MRNA capable of efficiently expressing GBA1 and application of mRNA

Abstract

The invention discloses mRNA (messenger Ribonucleic Acid) capable of efficiently expressing GBA1 (Glutathione Associated The mRNA capable of efficiently expressing the GBA1 sequentially comprises a 5'methylation cap, a 5 'UTR, a target gene sequence, a 3' UTR and a polyA tail from the 5'end to the 3 'end, and the target gene sequence is a sequence for coding GBA1 protein or GBA1 mutant protein.The mRNA capable of efficiently expressing the GBA1 is capable of obtaining the GBA1 mRNA which is more stable and higher in expression efficiency by modifying different elements of the mRNA.

Description

Technical Field

[0001] The present application relates to the technical field of glucocerebrosidase, and in particular to an mRNA for efficiently expressing GBA1 and its application. Background Art

[0002] Gaucher disease is a rare autosomal recessive genetic disease caused by a mutation in the lysosomal hydrolase, glucocerebrosidase (GCAse). It is one of the most common lysosomal storage diseases. Glucosylceramide (GluCer) is an important component of the cell membrane and is widely present in circulating blood cells. Changes in its content will have a significant impact on the physiological functions of cells. Under normal circumstances, GCAse performs a hydrolysis function in lysosomes, hydrolyzing GluCer into glucose and ceramide. When the gene GBA1 encoding GCAse mutates or is defective, the enzymatic activity of GCAse decreases, resulting in the inability to metabolize the substrate GluCer, which is mainly stored in the lysosomes of macrophages to form Gaucher cells. Gaucher cells infiltrate organs such as the bone marrow, liver, and spleen, causing organomegaly in patients with Gaucher disease and causing a series of adverse reactions.

[0003] At present, the main clinical treatment for Gaucher disease is enzyme replacement therapy, which is to generate GCAse protein in vitro and inject it into the patient to supplement the missing GCAse activity. However, GCAse protein drugs generated in vitro are expensive, have short half-life, and long injection time. In addition, existing GCAse recombinant protein drugs are ineffective in treating neurological Gaucher disease, which greatly limits the widespread application of enzyme replacement therapy. In addition to Gaucher disease, reduced glucocerebrosidase activity may be associated with the development of Parkinson's disease, Lewy body dementia, and some lipid metabolism disorders.

[0004] Therefore, developing new methods to efficiently express GBA1 is of great value and significance for the treatment of diseases related to reduced glucocerebrosidase activity. Summary of the invention

[0005] The purpose of the present application is to provide a novel mRNA for efficiently expressing GBA1 and its application.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions:

[0007] The first aspect of the present application discloses an mRNA for efficiently expressing GBA1, wherein the mRNA comprises a 5' methylated cap, a 5' UTR, a target gene sequence, a 3' UTR and a polyA tail from the 5' end to the 3' end; wherein the target gene sequence is a sequence encoding a GBA1 protein or a GBA1 mutant protein.

[0008] In one implementation of the present application, the DNA sequence encoding the GBA1 protein or the GBA1 mutant protein is at least one of the sequences shown in Seq ID No.1, Seq ID No.62 and Seq ID No.63, that is, at least one of G01, G02 and G04.

[0009] In one implementation of the present application, the 5'UTR is at least one of F00, F03, F04, F05, F06, F07, F08, F09, F10 and F11, and F00, F03 to F11 are, in sequence, the sequence shown in Seq ID No. 2 to the sequence shown in Seq ID No. 11.

[0010] In one implementation of the present application, the 3'UTR is at least one of T01, T02, T03, T04, T05, T06, T07 and T08, and T01 to T08 are, in sequence, the sequence shown in Seq ID No. 12 to the sequence shown in Seq ID No. 19.

[0011] In one implementation of the present application, the polyA tail is at least one of P02, P03, P04, P05 and P06, and P02 to P06 are, in sequence, the sequence shown in Seq ID No.20 to the sequence shown in Seq ID No.24.

[0012] The second aspect of the present application discloses a recombinant plasmid containing the mRNA or DNA thereof of the present application.

[0013] The third aspect of the present application discloses a recombinant cell containing the mRNA of the present application or the recombinant plasmid according to claim 5.

[0014] The fourth aspect of the present application discloses a gene medicine containing the mRNA of the present application, the recombinant plasmid of the present application or the recombinant cell of the present application.

[0015] The fifth aspect of the present application discloses a liposome nanoparticle containing the mRNA of the present application.

[0016] In one implementation of the present application, liposome nanoparticles are formed by nanocarriers encapsulating mRNA, and the nanocarriers are formed by cationic lipids, 1,2-distearoylglycerol-3-phosphocholine, cholesterol and polyethylene glycol lipids.

[0017] In one implementation of the present application, in the nanocarrier, the molar ratio of cationic lipid, 1,2-distearoylglycerol-3-phosphocholine, cholesterol and polyethylene glycol lipid is 50:10:38.5:1.5.

[0018] In one implementation of the present application, the volume ratio of the nanocarrier to the mRNA is 1:3.

[0019] In one implementation of the present application, the particle size of the liposome nanoparticles is 90-180 nm.

[0020] Due to the adoption of the above technical solution, the beneficial effects of this application are:

[0021] The present application efficiently expresses GBA1 mRNA, and obtains a more stable GBA1 mRNA with higher expression efficiency by modifying different elements of mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the full sequence of the GBA1-01 (F00-G01-T04-P02) vector in the examples of this application;

[0023] Figure 2 This is a flow chart of vector construction in the embodiments of this application;

[0024] Figure 3 This is the electrophoresis diagram of double enzyme digestion of plasmid DNA in the embodiment of the present application;

[0025] Figure 4 This is a result diagram of agarose gel electrophoresis to verify the purity of mRNA products in the examples of this application;

[0026] Figure 5 This is the WB detection result of GBA1 protein expression in the example of this application;

[0027] Figure 6 1 is a diagram showing the results of enzyme activity detection of mRNA using different elements in the examples of the present application;

[0028] Figure 7 This is a graph showing the results of liver protein WB in the examples of this application;

[0029] Figure 8 is the gray value of the WB result of liver protein in the embodiment of the present application;

[0030] Fig. 9 This is a WB result diagram of spleen protein in the examples of this application;

[0031] Fig.10 is the gray value of the WB result of spleen protein in the embodiment of the present application;

[0032] Fig.11 This is the test result of the effect of replacing different 5'UTRs on the expression stability of GBA1-01 in different time periods in the examples of this application;

[0033] Fig.12 This is the test result of the effect of replacing different 3'UTRs on the expression stability of GBA1-01 in different time periods in the examples of this application;

[0034] Fig.13 This is the experimental result of knocking out GBA1 cell line to verify the effect of GBA1 mRNA expression in the examples of this application;

[0035] Fig.14 These are the test results of enzyme activities expressed by different ORFs in the examples of this application. DETAILED DESCRIPTION

[0036] The elements of the plasmid designed with pVAX1 as the vector mainly include CMV promoter, T7 promoter, target gene ORF, upstream and downstream non-coding regions 5'UTR and 3'UTR, polyA tail and the linearization site immediately thereafter.

[0037] pVAX1 is a commonly used mammalian cell expression vector with a high expression level. It allows high-copy replication in E. coli and high-level transient protein expression in mammals. It is a nucleic acid vaccine vector approved by the US FDA. This vector has the following characteristics: ① The eukaryotic DNA sequence is limited to the eukaryotic DNA sequence required for expression to minimize the possibility of chromosomal integration; ② The kanamycin resistance gene can be used for E. coli screening; ③ The small size and multiple unique cloning sites can simplify the subcloning of even very large DNA fragments; ④ The CMV promoter is a human cytomegalovirus promoter, which allows this vector to express high-level genes in most mammalian cells; ⑤ The bovine growth hormone (BGH) polyadenylation signal can ensure efficient transcription termination and the polyA structure of mRNA.

[0038] By modifying different elements of mRNA, a more stable GBA1 mRNA with higher expression efficiency can be obtained.

[0039] mRNA is composed of a 5' methylated cap, 5' UTR, target gene sequence, 3' UTR and poly A tail. The GBA1 target gene sequence is combined with different 5' UTR, 3' UTR and poly A to construct different mRNA sequences in order to optimize mRNA stability and expression efficiency.

[0040] The present application is further described in detail below by specific examples. The following examples are only further described for the present application and should not be construed as limiting the present application. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can effortlessly recognize that some of the features can be omitted in different situations, or can be replaced by other kits, materials, and methods. In some cases, some operations related to the present application are not shown or described in the specification, and this is to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and the related operations can be fully understood according to the description in the specification and the general technical knowledge in the art. In the following embodiments, the experimental method without specifying specific conditions is usually in accordance with conventional conditions, or in accordance with the conditions recommended by the manufacturer. In the following embodiments, the materials, reagents, or instruments used, if not otherwise specified, can all be purchased from commercial sources.

[0041] Example

[0042] The sequence optimization method of this example will be carried out in the manner of Table 1. GBA1-01 (i.e., F00-G01-T04-P02) is obtained by optimizing the codons of the original sequence GBA1, and then the sequence is further optimized based on GBA1-01.

[0043] 1. Design of vector plasmid for in vitro transcription of glucocerebrosidase mRNA

[0044] Target gene: GBA1 encodes glucocerebrosidase (GCAse), a lysosomal membrane protein consisting of 536 amino acids, i.e., Seq ID No. 26:

[0045] MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ

[0046] The corresponding DNA sequence is Seq ID No. 27:

[0047]

[0048] The molecular weight is about 60kDA; it is able to hydrolyze GluCer into glucose and ceramide in the lysosome. GCAse requires the cofactor SAposin C and negatively charged lipids for maximum catalytic activity. SAposin C is a lysosomal membrane protein with a molecular weight of 9kDA that binds to the lysosomal membrane and the lipid portion of GluCer, thereby recruiting GluCer to the active site of the enzyme.

[0049] A FlAG tag was added after the GBA1 gene sequence, namely Seq ID No. 28: DYKDDDDK, to verify whether the gene sequence can be successfully translated and expressed.

[0050] 3'UTR: untranslated region

[0051] Poly(A): is the tail at the 3' end of most eukaryotic mRNAs and helps regulate mRNA stability, transport, and translation.

[0052] Linearization site: Xhol restriction site. Xhol restriction endonuclease recognizes the 5'C^TCGAG 3' sequence in the vector to generate linearized DNA as a template for in vitro transcription.

[0053] 2. GBA1 mRNA in vitro expression optimization experiment

[0054] Different vector elements of F00-G01-T04-P02 were replaced, such as 5'UTR, ORF, 3'UTR, polyA, etc., to obtain different sequence information to construct different mRNA sequences, so as to optimize mRNA stability and expression efficiency.

[0055] Table 1

[0056]

[0057]

[0058]

[0059]

[0060] In Table 1, 5'UTR is represented by F, ORF codon optimized GBA1 is represented by G01, 3'UTR is represented by T, polyA tail is represented by the letter P, and the enzyme activity results are shown in Figure 6 As shown. It can be understood that for mRNA, all T in the sequence are U.

[0061] 2.1. Vector construction

[0062] The full sequence of GBA1-01 (F00-G01-T04-P02) vector is Seq ID No. 29:

[0063]

[0064] The sequence shown in Seq ID No.29 is the mRNA recombinant plasmid in this example.

[0065] Among them, the F00 sequence is Seq ID No.2:

[0066] GAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC

[0067] The G01 sequence was optimized based on hGBA1-Flag, and the specific sequence is Seq ID No.1:

[0068]

[0069] The T04 sequence is Seq ID No.15:

[0070] CGTCTGCATAACTTTTATTATTTCTTTTATTAATCAACAAAATTTTGTTTTTAACATTTC

[0071] The P02 sequence is Seq ID No. 20:

[0072] AAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0073] The flag tag is Seq ID No.25: GATTACAAGGACGACGATGACAAG

[0074] The carrier structure is as follows Figure 1 The vector construction process is as shown in Figure 2 shown.

[0075] The 5'UTR of hGBA1 flAG TAG-T7AGG-PVAX1 was double-digested with NheI and BamHI, and the 5'UTR was synthesized with primers to add restriction sites, then annealed and connected to the vector, and then the enzyme activity was screened.

[0076] Experimental procedures

[0077] a) Enzyme Digestion System

[0078] hGBA-FlAG 1μL, BAmH11μL, NheI 1μL, 10×CuTBuffer 2μL, H2O 15μL.

[0079] Reaction conditions: 37°C, overnight culture

[0080] b) Agarose gel

[0081] Prepare 0.8% agarose gel

[0082] ① Weigh 0.4g agarose and place it in a conical bottle, add 50ml 1×TAE, and turn the bottle upside down with a small beaker. Heat and boil twice in a microwave oven until the agarose is completely melted, shake well to make 1.0% agarose gel solution, cool to about 65℃ and add 5μL Gel-Red 10000×.

[0083] ② Preparation of gel plate: Take the organic glass inner tank (gel making tank), wash it, dry it, put in the gel making glass plate and then put the comb. Mix the agarose gel solution cooled to about 65°C and carefully pour it onto the inner tank glass plate, allowing the gel solution to slowly spread until a uniform gel layer is formed on the entire surface of the glass plate. Let it stand at room temperature until the gel is completely solidified, gently pull out the comb vertically, remove the tape, and place the gel and inner tank into the electrophoresis tank. Add 1×TAE electrophoresis buffer until the gel plate is covered. If the gel is not used in a short period of time, it can be wrapped with plastic film and stored at 4°C until use.

[0084] ③ Run glue

[0085] Add 6×DNA load buffer to the sample DNA, slowly and carefully add the DNA sample to the gel sample slot, add DNA Marker, record the sample spotting order and sample addition amount, install the electrode wires, black to black and red to red, with one end of the spotting hole close to the negative electrode, turn on the power, adjust the voltage to 130mV, time 30min, when the blue band moves to 1-2cm away from the front of the gel, stop electrophoresis.

[0086] ④Take photos and check the molecular weight.

[0087] c) Rubber cutting and recycling.

[0088] ① Before first use, add 80 mL of anhydrous ethanol to dilute Buffer GW as indicated on the Buffer GW reagent bottle label and store at room temperature.

[0089] ②After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under UV light. It is recommended to use a paper towel to absorb the liquid on the surface of the gel and cut it into pieces, and try to remove excess gel. Weigh the gel (excluding the weight of the empty tube). 100mG gel is equivalent to 100μL volume, which is considered as a gel volume.

[0090] ③ Add an equal volume of Buffer GDP and incubate in a 50-55℃ water bath for 7-10 minutes. Adjust the time appropriately according to the size of the gel to ensure that the gel is completely dissolved. Invert and mix twice during the water bath to accelerate the dissolution.

[0091] ④ Briefly centrifuge to collect the droplets on the tube wall, place the FAsTPure DNAMini Columns-G adsorption column in the Collection Tubes 2ml collection tube, transfer the sol to the adsorption column, and centrifuge at 12,000rpm (13,800×G) for 30-60s.

[0092] ⑤Discard the filtrate and place the adsorption column in a collection tube. Add 300μL Buffer GDP to the adsorption column. Let stand for 1min. Centrifuge at 12,000rpm (13,800×G) for 30-60s.

[0093] ⑥Discard the filtrate and place the adsorption column in a collection tube. Add 700 μl Buffer GW (with anhydrous ethanol) to the adsorption column. Centrifuge at 12,000 rpm (13,800 × G) for 30-60 seconds.

[0094] Repeat step ⑥.

[0095] ⑦Discard the filtrate and place the adsorption column back into the collection tube. Centrifuge at 12,000 rpm (13,800×G) for 2 min.

[0096] ⑧ Place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 20 μL ddH2O to the center of the adsorption column, and place for 2 minutes. Centrifuge at 12,000 rpm (13,800 × G) for 1 minute. Discard the adsorption column and store the DNA at -20°C.

[0097] d) Experimental results

[0098] Double enzyme digestion verification, the enzyme digestion system is: EcoR1-HF 2μL, Not1-HF 2μL, 10× buffer 2.5μL, plasmid 0.5μg, water 20.5μL.

[0099] Plasmid digestion results Figure 3 shown.

[0100] e) Annealing

[0101] ① Dilute the substance to 10μM with TE buffer.

[0102] 10 μL of forward primer, 10 μL of reverse primer, total 20 μL.

[0103] Table 2

[0104]

[0105]

[0106] ② Use the PCR instrument to set the temperature at 95℃ for 30s, then 90℃ for 30s and then decrease to 45℃ for 30s.

[0107] f) Connection system

[0108] 10×T4 DNA buffer 1μL, T4 DNA 0.5μL, vector 1μL (about 30ng), plasmid 2μL, water 5.5μL.

[0109] g) Conversion

[0110] ① Take 50 μL of E. coli competent cells DH5a and dissolve for 10 minutes.

[0111] ② Add appropriate amount of plasmid (volume should not exceed 10 μL) and place on ice for 30 minutes.

[0112] ③ Heat shock at 42℃ for 45s, immediately put back on ice and ice bath for 2min; add 700μLLB medium and culture in a 37℃ shaker with slow shaking for 45-60min;

[0113] ④ Take 50-100 μL and apply it on LB solid medium containing ampicillin (100 μg / mL), and invert and culture at 37°C overnight

[0114] h) Pick a single colony, send it for sequencing, and preserve the bacteria.

[0115] ① Pick half of the single colony to shake the bacteria.

[0116] Use a 15 mL centrifuge tube, add 2 mL of LB medium containing kanamycin, culture at 37°C overnight, and use 50% glycerol to preserve the bacteria.

[0117] ② Half is sent for sequencing

[0118] Use a 1.5 mL centrifuge tube, add 500 μL of LB medium containing kanamycin, and incubate at 37°C for 3 h.

[0119] i) Design 4 polyA sequences and send them to Genewise for synthesis

[0120] Table 3

[0121]

[0122] 2.2. Preparation of mRNA

[0123] Experimental Materials

[0124] XhoI (10U / μL), 10×Buffer, sterile enzyme-free water, anhydrous ethanol, 3M sodium acetate, Nuclease-freewater, 10×Reaction Buffer, ATP (100mM), GTP (100mM), N1-pUTP (100mM), CTP (100mM), Cleancap AG (Cap1) (100mM), T7 RNA Polymerase (50U / μl), RNAase inhibitor (40U / μl), inorganic pyrophosphatase (0.1U / μl), DNase I (1U / μl), LiCl precipitation solution (7.5M), agarose, 20X TAE, GelRed (10,000×), DNAloading Buffer (6×), RNAloading Buffer (2×), DL5000 DNAMarker.

[0125] Experimental methods

[0126] 1. Extraction of template plasmid: Add kanamycin to LB medium, inoculate E. coli carrying the target plasmid into the medium (16-19h, 37°C), collect the bacteria by centrifugation, and extract the plasmid using a kit.

[0127] 2. Linearization of template plasmid: The template plasmid was digested with XhoI for 4 h (1 μL restriction enzyme for every 10 μG plasmid), and the linearized plasmid was directly purified by sodium acetate precipitation method

[0128] ① Take a 1.5mL EP tube, add 50uG plasmid, water (calculate the volume to 100μL), 10μL Buffer, 5μL XhoI enzyme, and operate on ice.

[0129] ②After incubation at 37℃ for 4h, add 200μL of anhydrous ethanol and 10μL of sodium acetate to each tube, mix well, and place on ice at -20℃ for 1h

[0130] ③ Centrifuge at 4℃13000rpm for 10min and discard the supernatant

[0131] ④Add 1ml 70% ethanol and mix by inverting

[0132] ⑤ Centrifuge at 4℃13000rpm for 10min and discard the supernatant

[0133] ⑥Put it in a 55℃ metal bath for drying

[0134] ⑦ Add 50 μL of sterile enzyme-free water, mix well and measure the concentration and purity

[0135] 3. In vitro transcription synthesis of mRNA: Take a certain amount of linearized DNA template, add reaction buffer, ATP, CTP, GTP, N1-pUTP (N1-methylpseudouridine triphosphate; introducing pseudouridine or N1-methylpseudouridine into mRNA may effectively reduce the immunogenicity of mRNA vaccines, enhance the stability of mRNA, and enhance its protein expression ability), AG(hat structure ReAGenTAG|TriLink BioTeChnoloGies), T7 RNA PolymerAse, RNAAseinhibiTor and InorGAniC pyrophosphATAse (the proportions are shown in Table 2). The reaction was placed at 37°C for 2 hours. After the reaction was completed, the corresponding amount of DNA hydrolase (1 μL DNAse I was added to every 20 μL reaction system) was added to hydrolyze the DNA template, and the mRNA product was purified with lithium chloride and the concentration was measured.

[0136] ① Take a 1.5mL EP tube and add each component in sequence according to the table below. Operate on ice (20μL system)

[0137] Table 4

[0138]

[0139]

[0140] ②After incubation at 37℃ for 2h, add 2μL DNAse I to each tube, mix well and incubate at 37℃ for 15min

[0141] ③Add 28μL sterile enzyme-free water and 25μL lithium chloride to each tube, mix well and place in -20℃ refrigerator for 30 minutes

[0142] ④ Centrifuge at 4℃12000rpm for 15min and discard the supernatant

[0143] ⑤ Add 1 mL of 70% ethanol and mix by inverting

[0144] ⑥ Centrifuge at 4℃12000rpm for 15min and discard the supernatant

[0145] ⑦ Dry in a 55℃ metal bath

[0146] ⑧Add 80 μL of sterile enzyme-free water, mix well and measure the concentration and purity

[0147] 4. Linearized plasmid electrophoresis and mRNA electrophoresis steps:

[0148] ① Prepare 0.8% agarose gel:

[0149] Weigh 0.4 g agarose and place it in a conical flask, add 50 mL 1×TAE, and place a small beaker upside down on the flask. Boil in a microwave oven for 3 times until the agarose is completely melted, shake well, and a 0.8% agarose gel solution is obtained. Cool to about 65°C and add 5 μL Gel-Red

[0150] ② Preparation of rubber sheet:

[0151] Take the organic glass inner tank (gel tank), clean it, dry it, put the gel glass plate in it and then put the comb in place. Mix the agarose gel solution cooled to about 65℃ and carefully pour it onto the inner tank glass plate, allowing the gel solution to slowly spread until a uniform gel layer is formed on the entire surface of the glass plate. Let it stand at room temperature until the gel is completely solidified, pull the comb vertically and gently, remove the tape, and put the gel and inner tank into the electrophoresis tank. Add 1×TAE electrophoresis buffer until the gel plate is submerged.

[0152] ③Add sample:

[0153] Pipette 1 μg plasmid into each linearized plasmid tube and add 1 μL DNA loading buffer (6×), and add sterile enzyme-free water to make up the volume to 6 μL

[0154] Pipette 1 μg mRNA into each mRNA tube and add 3 μL RNA loading buffer (2×), and add sterile enzyme-free water to make up the volume to 6 μL

[0155] Add 6 μL of sample and 6 μL of DNA Marker to the sample well.

[0156] ④Electrophoresis: constant voltage 120V electrophoresis for 35min

[0157] ⑤Take photos to see the results

[0158] The results of partial linearized plasmid electrophoresis and mRNA electrophoresis are as follows Figure 4 shown.

[0159] 2.3 LNP packaging of mRNA

[0160] Purpose

[0161] The mRNA chain is unstable and carries a negative charge. The cell membrane surface also carries a negative charge. Electrostatic repulsion makes it difficult for mRNA molecules to pass through the cell membrane and enter the cell. Therefore, a special modification or packaging delivery system is required to achieve intracellular expression of mRNA. Liposome nanoparticles (LNP) have become the most popular delivery technology due to their high delivery efficiency and good safety. This project intends to use the LNP delivery system to deliver mRNA into cells.

[0162] Material preparation

[0163] 1. mRNA Preparation

[0164] The mixed GBA1 mRNA synthesized in vitro was diluted to 150 μg / ml using CiTrATubffer (20 mM sodium citrate, pH 4.0).

[0165] 2. LNP Component Preparation

[0166] Weigh relevant lipids for preparing LNP and dissolve them in ethanol, including ionizable cationic lipids (SM102, C 44 H 87 NO5), auxiliary phospholipid 1,2-distearoylglycerol-3-phosphocholine (DSPC), cholesterol and polyethylene glycol lipid, with a molar ratio of 50:10:38.5:1.5.

[0167] Experimental methods

[0168] 1.LNP Package

[0169] ①LNP and mRNA were passed through INAno at a volume ratio of 1:3 TM The microfluidic chip of the rapid nano-drug preparation system was mixed (chip type: prescription screening-SHM), with a total mixing flow rate of 12mL / min to obtain the encapsulated nucleic acid lipid nanoparticles mRNA-LNP. The starting and ending waste liquids were 50μL, and the chip was finally washed with water and emptied.

[0170] ② After the samples were collected, the ethanol in the samples was replaced with PBS by ultrafiltration. Ultrafiltration was performed using a 10kD molecular weight cutoff ultrafiltration tube at 4000×G for 30 min, and repeated three times.

[0171] ③ Collect the samples after ultrafiltration and filter them. Store them at -4℃ for short term or replace them with freezing solution and store them at -80℃ for long term.

[0172] 2. Encapsulation Efficiency Determination

[0173] ① Take 5 μL mRNA-LNP sample

[0174] ② Add 20×TE buffer to the sample and dilute it with DEPC water, divide it equally into two tubes, add Triton-X100 to one tube to lyse LNP

[0175] ③Add two samples to a 96-well plate, replicate each sample three times, and add 100 μL to each well.

[0176] ④ Use DEPC water to prepare standards with concentrations of 1μg / mL, 0.5μg / mL, 0.25μg / mL, and 0.125μg / mL. Repeat 3 times for each concentration and add 100μL to each well.

[0177] ⑤ Dilute the dye in the kit 200 times, and then add it to the 96-well plate with samples in equal proportions, and protect from light.

[0178] ⑥ Read the plate with an ELISA reader, select the fluorescence mode, excitation light 485nm, emission light 528nm, and read the number.

[0179] ⑦Data processing

[0180] 3. LNP Particle Size Determination

[0181] 5 μL of encapsulated GBA1-LNP was diluted with 1.2 mL of PBS, and the particle size and pDI value were detected using a nanoparticle size and Zeta potential analyzer.

[0182] The experimental results of this example using hGBA1-Flag mRNA sequence are as follows

[0183] Table 5

[0184] Particle size / nm PDI Encapsulation efficiency mRNA-LNP Batch 1 119.2±4.8 0.225±0.016 97.2％ mRNA-LNP Batch 2 103.6±3.1 0.154±0.012 96.3％

[0185] Protein expression experiments

[0186] Purpose

[0187] Verify whether the designed mRNA and mRNA-LNP encapsulated by liposomes can be translated and expressed at the cellular level.

[0188] Experimental methods

[0189] Exogenous mRNA was introduced into 293T cells by liposome method. After 24h and 48h, the cells were collected and lysed, and the intracellular protein expression was detected by western blot.

[0190] The loading groups for western blot are as follows

[0191] Table 6

[0192] Sample loading group 1.Marker 2. NC group Protein collected by lysis of blank 293T cells 3.5ug GBA1-mRNA After transfection of 5ug mRNA into 293T cells, the collected proteins were lysed 4.5ugGBA1-mRNA-LNP Protein after transfection of liposome-encapsulated 5ug mRNA into 293T cells and lysis

[0193] Material preparation

[0194] 1. Cell Culture

[0195] Digest the 293T cells in a 10cm culture dish, resuspend them in 1mL DMEM medium after centrifugation, and take a six-well plate for seeding. Determine the cell seeding density according to the cell growth rate; add 2mL DMEM medium containing 10% serum and 1% double antibody to each well. Cultivate in a 37℃ CO2 incubator until the density reaches 60% to 70%.

[0196] 2. Reagents

[0197] ①Lipofectamine 2000 transfection reagent

[0198] ②Opti-MEM Medium

[0199] ③1×TBST solution

[0200] ④5% BSA solution (2.5 BSA dissolved in 50 mL TBST)

[0201] ⑤HRP-labeled AnTi-FlAG antibody (diluted in 5% BSA solution at a ratio of 1:10000)

[0202] Anti-GAPDH (diluted 1:10000 in 5% BSA solution) (HRP-conjugated GAPDH Mouse McAb)

[0203] Experimental procedures

[0204] 1. Transfection

[0205] ① Prepare the transfection solution, taking the amount for one well of a six-well plate as an example:

[0206] Solution A: dilute 3 or 6 μL lipo2000 with 125 μL OpTi-MEM

[0207] Solution B: dilute 2.5 or 5 μg mRNA with 125 μL OpTi-MEM

[0208] ② Add all of solution B to solution A, mix gently, and let stand at room temperature for 10 minutes

[0209] ③ Add the transfection mixture evenly to the wells, mix well, and culture in a 37℃ CO2 incubator for 24h-48h

[0210] 2. Western BloT

[0211] ①Sample preparation for electrophoresis

[0212] Pipette the supernatant from the six-well plate, centrifuge, take a portion of the supernatant and add 5× loading buffer at a ratio of 1:1, and place in a metal bath at 95-100℃ for 10 minutes

[0213] After rinsing the cultured cells with PBS, aspirate the PBS, add 150 μL of lysis buffer to each well, scrape the cells with a cell scraper on ice, transfer the cells and lysis buffer to a centrifuge tube, and incubate at 95-100℃ in a metal bath for 10 min.

[0214] ② Sample electrophoresis

[0215] Protein loading 10 μL

[0216] Precast gel: GenScript ExpressPlus TM Protein precast gel

[0217] ③Transfer

[0218] PVDF membrane, soak in anhydrous methanol before use; place sponge gasket, glue, membrane, and sponge gasket on the transfer device from the negative electrode to the positive electrode.

[0219] Transfer device: eBloT L1 fast wet transfer device

[0220] ④ Closed

[0221] Block with 5% BSA solution at room temperature for 2 h;

[0222] After blocking, the cells were washed three times with TBST, each time for 5 min.

[0223] ⑤ Primary antibody incubation (HRP-Conjugated DYKDDDDKTag mouse McAb)

[0224] GBA1 was incubated with 1:10000 HRP-labeled Anti-FlAG for 1 h

[0225] Wash three times with TBST, 5 min each time

[0226] ⑥ Incubation with colorimetric solution

[0227] BIO-RAD ClariTypical ECL Substrates, A solution: B solution = 1:1, ready for use.

[0228] ⑦Result analysis and testing

[0229] Gel imaging system detection

[0230] ⑧ Antibody clearance 15min

[0231] ⑨ Closed

[0232] ⑩Incubate with internal reference protein (AnTi-GAPDH) for 1 hour, wash three times with TBST, 5 minutes each time, incubate with substrate, and detect with gel imaging system

[0233] Experimental Results

[0234] Transfection of 5uG GBA1-mRNA and GBA1-mRNA-LNP can express in 293T cells, and WB verifies that protein is expressed in both experimental groups. Figure 5 shown.

[0235] 2.5 In vitro enzyme activity assay

[0236] Purpose

[0237] Detection of GCAse protease activity in cells.

[0238] Material preparation

[0239] 1. Cell culture:

[0240] Prepare DMEM medium containing 10% FBS and 1% double antibody. Resuscitate 293T cells and plate them when the cell confluence reaches 70% to 80%.

[0241] 2. GBA1 mRNA Transfection

[0242] ① Prepare the transfection solution, taking the amount for one well of a six-well plate as an example:

[0243] Solution A: dilute 2 μL lipo2000 with 50 μL OpTi-MEM

[0244] Solution B: dilute 1 μg mRNA with 50 μL OpTi-MEM

[0245] ② Add all of solution B to solution A, mix gently, and let stand at room temperature for 15 minutes. The control group was treated with only the same volume of lipo2000

[0246] 3. Reagent Preparation

[0247] RipA Lysate

[0248] GCAse enzyme activity buffer (AssAy buffer): 0.1M sodium phosphATe dibAsiCdihydrATe, 0.05M CiTriCACid, pH 5.0

[0249] GCAse enzyme inhibitor CBE (ConduriTol-β-epoxide): 10mM

[0250] GCAse enzyme substrate 4MUG (4-meThyl-umbelliferyl-β-D-GluCopyrAnoside): 10mM

[0251] GCAse enzyme activity reaction termination solution (sTop buffer): 0.3M GlyCine, 0.2M sodium CArbonATe, PH 10.7

[0252] BCA reagent: Use DEPC water to prepare BCA standard with concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0 mg / mL.

[0253] Experimental methods

[0254] Enzyme activity detection steps

[0255] 1.293T cells were plated, 5×10 cells per well of a 12-well plate 5 cells, 1 mL of culture medium per well

[0256] 2.24h later, transfect 1 μg mRNA per well (transfected with lipo2000)

[0257] 3. Collect cells and wash once with 600μL PBS, add 200μL RIPA, and break with magnetic beads

[0258] 4. Add 10 μL PBS and 5 μL cell lysis buffer to the PBS group, and 10 μL CBE and 5 μL cell lysis buffer to the CBE group. Set up three replicate wells.

[0259] 5. After 45 minutes at room temperature, add 50 μL Assay buffer and 70 μL 4 MUG to each well.

[0260] 6. After 2 hours at 37℃, add 100μL sTop buffer

[0261] 7. Measure fluorescence at 350 / 450nm using a fluorescence microplate reader

[0262] BCA Steps (Protect from light)

[0263] 1. Prepare BCA test reagent according to the instructions

[0264] 2. Add 200 μL BCA detection reagent to each well of the standard sample well

[0265] 3. Add 205 μL BCA detection reagent to each sample well

[0266] 4. Add 10μL of standard product and 5μL of sample

[0267] 5. Measure OD value at 562nm after 1h

[0268] Data processing: The fluorescence value obtained from the GCAse enzyme activity test was divided by the protein content of the measured enzyme activity to obtain the F00 enzyme activity.

[0269] The test results are as follows Figure 6 shown. Figure 6 The results showed that in 293T cells, the F00-G01-T07-P02 sequence had the best expression effect and the highest enzyme activity.

[0270] 2.6 Stability test

[0271] We detected the cellular content of hGBA1-mRNA in HEK293T cells at different time points after transfection, and examined the effects of substitutions of different 5'UTRs and different 3'UTRs on stability. Specifically, the expression of GCase mRNA was detected by qRT-PCR.

[0272] qRT-PCR method:

[0273] After transfection of different GCase mRNAs of 0.4 ng / ml in 12-well plates, cell lysates were collected at 6 hours, 12 hours, 32 hours and 54 hours, respectively. Total RNA was extracted using an RNA extraction kit according to the manufacturer's manual (Vazyme, Nanjing, China, CAT#RC112). 500 ng of total RNA was synthesized into cDNA using a reverse transcription kit according to the manufacturer's manual (Cwbio, Jiangsu, China, CAT#CW3371M).

[0274] The quantification of intracellular GCase mRNA levels was performed using a standard SYBR Green qRT-PCR method (Cwbio, Jiangsu, China, CAT#CW3360S) and primers for GCase, GCase-O1, GCase-O2, and GAPDH (primer sequences are shown in Supplementary Table 1). 40 ng of cDNA was used for 35 cycles of amplification on a qTOWER3 (Analytik Jena, Germany) instrument. Threshold cycle (Ct) values ​​were processed and analyzed by qPCRsoft software. The Ct values ​​of GCase mRNA were normalized to those of GAPDH mRNA, and the data were expressed as relative mRNA amounts calculated by the delta-delta Ct method.

[0275] The quantitative PCR primer sequences are shown in Table 7, and the results are shown in Fig.11 and Fig.12 shown.

[0276] Table 7

[0277]

[0278] Fig.11 and Fig.12 The results showed that the replacement of 5'UTR affected the stability of hGBA1-mRNA, thus affecting the GCase activity. The higher stability of F00 and F01 was consistent with the data of total intracellular GCase activity. However, the replacement of 3'UTR did not affect the total intracellular GCase activity mainly by affecting the stability of mRNA.

[0279] 2.7 Gene knockout verification test

[0280] We evaluated the subcellular localization of exogenous hGBA1-mRNA in wild-type and GBA1-KO HEK293T cell lines. In HEK293T cells, hGBA1-mRNA fused to red fluorescent protein RFP was analyzed by confocal immunofluorescence analysis 24 hours after transfection using Lyso Tracker. Fig.13 At the same time, the GCase activity was tested using the same method as in “2.5 In vitro enzyme activity test”. The results are shown in Figure c. Fig.13 As shown in Figure d and Figure f. Confocal immunofluorescence analysis of GBA1-KO HEK293T cells 24 hours after transfection of hGBA1-mRNA. In this experiment, wild-type, GBA1-KO HEK293T cells, and GBA1-KO HEK293T cells transfected with hGBA1-mRNA were subjected to triple-label immunofluorescence treatment with anti-GBA1, anti-flag, EGFP-RAB5B (endolysosomal marker) and DAPI. The results are shown in Figure d and Figure f. Fig.13 As shown in Figure e.

[0281] Fig.13 The results showed that the co-localization of hGCase protein and LAMP-1 (lysosomal marker) confirmed that GCase encoded by hGBA1-mRNA was correctly localized in lysosomes after intracellular translation ( Fig.13 c). By knocking out the GBA1 gene, we analyzed the effect of hGBA1-mRNA in alleviating lysosomal aggregation in HEK293T cells. The results showed that knocking out the GBA1 gene would lead to an increase in lysosomal volume, i.e., lysosomal storage phenomenon, such as Fig.13 As shown in Figure e; and the activity of GCase is less than 20% of the wild-type GCase ( Fig.13 Next, we transfected 1 μg / ml hGBA1 plasmid and hGBA1-mRNA into wild-type and HEK293T KO GBA1 cells. Compared with untransfected cells, the expression and activity of GCase were significantly increased, and the lysosomal volume was restored to normal, and the storage was improved ( Fig.13 e Figure ).

[0282] Fig.13 In Figure e, row 1: wild-type HEK293T; row 2: GBA1-KO HEK293T; row 3: GBA1-KO HEK293T transfected with GBA1 plasmid (1 μg / ml); row 4: GBA1-KO HEK293T transfected with hGBA1-mRNA (1 μg / ml). The right column shows representative confocal images of single cells and their merged images.

[0283] 2.8 Effects of different optimized sequences on enzyme activity

[0284] This experiment further used different ORF sequences to replace G01 and tested its effect on enzyme activity. Specifically, this example used G02, G04 and G05 for the experiment. Among them, G02 is the sequence shown in Seq ID No.62, and the encoded amino acid is the sequence shown in Seq ID No.26; G04 is the sequence shown in Seq ID No.63, which encodes a GBA1 mutant protein in which the 534th R of the wild-type GBA1 is mutated to H, and the specific sequence is the sequence shown in Seq ID No.64; G05 is the sequence shown in Seq ID No.65, which encodes a GBA1 protein with 38 amino acids truncated from the N-terminus of the wild-type GBA1, as shown in Seq ID No.66. The activity test results of the enzymes expressed by different ORFs are shown in the figure. Fig.14 shown.

[0285] Seq ID No.62:

[0286]

[0287] Seq ID No.63:

[0288]

[0289] Seq ID No.64:

[0290] MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWHRQ

[0291] Seq ID No.65:

[0292]

[0293] Seq ID No.66:

[0294] MARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPM ASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQC LGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLL YHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ

[0295] Fig.14 The results showed that different ORFs had a great influence on enzyme activity, among which G01 had the highest enzyme activity, followed by G02 and G04, and G05 had the poorest enzyme activity.

[0296] 3. In vivo enzyme activity test

[0297] Purpose

[0298] To test whether the LNP-encapsulated glycosyltransferase mRNA can express active GCAse protease activity when injected into the body. In this case, F00-G01-T07-P02, referred to as T07, was used for animal testing.

[0299] Material preparation

[0300] Species & strain: FVB mice

[0301] Animal age: 4-6 weeks

[0302] Animal Grade: SPF

[0303] Source of experimental animals: Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0304] Number of animals & gender: 40 males

[0305] Treatment of excess animals: Excess animals were euthanized 2 days after the first administration:

[0306] Experimental methods

[0307] After one week of adaptive feeding of mice, the experiment began. All mice were randomly divided into 2 groups according to their body weight, 3 mice in the control group and 15 mice in the model group.

[0308] The model group mice were injected with 2mg / kg GBA1-mRNA-LNP by tail vein according to body weight, and the control group mice were injected with an equal volume of PBS by tail vein. Three mice in the control group were killed at 0 hours after the injection. Three mice in the model group were randomly euthanized at each time point of 6h, 12h, 24h, 36h, and 72h after the modeling injection, and the liver, spleen, and plasma were collected. The collected organs were preserved and tested uniformly. If the experimental conditions are limited, please use liquid nitrogen for storage.

[0309] The liver and spleen were collected and homogenized by ultrasound. After centrifugation, the supernatant was collected for protein quantification and GCase enzyme activity test.

[0310] The detailed experimental grouping and dosing schedule are shown in Table 8.

[0311] Table 8

[0312]

[0313]

[0314] Experimental Results

[0315] Verification of GCAse protein expression (wb): 4-20% SDS-PAGE (BYT), 60V 130min. The experimental method refers to the West Blot method under 2.4 Protein Expression Experiment.

[0316] Transfer: semi-dry transfer apparatus, 20V 80min.

[0317] The results are as follows Figure 7 , Figure 8 , Fig. 9 and Fig.10 shown. Figures 7 to 10 The results showed that compared with 0h, both liver protein and spleen protein showed an upward trend, with the highest expression of liver protein at 24h and spleen protein at 12h, indicating that the T07 sequence was successfully expressed in vivo.

[0318] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. An mRNA for efficiently expressing GBA1, characterized in that: The mRNA includes a 5' methylated cap, a 5' UTR, a target gene sequence, a 3' UTR and a polyA tail in sequence from the 5' end to the 3' end; wherein the target gene sequence is a sequence encoding a GBA1 protein or a GBA1 mutant protein.

2. The mRNA according to claim 1, characterized in that: The DNA sequence encoding the GBA1 protein or the GBA1 mutant protein is at least one of the sequences shown in Seq ID No.1, Seq ID No.62 and Seq ID No.63; Preferably, the 5'UTR is at least one of F00, F03, F04, F05, F06, F07, F08, F09, F10 and F11, and F00, F03 to F11 are, in sequence, the sequence shown in Seq ID No. 2 to the sequence shown in Seq ID No.

11.

3. The mRNA according to claim 1, characterized in that: The 3'UTR is at least one of T01, T02, T03, T04, T05, T06, T07 and T08, and T01 to T08 are sequentially the sequence shown in Seq ID No. 12 to the sequence shown in Seq ID No.

19.

4. The mRNA according to any one of claims 1 to 3, characterized in that: The polyA tail is at least one of P02, P03, P04, P05 and P06, and P02 to P06 are sequentially the sequence shown in Seq ID No.20 to the sequence shown in Seq ID No.

24.

5. A recombinant plasmid containing the mRNA or DNA thereof according to any one of claims 1 to 4.

6. A recombinant cell comprising the mRNA according to any one of claims 1 to 4 or the recombinant plasmid according to claim 5.

7. A gene medicine comprising the mRNA according to any one of claims 1 to 4, the recombinant plasmid according to claim 5 or the recombinant cell according to claim 6.

8. A liposome nanoparticle containing the mRNA according to any one of claims 1-4.

9. The liposome nanoparticle according to claim 8, characterized in that: The liposome nanoparticles are formed by nanocarriers encapsulating mRNA; The nanocarrier is formed by cationic lipid, 1,2-distearoylglycerol-3-phosphocholine, cholesterol and polyethylene glycol lipid.

10. The liposome nanoparticle according to claim 9, characterized in that: In the nanocarrier, the molar ratio of cationic lipid, 1,2-distearoylglycerol-3-phosphocholine, cholesterol and polyethylene glycol lipid is 50:10:38.5:1.5; Preferably, the volume ratio of nanocarrier to mRNA is 1:3; Preferably, the particle size of the liposome nanoparticles is 90-180 nm.

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