and pathologically identify a non-alcoholic fatty liver disease phenotype in the liver at 15 weeks of age. The entire 8-week modeling process does not require ABSL-2 manipulation. Finally, the model disclosed in this invention has the potential to be applied to large animal models.

[0051] Explanation

[0052] A coding sequence (CDS) is a DNA sequence that encodes a protein product and corresponds one-to-one with the protein codons.

[0053] The 3'UTR refers to the 3' untranslated region in a ribonucleic acid (RNA) molecule, which is the part of the protein-coding sequence that has not been translated into protein. It usually contains sequence and structural information about RNA stability, degradation and transport, and may also contain functional elements related to gene expression regulation.

[0054] siRNA refers to small interfering RNA, sometimes called short interfering RNA or silencing RNA, which is a double-stranded RNA that is 20 to 25 nucleotides long.

[0055] P2A, or self-cleaving 2A peptide, is a tool used in molecular biology research. Its principle is to utilize a special peptide chain to cleave protein molecules into two parts. The P2A sequence itself contains a specific nucleic acid sequence. During translation, this nucleic acid sequence is transcribed into a special peptide chain. This peptide chain has an automatic cleaving function, capable of cutting the protein molecule into two parts. The cleaving function of the P2A sequence is achieved through the special structure of the peptide chain, which enables automatic cleavage during translation. Attached Figure Description

[0056] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0057] Figure 1 To construct a plasmid atlas of liver-targeting, in vivo, visually humanized PNPLA3 I148M transgenic mice, among which... Figure 1 A uses the Luc2 gene as a reporter gene. Figure 1 B uses iRFP as a reporter gene, which is expressed in the gene plasmid DNA used to construct the model.

[0058] Figure 2 Real-time images of humanized PNPLA3 I148M transgenic mice were detected using a small animal live imaging system.

[0059] Figure 3 The expression level of human protein in the serum of humanized PNPLA3 I148M transgenic mice was detected by ELISA.

[0060] Figure 4 To detect the mRNA expression level in the liver of humanized PNPLA3 I148M transgenic mice by RT-qPCR.

[0061] Figure 5The pathological phenotype of the liver in humanized PNPLA3 I148M transgenic mice. Figure 5 A shows that the liver structure of the wild-type negative control is clear, the portal area structure is normal, the central vein is visible, the hepatocyte cords are neatly arranged, no inflammatory cell infiltration is seen, and no hepatocyte ballooning degeneration is seen, with a score of 0. Figure 5 BD showed that in the transiently transfected plasmid DNA models of 2.5, 5, and 10 pmol / animal, the hepatic sinusoids were open with mild congestion, irregularly arranged hepatocyte cords, multiple localized focal inflammatory cell infiltrations, and multiple hepatocyte ballooning degenerations. Based on the SAF hepatocyte ballooning degeneration score, Figure 5 B and Figure 5 C shows that plasmid DNA levels of 2.5 and 5 pmol / animal were scored as 1 point in the model groups, respectively. Figure 5 D shows that the plasmid DNA of 10 pmol / animal in the model group scored 2 points. Detailed Implementation

[0062] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0063] Example 1: Design and Construction of Plasmid DNA

[0064] (1) Design of plasmid DNA based on the I148M point mutation of humanized PNPLA3 gene CDS

[0065] PNPLA3 I148M is a publicly recognized genetic diagnostic marker for NAFLD patients. In healthy individuals, the 148th amino acid of PNPLA3 is expressed as isoleucine (Ile, I), nucleotide-coded as AT "C" (Genebank ID: NM_025225.3). However, in patients with non-alcoholic fatty liver disease, this nucleotide-coded sequence is AT "G," and the amino acid expressed is methionine (Met, M). Therefore, the humanized transgenic mouse model of fatty liver disease of this invention must incorporate the CDS of the humanized PNPLA3 gene I148M point mutation into the plasmid DNA used to construct the model.

[0066] (2) Design of plasmid DNA with the 3'UTR of humanized PNPLA3 I148M

[0067] Among the several siRNA small nucleic acid drugs already on the market, the double-stranded siRNA sequences of the finished drugs are mostly located in the 3'UTR, for example... (Patisiran) (Inclisiran) (Lumasiran) and, respectively, target the 3'UTR of the TTR, PCSK9 and HAO1 genes. Small nucleic acid drug sequences designed in the 3'UTR often solve the off-target effect problem better than the CDS region, and the 3'UTR can often screen out small nucleic acid drugs with higher efficacy than the CDS region. Therefore, if the invented model is to be used to evaluate the in vivo efficacy of small nucleic acid drugs in addition to protein drugs, the construction of humanized transgenic animal models must consider the introduction of the 3'UTR gene into the model.

[0068] However, past methods of replacing the 3'UTR in mouse genomic DNA with human-derived DNA via homologous recombination have proven difficult to achieve in stable transformation models due to their excessively long sequences. This invention incorporates the CDS of the human PNPLA3 I148M gene and the crucial 3'UTR element for small nucleic acid drug development, totaling 2,646 bp, with restriction enzyme sites MluI and SacI designed at the 5' and 3' ends respectively. These elements are incorporated into the gene plasmid DNA used to construct the model, enabling the expression of both the CDS and 3'UTR genes in a humanized mouse model.

[0069] (3) The design of reporter genes in plasmid DNA that can be detected by a small animal in vivo imaging instrument that can penetrate the body surface.

[0070] After administering D-Luciferin potassium (Maclean D812647-1g), a luciferase substrate, bioluminescence can be detected by in vivo imaging of small animals using the Luciferase (Luc2, Genebank ID: AY738225.1) gene, which is currently the most commonly used reporter gene for in vivo detection. This invention, in addition to using the Luc2 gene as a reporter gene... Figure 1 In addition to A), a near-infrared fluorescent protein (iRFP, Genebank ID: JN247409.1) was selected as the reporter gene, which is chemiluminescent, does not require substrate application, or can reduce costs. Figure 1 B), each is expressed in the DNA plasmid of the gene used to construct the model.

[0071] Considering that reporter genes require appropriate and correct protein folding to penetrate the body surface and detect reporter genes deep within organs using small animal in vivo imaging, expressing the reporter gene and human gene together as a single multiprotein could increase the risk of incorrect protein folding preventing real-time detection in vivo. Furthermore, by pre-designing the reporter gene-P2A-MCS plasmid vector DNA, the cost of gene synthesis can be reduced by simply replacing the humanized gene with the MCS. Therefore, P2A was designed between the reporter gene and the humanized gene. When the reporter gene-P2A-humanized gene DNA is transcribed into mRNA and translated into a multiprotein of reporter protein-P2A-humanized protein, the P2A protein will self-recognize and cleave, allowing the reporter protein and humanized protein to be expressed separately. The plasmid DNA design described above was outsourced to gene synthesis (Nanjing Genscript Biotech Co., Ltd.).

[0072] Example 2: Scale-up, purification and quality control of plasmid DNA

[0073] Plasmid DNA was transformed into competent *E. coli* cells (DH5α, Novizan C502-03) using the heat shock method. The transformed cells were plated on LB agar plates containing 30 μg / ml kanamycin sulfate (Kan, Aladdin K103024-25g). Positive clones were picked and inoculated into LB broth (Huankai Microbial 28324) containing 30 μg / ml Kanamycin sulfate. The culture was then incubated at 37°C for 3 hours. A 1000-fold dilution was then inoculated, and the culture was incubated overnight at 37°C for large-scale amplification, yielding six 100ml bottles of amplified bacterial culture.

[0074] Plasmid DNA was then purified using a plasmid extraction kit (MACHEREY-NAGEL 740410.50) with six reactions. Following the manual, the high-speed benchtop refrigerated centrifuge (Hunan Xiangyi H2050R) was first replaced with a 50ml rotor and pre-cooled at 4℃; the ELU reagent was preheated in a 37℃ water bath (Shanghai Jinghong DK-S22). The overnight bacterial culture was centrifuged at 6,000g in a high-speed benchtop refrigerated centrifuge for 15 minutes, the supernatant was discarded, and the cells were lysed and resuspended using RES reagent containing RNase A. Cell lysis was further performed using LYS reagent; then NEU reagent was added for acid-base neutralization. The white, egg-drop-like E. coli genomic DNA and plasmid DNA were then purified using… Plasmid DNA was obtained through Xtra column purification. After washing twice, 5 ml of plasmid DNA from each reaction was extracted using preheated ELU reagent. The plasmid DNA was then precipitated and concentrated with isopropanol. The six reactions were collected together and finally quantified to 1 mg / ml and stored at -20°C.

[0075] For the preliminary quality control of plasmid DNA, a NanoDrop spectrophotometer (Aosheng Nano-300) was used to measure the A260 / 280 value of the plasmid DNA, which should be between 1.8 and 2.0. Further, multiple primers were used, and sequencing was commissioned (Youkang Biotechnology). After the DNA fragments obtained by sequencing were assembled and compared with the original design, if they were consistent, the quality control was judged to be qualified.

[0076] Example 3: Method for transient transfection of plasmid DNA combined with a high-fat diet-induced fatty liver disease model

[0077] Taking plasmid DNA pLC-Luc2-P2A-hPNPLA3 I148M-3UTR as an example, the full length is 7,766 bp ( Figure 1 A), with a molecular weight of 4.8 x 10 6 Da, and 1 pmol is equivalent to 4.801 μg of plasmid DNA. The plasmid DNA was designed to be modeled at three concentrations of 2.5, 5, and 10 pmol / mouse, and the unit conversions were 12, 24, and 48 μg / mouse in sequence. Using DNA / RNA-free sterile saline (0.9% NaCl, Meilun MA0083-D) as the solvent, a 10% plasmid DNA solution was prepared according to the mouse body weight. For example, if the body weight was 22 g, 2.2 ml of saline was used to dissolve the plasmid DNA at three concentrations of 2.5, 5, and 10 pmol. Using 7-week-old wild-type (WT) C57BL / 6, BALB / c, or ICR mice, based on the principle of high-speed hydrodynamics, plasmid DNA was transiently transfected into the mice through the tail vein in an average of 6 seconds. For the same plasmid DNA concentration, according to the mouse body weight at that time, a supplementary injection of pLC-Luc2-P2A-hPNPLA3 I148M-3UTR plasmid DNA was given by high-speed hydrodynamic tail vein injection at 11 weeks of age, with a 4-week interval.

[0078] In terms of diet, during the one-week adaptation feeding of 6-week-old mice, regular feed was used to avoid excessive weight gain, which could lead to difficulties in the first high-speed hydrodynamic tail vein injection for modeling. After transient gene transfection of 7-week-old mice, the diet was changed to a 45% high-fat diet (Rodent diet with 45% calories from fat, XTHF45-1 from Xietong Biotechnology). In addition to 43.5% of the maintenance basal feed, its composition was supplemented with 17.5% lard, 12% sucrose, 10% whole milk powder, 13% casein, 2% calcium hydrogen phosphate, etc. The high-fat diet continued until the end of the experiment at 15 to 16 weeks of age. The method of transient transfection of plasmid DNA combined with a 45% high-fat diet-induced modeling was used to obtain a complex mouse model that was both humanized PNPLA3 I148M CDS and 3'UTR transgenic and had non-alcoholic fatty liver disease.

[0079] Example 4: Identification of plasmid DNA-specific expression in mouse liver using small animal in vivo imaging.

[0080] After 8 weeks of feeding on a high-fat diet, the mice were fasted overnight but allowed free access to water the night before in vivo imaging. The following morning, fresh D-fluorescein potassium salt (Axygen E011306-1g) powder was prepared and dissolved in D-PBS (magnesium and calcium ion-free, Biyuntian C0221D) to a concentration of 15mg / ml. The solution was then sterilized through a 0.2μm filter and dispensed into brown 1.5ml EP tubes (Axygen MCT-150-X).

[0081] Mice were anesthetized, and their chest hair was shaved. Mice weighing 25g were injected intraperitoneally with 250μl of D-fluorescein potassium solution. Three concentrations were used: non-transgenic wild-type mice (WT blank), and transiently transfected plasmid DNA at concentrations of 2.5, 5, and 10 pmol / mouse. The mice were placed in a supine position with their chests facing upwards on a small animal in vivo imaging system (Tianneng X6) with a gas-anesthetized hood. The Luc2 signal was captured in bioluminescent mode. Figure 2 As shown, the plasmid DNA pLC-Luc2-P2A-hPNPLA3 I148M-3UTR ( Figure 1 A) The Luc2 protein was successfully expressed specifically in mouse liver, and the signal showed a dose-dependent effect visible to the naked eye, meaning that the intensity of the Luc2 signal was consistent with the trend of plasmid DNA concentration.

[0082] However, under the same experimental conditions, using plasmid DNA pLC-iRFP-P2A-hPNPLA3 I148M-3UTR ( Figure 1 B) Modeling: Neither the Tianneng X6 nor the PE IVIS small animal in vivo imaging system could detect the signal of the iRFP reporter gene. Therefore, iRFP is not suitable for constructing an in vivo visualized humanized transgenic liver model. This invention... Figures 2 to 5 The data presented is based on plasmid DNA pLC-Luc2-P2A-hPNPLA3I148M-3UTR ( Figure 1 A) The constructed transient transgenic mouse model.

[0083] Small animal in vivo imaging devices can acquire images, locate organs, and perform qualitative analysis. However, quantitative analysis is limited due to a high background value. For example, in this case study, even in non-transgenic wild-type mice (WT blanks) without any exogenous Luc2 gene introduction, a background value as high as 5.4 x 10⁻⁶ was still detected. 8 Light intensity (p / sec / cm) 2 The signal of / sr (Table 1) was obtained by transfecting plasmid DNA at three concentrations: 2.5, 5, and 10 pmol / animal, with the concentrations being 9.5 x 10^6 pmol / animal in that order.8 10.8 x 10 8 14.9 x 10 8 Light intensity. If only WT blank and plasmid DNA (2.5 pmol / animal) are compared, the signal intensity for both is 5.4 x 10⁻⁶. 8 and 9.5 x 10 8 In terms of light intensity, the difference between the wild type and the model is indeed not significant in terms of numerical values, so small animal live imaging instruments are not suitable for quantitative analysis.

[0084] Table 1. Raw data from small animal live imaging cameras after background subtraction.

[0085]

[0086] Therefore, in addition to the classic and more expensive PE model, some subsequent more economical small animal in vivo imaging instruments have added the function of automatically removing background values ​​during development. The operation involves setting up a blank mouse, such as the aforementioned WTblank, as a negative control during data analysis. Besides intuitively identifying the specific location of signals in organs during image acquisition, it also allows for the selection of regions with consistent area for comparison to remove background values. The mean and standard deviation can also be obtained by dividing the total photon count by the area. As shown in Table 2, the WT blank, transiently transfected plasmid DNA concentrations are 2.5, 5, and 10 pmol / mouse, respectively, which are 0, 6.7 x 10^6 pmol / mouse. 7 21.7 x 10 7 47.4x10 7 Light intensity, the data in Table 2 after background subtraction, and... Figure 2 The Luc2 images of small animal live imaging are quite similar, and Table 2 seems more reasonable than Table 1.

[0087] Table 2. Data from small animal liveness imaging system with automatic background subtraction.

[0088]

[0089]

[0090] The transgenic model of this invention can be tissue-specifically limited to expression in the mouse liver. Figure 2 This invention avoids the expression of the pathogenic PNPLA3 I148M gene in other organs, solving the challenge of non-specific expression of the exogenous gene in organs and tissues such as the kidney, epididymal fat, and subcutaneous fat in domestically available stable transgenic humanized PNPLA3 I148M mouse models, in addition to expression in the liver. Furthermore, this invention's model enables real-time, in vivo visual detection of the humanized PNPLA3 I148M gene without sacrificing the mouse, filling a gap in domestic research.

[0091] Example 5: Detection of human PNPLA3 protein expression level in model serum using ELISA

[0092] The night before ELISA was performed on mice, the mice were fasted but allowed free access to water. Early the next morning, humanized PNPLA3I148M transgenic mice were used. Blood was collected from the orbital rim of the mice, and the serum was separated and diluted 10-fold. Using a human PNPLA3 kit (Vancovel, F0023-H), the double-antibody sandwich method was followed according to the instructions. Standards were prepared sequentially, and the standards and test samples were added to a microplate coated with PNPLA3 antibody. HRP was then added, and after color development, the OD value was read at 450 nm using a microplate reader. The human PNPLA3 protein level of the analyte was calculated using a standard curve. Figure 3 The levels of human PNPLA3 protein in the WT blank, transiently transfected plasmid DNA (2.5, 5, and 10 pmol / mouse) models were 322.9±53.05, 482.4±68.57, 815.7±104.41, and 1,361.0±176.54 pg / ml, respectively. The trend of the transiently transfected plasmid DNA (2.5, 5, and 10 pmol / mouse) model groups was consistent with the Luc2 signal. However, the WT blank detected a human PNPLA3 protein level of 322.9±53.05 pg / ml, which may be due to the 79% amino acid sequence similarity between human PNPLA3 (Genebank ID: NP_079501.2) and mouse PNPLA3 (Genebank ID: NP_473429.2), and the insufficient specificity of the human PNPLA3 antibody in the human PNPLA3 ELISA kit to distinguish mouse PNPLA3 protein.

[0093] Example 6: Identification of human PNPLA3 mRNA expression level in model liver using RT-qPCR

[0094] Humanized PNPLA3 I148M transgenic mice were fed a high-fat diet for 8 weeks, then fasted overnight with free access to water. The 15-week-old mice were euthanized the following morning to reach the experimental endpoint. After homogenizing liver tissue, 0.2g of the liver homogenate was weighed and total RNA was extracted using the Trizol method (Invitrogen 15596018), and reverse transcribed (RT) into 1st-cDNA (Tuosu TR0101). A forward primer was designed located at the 2nd exon (nt 313-333) of the human PNPLA3 coding region, with the sequence 5'-aatgtccaccagctcatctcc-3'. A reverse primer was designed located at the human PNPLA3 I148M mutation site, specifically amplifying cDNA containing the human gene I148M mutation rather than the wild-type 148I, with the sequence 5'-ataaggccactgtagaaggg“C”-3'. Primers were synthesized by Youkang Biotechnology. The cDNA template was amplified using this specific primer, and the mRNA level was identified by qPCR (TRO204-5). The results were then analyzed using qPCRsoft 4.1 software (Jena qTOWER). 3 G) analysis, relative quantification with murine Actin, with WT blank considered as 1-fold, showed that the humanized PNPLA3 I148M mRNA levels in the transiently transfected plasmid DNA model groups of 2.5, 5, and 10 pmol / mouse were 3.1±0.22, 21.8±2.10, and 33.6±2.61-fold respectively, higher than the wild-type negative control and showing a dose-dependent relationship. Figure 4 The overall trend is similar to that of human PNPLA3 protein detected by ELISA. Figure 3 ), and Luc2 signal ( Figure 2 The trends are consistent with those in Tables 1-2.

[0095] Example 7: Identification of lipid markers in model serum using an automated biochemical analyzer

[0096] Humanized PNPLA3 I148M transgenic mice were fed a high-fat diet for 8 weeks, then fasted overnight with free access to water. The 15-week-old mice were euthanized the following morning to complete the experiment. Serum samples were collected from whole blood of the mice, and three major lipid biochemical indicators—TG, total cholesterol (CHOL), and LDL-C—were measured using a fully automated biochemical analyzer (Hitachi, 3110) (Table 3). All three indicators in the model group were higher than those in the wild-type negative control. Specifically, the TG levels in the wild-type negative control, the transiently transfected plasmid DNA (2.5, 5, and 10 pmol / mouse model groups) were 0.58, 0.70, 0.84, and 1.29 mmol / L, respectively, indicating a dose-dependent relationship between TG values ​​and plasmid DNA concentration.

[0097] The transgenic model group with 10 pmol / animal of humanized PNPLA3 I148M plasmid DNA of this invention had a TG value of 1.29 mmol / L, a CHOL value of 2.97 mmol / L, and an LDL-C value of 0.34 mmol / L, which were much higher than the wild-type negative control with a TG value of 0.58 mmol / L, a CHOL value of 2.02 mmol / L, and an LDL-C value of 0.20 mmol / L. Therefore, the serum of this model was identified as having a hyperlipidemic phenotype by an automated biochemical analyzer.

[0098] Table 3. Fold change of human PNPLA3 mRNA in model liver and serum lipid biochemical values

[0099]

[0100] Example 8: Pathological identification of non-alcoholic fatty liver disease phenotype in humanized PNPLA3 I148M transgenic mice

[0101] Humanized PNPLA3 I148M transgenic mice were fed a high-fat diet for 8 weeks, then fasted overnight with free access to water. The 15-week-old mice were euthanized the following morning to reach the experimental endpoint. The livers were dissected, washed twice with PBS, and a small, comparable section of liver tissue was fixed. Hematoxylin-eosin staining (HE) revealed vacuoles (clear, round) caused by lipid droplets (LD), exhibiting the typical pathological features of ballooning hepatocytes in non-alcoholic fatty liver disease.

[0102] The liver structure of the wild-type negative control was clear, with no abnormalities in the portal area structure, a central vein visible, and hepatocyte cords arranged neatly. No inflammatory cell infiltration or hepatocyte ballooning degeneration was observed. Figure 5 A), scored 0 points; however, in the transient transfected plasmid DNA 2.5, 5, and 10 pmol / animal model groups, the liver sinusoids were open, with mild congestion, irregular arrangement of hepatocyte cords, multiple localized small focal inflammatory cell infiltrations, and multiple hepatocyte ballooning degenerations ( Figure 5 BD). According to the SAF hepatocyte ballooning degeneration score, the plasmid DNA 2.5 and 5 pmol / animal model groups scored 1 point ( Figure 5 BC), the plasmid DNA 10 pmol / mouse model group scored 2 points ( Figure 5 D). Hepatocellular steatosis scores were 0, 1, 1, and 2 for wild-type negative control, transiently transfected plasmid DNA 2.5, 5, and 10 pmol / animal model groups, respectively. Figure 5The liver pathology of the transgenic model group with 10 pmol / animal of humanized PNPLA3 I148M plasmid DNA in this invention showed hepatocellular ballooning degeneration, hepatocellular steatosis and mild inflammation, thus identifying a pathological phenotype with NAFLD.

Claims

1. A method for constructing a mouse model of non-alcoholic fatty liver disease based on humanized PNPLA3 I148M transgenic, comprising the following steps: Step i: Design the CDS of the human gene and the 3'UTR element crucial for the druggability of small nucleic acid drugs in the gene plasmid DNA for constructing the model; Step ii: Express the Luc2 or iRFP reporter gene in the gene plasmid DNA for constructing the model; Step iii: Use plasmid DNA transient transfection combined with a high-fat diet to induce a disease model of fatty liver disease.

2. The construction method according to claim 1, wherein the CDS of the human gene can stably express human PNPLA3 I148M CDS in the mouse liver for a long term.

3. A method for constructing a mouse model of non-alcoholic fatty liver disease based on humanized PNPLA3 I148M transgenic, comprising the steps: Step a: Design the CDS with a point mutation of I148M of the humanized PNPLA3 gene in the gene plasmid DNA for constructing the model; Step b: Design the 3'UTR with a point mutation of I148M of the humanized PNPLA3 gene in the gene plasmid DNA for constructing the model; Step c: Express the Luc2 or iRFP reporter gene in the gene plasmid DNA for constructing the model respectively; Step d: Amplification, purification and quality control of the gene plasmid DNA; Step e: Use the plasmid DNA transient transfection combined with a high-fat diet to induce a disease model of fatty liver disease.

4. According to the construction method of claim 3, restriction enzyme cutting sites MluI and SacI are designed at the 5' and 3' ends of the humanized PNPLA3 I148M gene respectively.

5. According to the construction method of claim 3, a sequence P2A is designed between the reporter gene and the humanized gene.

6. According to the construction method of claim 3, in step d, the amplification step is to transform the plasmid DNA into Escherichia coli competent cell DH5α by heat shock method, coat it on an LB plate containing 30 μg / ml antibiotic kanamycin sulfate, pick a positive clone and inoculate it into an LB broth containing 30 μg / ml Kan, shake the bacteria at 37°C on a shaker for small-scale culture, dilute and inoculate, and shake the bacteria at 37°C on a shaker overnight for large-scale culture and amplification to obtain the amplified bacterial solution.

7. According to the construction method of claim 3, in step d, the purification step is to extract and purify the plasmid DNA using a plasmid extraction kit to obtain the gene plasmid DNA; the quality control step is to measure the A260 / 280 value of the plasmid DNA using a micro-spectrophotometer to be between 1.8 and 2.

0. After the DNA fragments obtained by sequencing are spliced, compare with the original design, and if the two are consistent, it is judged that the quality control is qualified.

8. According to the construction method of claim 7, the gene plasmid DNA is pLC-Luc2-P2A-hPNPLA3I148M-3UTR.

9. The construction method according to claim 3, in step e, the plasmid DNA transient transfer step includes designing plasmid DNA to create a model using three concentrations of 2.5, 5, and 10 pmol / mouse; using sterile physiological saline without DNA / RNase as a solvent; preparing a 10% plasmid DNA solution according to the mouse's body weight; and using 7-week-old wild-type mice (C57BL / 6, BALB / c, or ICR) to transiently transfer the plasmid DNA into the mice via the tail vein using high-speed fluid dynamics principles.

10. The construction method according to claim 9 further comprises, at 4-week intervals, injecting the plasmid DNA solution into the mouse via a high-velocity hydrodynamic tail vein, with a 10% plasmid DNA solution.

11. A mouse model of non-alcoholic fatty liver disease based on humanized PNPLA3I148M transgene, obtained by the construction method provided in any one of claims 1-10.

12. The use of the mouse model of claim 11 in the preparation or screening of drugs for non-alcoholic fatty liver disease based on humanized PNPLA3 I148M transgene.

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