Recombinant genes for increasing protein expression level and expression duration of co-transfected mRNAs and applications thereof

Abstract

The application discloses a kind of recombinant genes for increasing the protein expression level and expression length of co-transfected mRNA, and belongs to mRNA genetic engineering technology;The polypeptide encoded by the recombinant gene is a polypeptide with ribonuclease III activity, which is not inhibited by ribonuclease inhibitor and does not degrade single-stranded RNA;The recombinant gene and the target gene can be constructed in series or in parallel to form a fusion protein or an independently expressed recombinant protein;Or isolated by 2A peptide, internal ribosome entry site, etc., to express independent recombinant enhancement protein and target protein;The recombinant enhancement protein inhibits the innate immune mechanism and mRNA degradation mechanism of the host cell by degrading dsRNA, tRNA, miRNA and its precursor molecules, so that the protein expression level of co-transfected gene is increased by 3-4 times, and the expression time is prolonged by nearly 1 time;In addition, the present application inhibits the innate immune response to degrade impurities by rR3GE, and promotes the storage time of LNP preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to mRNA genetic engineering technology, and in particular to a kind of co-transfection mRNA protein expression level and expression length of recombinant gene and its application. BACKGROUND

[0002] Eukaryotic messenger RNA (mRNA) is a single-stranded ribonucleic acid molecule composed of several different elements, including a 5'-m7G cap, a 5'-untranslated region (5'UTR), a translation initiation codon, a coding region, a stop codon, a 3'UTR, and a poly-A tail. These elements serve as templates and regulators for the translation of protein sequences. The translation efficiency of mRNA proteins is influenced by the components of mRNA and is related to the host cell type. mRNA-mediated gene therapy depends on the protein translation efficiency of the delivered gene and the pharmacokinetic and toxicological characteristics of the protein. The recombinant sequence of pharmaceutical mRNA must be optimized according to the host cell type. The challenges in the preparation of mRNA drugs are to avoid the immunogenicity of mRNA and delivery materials, as well as heat source impurities, while improving the intracellular stability of mRNA, protein translation efficiency, and protein expression kinetic properties. The immunogenicity of lipid excipients and their degradation products, as well as the immunogenicity of mRNA, can stimulate the innate immune mechanisms of cells, causing inflammatory and cellular immune responses in the body. Combined with the intracellular mRNA degradation mechanism, this leads to a decrease in the intracellular stability of mRNA. Therefore, in addition to optimizing the design of mRNA sequences, mRNA gene therapy drug development also needs to avoid the influence of host cell innate immune mechanisms and mRNA degradation mechanisms.

[0003] Current technical solutions for intracellular mRNA degradation mechanisms involve mRNA recombinant sequence optimization, such as coding optimization, poly-A extension, UTR optimization, and miRNA target sequence removal, as well as innate immune inhibitors. For delivery lipids, ionizable lipids and helper lipids with low immunogenicity have been developed. To address the immunogenicity of mRNA, nucleoside modification techniques, primarily pseudouridine modification, are used to reduce the activation of host cell innate immune responses by mRNA. These methods are the current mainstream technical approach to improving the stability and efficacy of mRNA-based therapies. In particular, the experience of Pfizer / BioNTech and Moderna in successfully developing the COVID-19 mRNA vaccine using methyl pseudouridine nucleoside modification technology, and the lessons learned from the failure of CureVac and others to develop vaccines using naturally unmodified mRNA, have established the key role of nucleoside modification technology in mRNA vaccine development. Nucleoside-modified mRNA not only increases protein expression levels, but more importantly, avoids the activation of cellular innate immune mechanisms, resulting in more specific binding antibodies and neutralizing antibodies.

[0004] However, the current technical means can only eliminate the immunogen from the perspective of preparation production, and partially alleviate the influence of innate immune mechanism and mRNA degradation mechanism on mRNA stability. The improvement effect is greatly restricted by factors such as cognitive level, complexity of nucleotide modification, production process difficulty, and storage conditions. SUMMARY

[0005] One of the purposes of the present application is to provide a kind of recombinant gene for increasing the protein expression level and expression duration of co-transfected mRNA, to solve the above problems.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The recombinant gene for increasing the protein expression level and expression duration of co-transfected mRNA, the polypeptide encoded by the recombinant gene is a polypeptide with ribonuclease III activity, which is not inhibited by ribonuclease inhibitor and does not degrade single-stranded RNA.

[0008] As a preferred: the polypeptide is a recombinant Amphinase or an Amphinase recombinant derivative with deletion, substitution, insertion or addition of one or more amino acids.

[0009] As a further preferred: the Amphinase recombinant derivative is substituted with amino acids Met and Ser at the N-terminus of Amphinase instead of Gln, which has an amino acid sequence as shown in SEQ ID NO. 1; the recombinant derivative of its human corresponding gene has an amino acid sequence as shown in SEQ ID NO. 2 and SEQ ID NO. 8, which are rAng (RNase 5) and rRanpK32R respectively.

[0010] As a preferred: the polypeptide is a recombinant Amphinase or an Amphinase recombinant derivative with deletion, substitution, insertion or addition of one or more amino acids.

[0011] As a further preferred: the Amphinase recombinant derivative has an amino acid sequence as shown in SEQ ID NO. 3-7, in order: rAmph1 (based on Amph-1 P85072), rAmph2 (based on Amph-2 P85073), rAmph3 (based on Amph-3 P85074), rAmph4 (based on Amph-4 P85075) and rBS-RNase.

[0012] The second object of the present application is to provide a method for expressing a target RNA in a body by using the recombinant gene, and the technical solution is as follows: the enhancing gene and the target gene are constructed in a tandem manner in the same mRNA expression framework to form a fusion protein; or are isolated by a 2A peptide or an internal ribosome entry site to express independent recombinant enhancing proteins and target proteins; or are independently constructed in the mRNA expression framework and mixed with the target gene mRNA to prepare and implement co-transfection.

[0013] As a preferred solution, the nucleotide sequence of the enhancing gene is added to any RNA vector for eukaryotic expression to express proteins in a manner of independent transfection or co-transfection, so as to improve the expression level of the exogenous target gene in the body and cells.

[0014] As a preferred solution, the introduction of the recombinant gene is combined with nucleotide modification. The recombinant enhancing gene of the present application is combined with the nucleotide modification technology to have a synergistic effect on mRNA expression enhancement.

[0015] Another object of the present application is to provide an mRNA preparation prepared by the above method.

[0016] As a preferred solution, the preparation is an injection.

[0017] Another object of the present application is to provide the use of the mRNA preparation in the preparation of biological vaccines and gene drugs.

[0018] The present inventors have found through a large number of experiments that the method for increasing protein translation efficiency by inhibiting the innate immune response mechanism and mRNA degradation mechanism of host cells can reduce the innate immune response of cells to lipids, mRNA and hydrolysates in a larger dimension, replace nucleotide modification, or be combined with existing nucleotide modification technology to produce a synergistic effect, thereby greatly improving the protein expression efficiency.

[0019] Nuclease III (RN3, RNase III) is a ribonuclease of prokaryotes, which degrades double-stranded RNA (dsRNA). Dicer and Drosha are human RN3-type nucleases, which cut miRNA precursors to participate in the maturation of miRNA, and further participate in the regulation of gene translation level and mRNA stability.

[0020] Specifically, the immunogenic substances contained in the mRNA vaccine preparation include:

[0021] LNP (Lipid Nanoparticle) is a complex formulation, its components are considered to be non-pharmacologically active and less toxic, but the components used, such as PEG, ionizable lipids have weak immunogenicity. Impurities and oxidative degradation products of lipid components during storage can stimulate immune response. These substances stimulate non-specific immune response, leading to side effects. The following is an analysis of the main immunogenic components and their impact on mRNA expression efficiency:

[0022] mRNA: Natural mRNA synthesized by eukaryotic cells after transcription modification has a large number of modified nucleotides, including pseudouracil and methylated adenosine, and has no immunogenicity. Unmodified mRNA molecules synthesized by in vitro transcription (IVT) are activators of immune pattern receptors (TLRs and RIG-1), which can activate intracellular TLR and RIG-I receptors in cells, activate cellular innate immune response, and induce inflammatory response, leading to translation inhibition and mRNA degradation. Nucleotide modifications such as N1-methyl pseudouridine (Ψ), pseudouridine and N-6-methyladenosine (m6A) in in vitro synthesis reduce the recognition of innate immune mechanisms, increase mRNA stability, and increase protein translation efficiency by more than 10 times.

[0023] dsRNA: RIG-I and MDA5 are two major intracellular dsRNA receptors, which will activate the antiviral signaling pathway and produce anti-inflammatory factors such as interferon once they recognize exogenous dsRNA. IVT-produced mRNA is accompanied by dsRNA, which stimulates dsRNA receptors and produces stress response, leading to translation inhibition and mRNA degradation.

[0024] 5'-Triphosphate Motif: After the capping modification step of IVT-produced mRNA, the uncapped 5'-triphosphate motif has immunogenicity, which activates cellular innate immune response by binding to RIG-I;

[0025] Lipids and impurities: PEG2000-DMG. The impurities present in the PEG group are the source of toxic substances that cause DSPC degradation. Cholesterol is easily oxidized, leading to a series of cholesterol oxidation products;

[0026] Impurities generated during storage: Studies have shown that naked mRNA molecules can be stored stably in a water phase with nuclease inhibitor (RI) at 5℃ for more than 941 days at pH 7.4. In contrast, the shelf life of the marketed COVID-19 mRNA vaccines is less than a year when stored at ultra-low temperature (BNT162b2, -80℃; mRNA-1273, -20℃). By summarizing the different storage conditions and shelf life of existing mRNA-ionizable lipid LNPs and commercial mRNA-ionizable lipid LNPs, it is found that the excipients and manufacturing methods of mRNA-ionizable lipid LNPs have a significant impact on the storage stability. The fundamental reason for the short shelf life of mRNA-ionizable LNP is the internal water content and excipient composition in the LNP formulation. Ionizable LNPs will degrade over time due to aggregation, fusion, mRNA leakage, or lipid degradation. During the long-term storage of ionizable LNPs, the rate of mRNA decomposition and lipid denaturation is affected by storage temperature, storage time, organic solvents, lipids, and core lipids. The most common cause is the hydrolysis and oxidation of the nucleic acid phosphodiester backbone in the presence of water or acid / base. The PEG group of PEG2000-DMG and the carboxyl ester bond of DSPC are prone to hydrolysis during storage;

[0027] The lipids present in ionizable LNPs have an indirect impact on the stability and structural integrity of mRNA, and are one of the main reasons for the short shelf life.

[0028] Detailed description of Ranpirnase

[0029] Ranpirnase (Ranp) is a ribonuclease from amphibian, which belongs to the superfamily of pancreatic ribonuclease (RNaseA) proteins. N-pyroglutamyl residues are key components of Ranp cytotoxic activity, which contributes to its entry into cells and stable conformation. The C-terminal disulfide bond (87-104) is covalently bound to form a super-stable conformation of Ranp, which is resistant to endogenous proteases and reduces the affinity for RI, allowing it to remain active in cells, while most mammalian nucleases are inhibited by RI.

[0030] Ranp has ribonuclease III (RN3) activity, and the degradation substrates that have been confirmed include tRNA, dsRNA, miRNA precursor, while mRNA and rRNA are not affected.

[0031] The degradation of tRNA by Ranp leads to inhibition of protein synthesis; degradation of double-stranded nucleic acids reduces stimulation of the innate immune response of cells; and degradation of small RNA precursors reduces the degradation of RNA by interference mechanisms such as siRNA and miRNA, and produces miRNA and siRNA that affect gene expression.

[0032] The miRNA-mediated RNA silencing mechanism is one of the main pathways of mRNA degradation. Ranp degrades miRNA precursors, universally down-regulates high-abundance miRNAs, especially high-abundance immune modulators such as miR-155 and miR-21, to down-regulate the innate immune function and enhance mRNA stability.

[0033] Ranp has an immune regulation mechanism by interfering with the nuclear factor kappa light chain enhancer (NFκB) pathway of activated B cells. Ranp inhibits the translocation of NFκB to the nucleus and regulates the response of cells to stimuli such as stress, free radicals, bacteria and / or viral antigens. In addition, NFκB plays a key role in regulating the immune response to infection (κ light chain is an important component of immunoglobulin). By inhibiting the translocation of NF-κB into the nucleus, the inflammatory process will be inhibited.

[0034] The N-terminal of Ranp is a unique pyr1 modification of ring focal glutamic acid, which prefers uridine-guanine (UG) substrates and has anti-inflammatory and tumor cell growth inhibition effects. As one of the embodiments, the recombinant polypeptide expressed by the recombinant Ranp (rRanp) gene starts with Met, which reduces the thermal stability, catalytic activity and antigenicity of rRanp, and is mainly limited in the cytoplasm due to the loss of the secretion signal peptide. The pyr1 replacement variant maintains a similar secondary structure to wild-type Ranp, but has lower thermal stability and specific catalytic activity for the innate substrate UG.

[0035] Design and functional verification of rR3GE

[0036] As one of the embodiments, as a piece of artificially synthesized recombinant gene (rR3GE), the recombinant polypeptide protein encoded thereby improves the protein expression efficiency and prolongs the expression time of the target gene by inhibiting the innate immune response and mRNA degradation of the host cell.

[0037] The protein encoded by rR3GE has the following characteristics: 1. Nuclease III activity, degrading dsRNA; 2. Low immunogenicity, which can be soluble or non-soluble; 3. No cytotoxicity; 4. No hydrolytic activity on single-stranded RNA; 5. Low affinity to RI; 6. The enhancement function of rR3GE is independent of the type of target protein, the gene coding sequence, and the type of host cell.

[0038] One embodiment of the present application is to co-transfect rRanp mRNA synthesized in vitro with target protein mRNA. The polypeptide sequence (4-105) of rRanp with RNA hydrolysis activity is reverse coded into rR3GE gene, and the optimized gene code is recombined into plasmid DNA under the expression framework of T7 promoter to form mRNA production template with translation activity. mRNA is generated by in vitro transcription reaction, capped and modified by vaccinia capping enzyme to form mature mRNA with translation activity. rRanp mRNA and tracer protein mRNA are wrapped with traditional LNP, transfected into in vitro cultured cells or intramuscularly administered to mice, and compared with the control group, it is observed that rRanp significantly increases the expression amount of tracer protein and prolongs the expression time, as shown in Figure 3 ,4. After the LNP (LNP Å ) is modified by non-ionizing cations, the same expression enhancement and expression time prolongation are observed, confirming that the enhancement of rRanp is not related to the type of delivery medium and has universality. The rRanp is confirmed by in vitro cultured cell colony production experiment, and the rRanp gene has no obvious cytotoxicity, as shown in Table 1.

[0039] Another embodiment of the present application is to recombine rR3GE gene and target protein gene into the same mRNA with self-cleavage polypeptide (P2A) or IRES in between to simultaneously generate independent rR3GE polypeptide and target protein in cells. The rRanp polypeptide sequence is reverse coded into rRanp gene under the expression framework of T7 promoter, and the optimized target gene sequence, P2A gene sequence and rR3GE gene sequence are arranged in a linear manner and constructed into plasmid DNA to form mRNA production template. The mRNA is wrapped with LNP and intramuscularly administered to mice, and compared with the control group, it is observed that the rRanp gene significantly increases the expression amount of tracer protein and prolongs the expression time, as shown in Figure 6 .

[0040] Another embodiment of the present application is to recombine rR3GE gene and target protein gene into the same mRNA with self-cleavage polypeptide (P2A) in between to simultaneously generate independent rAmph1 protein and target protein. The polypeptide sequence (4-105) of Amph-1 with RNA hydrolysis activity is reverse coded into rR3GE gene under the expression framework of T7 promoter, and the optimized target gene sequence, P2A gene sequence and rAmph1 gene sequence are arranged in a linear manner and constructed into plasmid DNA to form mRNA production template with translation activity. The mRNA is wrapped with LNP and intramuscularly administered to mice, and compared with the control group, it is observed that the rAmph1 gene significantly increases the expression amount of tracer protein and prolongs the expression time, as shown in Table 1.

[0041] Another embodiment of the present application is to use the polypeptide sequence (4-105) with RNA hydrolysis activity of rRanp, add a translation initiation codon to the N-terminus of the polypeptide, form a recombinant gene sequence with expression activity in eukaryotic cells, place it in the expression frame of the CMV promoter, and recombine it in plasmid DNA. Use the plasmid DNA to transfect in vitro cultured cells, and express the rRanp protein containing different N-terminal amino acid sequences under the driving of CMV, as shown in Figure 1 Although the mechanism of action of rR3GE is not very clear, RN3 activity and low RI affinity are two necessary conditions, and there is no degradation effect on linear RNA. Based on these characteristics, those skilled in the art can easily recombine and construct recombinant gene fragments with similar functions but different sequences.

[0042] Potential mechanism of action of rR3GE:

[0043] (1) degrade tRNA, reduce the efficiency of cell translation, maintain low-level protein expression, and reduce the degradation of exogenous mRNA and protein;

[0044] (2) specifically cleave miRNA and its precursor, intervene in the maturation of miRNA / siRNA, reduce the innate immune response of cells, and weaken the RNA degradation mechanism of cells;

[0045] (3) knock out dsRNA to avoid stimulating the innate immune response of cells;

[0046] (4) intervene in the NF-κB pathway and MMP9 activity to reduce the innate immune response of cells

[0047] As a preferred embodiment, the mRNA expression enhancer gene of the present application can be an artificially synthesized short recombinant gene fragment encoding a recombinant polypeptide with nuclease III activity, which is co-transfected with a pharmacodynamic mRNA to significantly increase the expression efficiency and duration of pharmacodynamic proteins by inhibiting the innate immune response and mRNA degradation of host cells. The gene fragment with such a function is referred to as rR3GE, i.e. recombinant Ribonuclease Ⅲ-associated gene expression Enhancer.

[0048] In terms of mechanism, rR3GE reduces the recognition and degradation of mRNA by the innate immune mechanism of cells, thereby increasing the stability of mRNA and promoting protein expression and expression duration. These findings suggest that rR3GE has the potential to be used as a general booster for mRNA vaccines (drugs), which is of great significance for biomedical research and development of biological products, drugs and vaccines. The degree of this enhancement does not vary with protein type, cell density / function, transfection efficiency, delivery mechanism, reporter dose, secretion signal and 2A-mediated autocleavage efficiency, nor does it change the tissue distribution of gene expression.

[0049] Protein replacement therapy has a wide range of applications. For example, in the treatment of hemophilia, hemophilia patients lack blood clotting proteins due to genetic mutations, and because the half-life of proteins is short, usually only 12 hours, patients need to inject 3-7 times of blood clotting proteins per week. Preclinical studies in mice have shown that a dose of 0.2-0.5 mg / kg of nucleoside-modified mRNA injected once a week can maintain effective levels of clotting factor proteins. Hemophilia clinical trials using adeno-associated virus (AAV) have shown that protein expression is stable for 2 years after injection. However, some recent studies have shown that due to the rejection of the immune system to viral vectors, re-injection may be required after 5-7 years of injection, and viral vectors have their own safety issues, especially in pediatric disease treatment. In addition, patients with underlying immunity cannot use AAV gene therapy. Based on the rR3GE-enhanced clotting factor VⅢ mRNA therapy of the present application, it is possible to replace traditional therapy for the treatment of hemophilia.

[0050] Currently, in mRNA COVID-19 vaccination, low-dose mRNA local delivery has been observed to have inflammatory complications, while mRNA therapy is administered by high-dose long-term administration, which will amplify inflammatory complications and other side effects. However, the immunogenicity of the rR3GE of the present application is very low, and there is further room for improvement. Species source modification of the rR3GE protein sequence and structure can further reduce the immunogenicity of rR3GE in a specific species. For example, humanization recombination of the rR3GE protein can further reduce or eliminate its immunogenicity, which is beneficial for long-term repeated drug use.

[0051] CircRNA is a kind of circular RNA with a series of protein coding and non-coding functions, which does not contain 5'-triphosphate motif, so the RNA-mediated innate immune response is low, and the protein expression of circRNA shows higher stability than the uridine-modified linear mRNA in adipose tissue. Although circRNA does not contain a typical triphosphate motif required for RIG-I activation, RIG-I can interact with circRNA instantaneously without host nuclear protein protection, resulting in a typical RIG-I-mediated inflammatory response. CircRNA can also interact with other RNA sensors, such as endosomal TLR3, 7 and 8, and MDA5, etc., resulting in an inflammatory response. The protein expression of circRNA is initiated by IRES, and nucleotide modification affects the protein translation level, so it is not possible to reduce the host's innate immune response to circRNA by means of nucleotide modification. rR3GE is compatible with the IRES expression system of circRNA, and in the absence of nucleotide modification, rR3GE can be fully utilized to suppress the host's innate immune response and enhance the expression level and duration of the target gene.

[0052] Therefore, compared with the prior art, the advantages of the present application are that the mRNA expression enhancing gene and the method for enhancing intracellular gene expression provided by the present application can increase the expression level of co-transfected gene protein by 3-4 times by adding a small piece of enhancing gene, and the expression time is prolonged by nearly 1 times:

[0053] (1) Enhancing the expression level of co-transfected gene protein by 3-4 times, reducing the dosage of mRNA preparation;

[0054] (2) Reducing the overall level of cellular protein, reducing the degradation of exogenous mRNA, and prolonging the expression duration of the gene;

[0055] (3) Inhibiting the innate immune mechanism of cells, reducing the reactivity of cells to preparation heat sources (including impurities and degradation products); in specific applications, nucleotide modification can be replaced, and the destruction of the host's innate immune mechanism to mRNA can be avoided.

[0056] (4) rR3GE degrades dsRNA, reducing the difficulty of mRNA production and purification; reducing the threshold of mRNA production;

[0057] (5) Simplifying the mRNA-LNP production process; prolonging the storage time of mRNA-LNP;

[0058] (6) Reducing the innate immune response in muscle cells, making the presentation of specific proteins more specific. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 : rRanp enhancing gene recombinant sequence and co-expression mRNA molecule sequence analysis;

[0060] Figure 2 : rRanp enhanced co-expression mRNA recombinant molecule design;

[0061] Figure 3 : rRanp enhanced co-transfection nucleoside modified mRNA expression level and expression duration IVIS imaging results;

[0062] Figure 4 : rRanp enhanced co-transfection nucleoside modified mRNA expression level and expression duration analysis comparison;

[0063] Figure 5 : rRanp enhanced co-transfection unmodified mRNA expression level and expression duration IVIS imaging results;

[0064] Figure 6 : rRanp enhanced co-transfection unmodified mRNA expression level and expression duration analysis comparison;

[0065] Figure 7 : rRanp enhanced co-transfection different dsRNA content mRNA expression level and expression duration IVIS imaging results;

[0066] Figure 8 : rRanp enhanced co-transfection different dsRNA content mRNA expression level and expression duration analysis;

[0067] Figure 9 : rRanp enhanced co-transfection different dsRNA content mRNA expression level difference comparison;

[0068] Figure 10 : rR3GE gene variant mRNA design;

[0069] Figure 11 : rR3GE gene variant mRNA expression enhancement in vivo IVIS imaging results;

[0070] Figure 12 : rRanp recombinant gene colony formation inhibition rate in cultured cells;

[0071] Figure 13 : rR3GE promotes LNP preparation stability under 4℃ and -20℃ storage conditions. Embodiment

[0072] The application will be further described below with reference to the accompanying drawings.

[0073] Noun explanation: ionizable lipid: such as ALC-0315, MC3, DHA-1, L319, SM-102, etc. The ionizable lipid used in the embodiments of the present application is ALC-0315;

[0074] Non-ionizable cationic lipid: an amphipathic molecule with hydrophilic and hydrophobic groups, composed of a polar head (hydrophilic group), a connecting bond, and a hydrophobic tail. The hydrophilic head is a quaternary ammonium salt, which is a permanent cation and does not have ionizable characteristics. The non-ionizable cationic lipid used in the embodiments of the present application is DOTAP;

[0075] Nucleic acid: refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single or double stranded form, including DNA and RNA. RNA can be in the form of siRNA, microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, circular RNA and combinations thereof. Nucleic acids can be synthetic, naturally occurring and non-naturally occurring. Including but not limited to phosphorothioate, phosphoramidate and peptide nucleic acid (PNA), and including nucleic acids containing known natural nucleotide analogs and artificially modified nucleotides, such as pseudouracil, methylated, methyl pseudouracil modified nucleic acids. DNA can be double-stranded DNA, single-stranded DNA, and plasmid DNA, etc.

[0076] Example 1: Raw materials and preparation of formulations

[0077] 1.1 RNA preparation

[0078] The mRNA used in the embodiments of the present application is obtained by IVT reaction. The general process is: enzyme digestion of plasmid DNA template; column purification to obtain linearized plasmid DNA; IVT transcription production of RNA (Thermo, MEGAscript® Kit); after transcription is completed, the RNA is purified with oligo-dT column (Sartorius). Unless otherwise specified, the transcription reaction substrate UTP is replaced with N1-methylpseudouridine acid (ѱ).

[0079] The mRNA capping modification reaction is completed by Vaccinia Capping Enzyme of the nearshore protein. The mRNA capping modification reaction is set according to the recommended reaction system of the kit, and the reaction condition is 37℃ for 1 hour. After the reaction is completed, the capping product is purified with oligo-dT affinity column. The purified mRNA is dissolved in sterile water for injection, and analyzed by RNA gel electrophoresis and Qubit concentration identification.

[0080] 1.2 LNP, LNP ⊕ Preparation of formulations

[0081] LNP, LNP ⊕The preparation is composed of ionizable lipid, non-ionizable cationic lipid, DSPC, cholesterol (Chol) and PEG2000-DMG in a certain molar ratio, and the specific composition is as follows:

[0082] The molar ratio of LNP lipids is: ALC-0315: DSPC: Chol: PEG = 46.29: 9.4: 42.67: 1.64; and the N / P ratio is 6.09.

[0083] LNP ⊕ 46The molar ratio of lipids is: Dotap: ALC-0315: DSPC: Chol: PEG = 23.01: 23.01: 9.35: 42.43: 2.2; and the N / P ratio is 12.1.

[0084] The lipid material is dissolved in anhydrous ethanol, and the nucleic acid is dissolved in an aqueous citric acid solution (10 mM, pH 4.0). The aqueous solution and the organic solution are mixed at a volume ratio of 3:1 through a microfluidic chip (Shanghai Pengzan Biotechnology, luer connector microfluidic chip) at a total flow rate of 12 ml / min. The LNP preparation is dialyzed against 1x PBS solution overnight, and then transferred to a glass bottle and stored at 4°C or -20°C. The final concentration of mRNA is 0.1-0.375 µg / µl.

[0085] Example 2 P2A self-cleavage rRanp gene enhances the expression level and expression time of nucleoside-modified mRNA

[0086] This example compares the expression levels of nucleoside-modified mRNA delivered by LNP and LNP ⊕ 46The expression level of nucleoside-modified mRNA delivered by LNP is affected by rR3GE. In the IVT reaction, N1-methyl pseudouridine nucleotides are used instead of UTP as substrates to prepare ѱFluc, ѱFluc-rRanp mRNA. LNP and LNP ⊕ 46Lipid, respectively, is used to encapsulate mRNA to prepare a series of LNPs containing different mRNAs. The protein sequence of rRanp is designed as Figure 1 , and the composition of mRNA is Figure 2 .

[0087] As can be seen from Figure 1 , rRanp adopts the polypeptide sequence of mature Ranp, with Gln (Q) removed from the N-terminus and replaced by Met and Ser. The amino acid sequence of "rRanp" in the following examples is shown in SEQ ID NO. 1. The co-expression mRNA of Fluc (firefly luciferase gene) and rRanp is connected by a self-cleavage polypeptide P2A sequence to form a single-chain mRNA of Fluc-rRanp. When the gene is transcribed, the polypeptide is cleaved from the 21st position of P2A, dividing the transcript into two polypeptides: Fluc and rRanp.

[0088] Seven-week-old female Balb / c mice were divided into six groups, with three mice in each group. The mice were administered 5.0 µg / 30 µl of the test ѱFluc-LNP, ѱFluc-rRanp-LNP, ѱFluc-LNP ⊕ 46, ѱFluc-rRanp-LNP ⊕ 46, and ѱFluc-LNP by intramuscular injection (IM) in the right lower limb. Figure 3 .

[0089] The experimental results show that the trace genes in the mice of the LNP preparation group have high levels of expression in the intramuscular administration site and liver tissue, as shown in FIG. 4A. Figure 4 The trace protein expression of the mice in the ѱFluc-rRanp-LNP experimental group with the addition of the rRanp recombinant fragment is significantly increased. Calculated by the area under the curve (AUC), the trace protein expression of the mice in the ѱFluc-LNP control group is 6.14, 1.60, and 3.88 times that of the mice in the ѱFluc-rRanp-LNP experimental group in the liver, muscle, and whole body, respectively, as shown in FIG. 4C. Figure 4 The trace protein expression duration of the mice in the ѱFluc-rRanp-LNP experimental group is also significantly increased. The trace protein expression in the liver is prolonged by 48 hours, and the trace protein expression at the intramuscular administration site is prolonged by 96 hours. At the same time, the half-life of the trace protein at the muscle site is increased from 20.2 hours in the control group to 23.2 hours. The trace signal at the intramuscular administration site of the mice in the rRanp group decreases sharply at the initial stage of transfection, but turns around after 24 hours, and the decay rate of the trace protein signal slows down. The curvature of the trace protein expression dynamics after that changes significantly, as shown in FIG. 4B. Figure 4

[0090] Intramuscular LNP ⊕ The mice in the ѱFluc-rRanp-LNP ⊕ The trace protein expression of the mice in the ѱFluc-rRanp-LNP ⊕ The trace protein expression of the mice in the ѱFluc-rRanp-LNP ⊕ The trace protein expression of the mice in the ѱFluc-rRanp-LNP Figure 4 D shows. The trace protein expression duration of the mice in the ѱFluc-rRanp-LNP ⊕ The trace protein expression duration of the mice in the ѱFluc-rRanp-LNP ⊕ ​46control group. Meanwhile, the half-life of the tracer protein in the muscle site was increased from 27.8 hours in the control group to 37.2 hours; LNP ⊕ 46The tracer signal in the intramuscular injection site of the mice in the group decreased sharply at the beginning of the transfection, slowly rose at 24 to 48 hours, and then the expression kinetic curve of the tracer protein changed significantly, as shown in Figure 4 B.

[0091] Conclusion: rRanp has a significant effect on the expression kinetics of LNP and LNP ⊕ 46The expression kinetics of the nucleoside-modified mRNA delivered by lipids were significantly enhanced, and the protein expression was increased by 1.80 to 6.24 times, and the expression time was prolonged by about 1 time, depending on the different expression sites. The separation of the tracer protein and the rRanp protein by the P2A self-cleavage polypeptide did not affect each other's function. The expression kinetic characteristics of the tracer protein in the intramuscular injection site were superimposed by the effects of multiple modifications. The half-life of the tracer protein was increased from 20.2 hours to 23.2 hours by pseudouridine modification; LNP ⊕ 46to 27.8 hours, and the addition of rRanp increased the half-life to 37.2 hours. In comparison, the half-lives of the tracer proteins expressed by the same dose of nucleoside-modified mRNA by intramuscular injection and intradermal injection were 20.6 hours and 29.6 hours, respectively (Pardi, Tuyishime et al. 2015). Therefore, the expression enhancement effects of pseudouridine modification, LNP ⊕ 46formulation, and rRanp have a synergistic effect when used simultaneously. Even if the drug is administered by intramuscular injection, clear tracer protein signals can still be detected by the IVIS imager 15 days after administration. Unlike nucleoside modification, which takes effect immediately, rRanp needs about 24 hours to express and take effect, and produces a characteristic turning point in the expression level of the tracer protein between 24 to 48 hours, and changes the curvature of the subsequent protein expression kinetics. Example 3

[0092] This example compares the effect of rRanp on the expression level of LNP lipid-delivered mRNA without nucleoside modification. In the IVT reaction, UTP or N1-methyl pseudouridine nucleotide was used as a substrate instead of UTP to prepare Fluc-rRanp mRNA and ѱFluc mRNA, respectively. The mRNA was encapsulated with LNP lipids to prepare mRNA-LNPs.

[0093] The 7-week-old female Balb / c mice were divided into three groups, each group of 3, and were administered by intramuscular injection of the right lower limb. The nucleic acid modified ¾Fluc-LNP (hereinafter referred to as the "control group"), and the unmodified mRNA lipid particle Fluc-rRanp-LNP (hereinafter referred to as the "experimental group") (5 µg and 10 µg) lipid nanoparticle preparations were tested. After intramuscular administration, in vivo IVIS imaging analysis was performed at different time points. The results of in vivo IVIS imaging of mice are shown in Figure 5 .

[0094] The experimental results show that under intramuscular administration, the tracer gene in the LNP preparation group of mice has high expression in the intramuscular administration site and liver tissue. The total amount of tracer protein expression in the experimental group of mice with a 5 µg mRNA dose is significantly lower than that of the control group, and the liver, muscle, and whole body protein expression amounts are 49%, 15%, and 16% of that of the control group, Figure 6 A、 Figure 6 C. However, the expression duration of the tracer protein in the experimental group of mice is significantly increased, and the expression of the tracer protein in the liver is prolonged by 48 hours, and the expression of the tracer protein at the intramuscular injection site is prolonged by 96 hours, which is consistent with the experimental group of ¾Fluc-rRanp-LNP in Example 2 ( Figure 4 A). The tracer protein expression kinetics profile at the intramuscular injection site is significantly changed. The tracer protein of the control group in this embodiment is mainly expressed on the first day after administration, and the expression amount is much higher than that of the same dose group. However, the expression amount of the tracer protein in the experimental group of mice from the 3rd to the 13th day is 1.47 times that of the control group, and the half-life of the tracer protein is increased to 32.9 hours, Figure 6 A.

[0095] The total amount of tracer protein expression in the experimental group of mice with a 10 µg mRNA dose is increased compared to the low-dose (5 µg mRNA) experimental group, which is 1.73, 1.47, and 3.75 times the amount of tracer protein expression in the liver, muscle, and whole body, respectively ( Figure 6 D), and the half-life of the tracer protein is increased to 31.6 hours ( Figure 6 B). The tracer protein expression kinetics profile at the muscle site of the experimental group of mice under different doses is similar, and the protein expression amount is 2.21 times that of the control group from the 3rd to the 13th day. After experiencing a sharp decrease in the first 24 hours, the tracer signal at the intramuscular injection site slowly rises from 24 to 48 hours, and the experimental group is particularly obvious, and the rising intensity is proportional to the mRNA injection dose. Thereafter, the kinetics of tracer protein expression is significantly changed, Figure 6 A、 Figure 6 B.

[0096] Conclusion: At the same mRNA dose level, the total amount of tracer protein expression of unmodified rRanp enhanced mRNA is lower than that of nucleoside-modified mRNA, but the rRanp polypeptide affects the kinetic characteristics of tracer protein expression, prolonging protein expression time by more than one-fold. The rRanp has a positive dose relationship with the expression of tracer protein of unmodified mRNA. According to the different expression sites, the protein expression amount of 10 µg rRanp mRNA is 1.47 to 3.75 times that of 5 µg rRanp mRNA, but the mRNA dose has no effect on the expression kinetic profile, and increasing the mRNA dose does not prolong the protein expression time. Although Figure 4 LNP in B ⊕ 46The expression signal of the delivered tracer gene is extended on the second day after intramuscular injection, and the tracer protein signal at the intramuscular injection site rebounds on the first day after intramuscular injection, which is caused by the rRanp gene, and the rebound intensity is proportional to the mRNA dose, which is particularly evident in unmodified mRNA. The signal rebound period at the beginning of transfection is exactly the time when the rRanp gene is expressed and functions, which also confirms that the expression enhancement effect of nucleoside-modified mRNA and the expression enhancement effect of rR3GE are independent of each other, i.e., the mRNA that does not undergo nucleoside modification and only integrates rR3GE also shows a significant expression enhancement effect, and the two have a synergistic effect when used together. Example 4

[0097] Fluc mRNA containing different concentrations of dsRNA, Fluc-rRanp mRNA were used to prepare Fluc-LNP, Fluc-rRanp-LNP. 7-week-old female Balb / c mice were divided into four groups, 3 in each group, and were administered intramuscularly. The LNPs containing H-Fluc, H-Fluc-rRanp mRNA with high concentration of dsRNA (1.26%, hereinafter referred to as “H-Fluc-rRanp group”) and L-Fluc-rRanp mRNA with low concentration of dsRNA (0.14%, hereinafter referred to as “L-Fluc-rRanp group”) were tested. After intramuscular administration (5 µg or 10 µg mRNA), in vivo IVIS imaging analysis was performed at different time points. The results of in vivo IVIS imaging of mice are shown in Figure 7 .

[0098] The experimental results show that, under intramuscular administration, the mice in the group of mRNA-LNP preparation containing high-concentration dsRNA are all shown to have high expression of the trace protein in the intramuscular injection site and liver tissue, the total amount of the protein expressed in the liver and the intramuscular injection site of the mice in the rRanp-mRNA group is respectively 2.50 times and 2.90 times higher than that of the H-Fluc-rRanp group, and the expression time is respectively prolonged by 48 hours and 72 hours. The total amount of the protein expressed in the liver of the mice in the L-Fluc-rRanp group is 4.16 times higher than that of the H-Fluc-rRanp group, the expression time is prolonged by 24 hours, the total amount of the protein expressed in the intramuscular injection site is 1.72 times higher, but the expression time is not prolonged, as shown in Figure 8 and Figure 9 .

[0099] This embodiment proves that the high-concentration dsRNA and pseudouridine modification have no significant influence on the expression time of the rRanp-mRNA gene, but influence the protein expression amount at the initial stage of the transfection.

[0100] Example 5: rR3GE protein sequence analysis

[0101] According to the performance indicators such as RN3 activity and low RI binding that may be involved in the enhancement of gene expression, the protein amino acid sequence, structure and functional groups of rRanp are analyzed, and the genes with similar structures in the NIH-NCBI gene library are searched by taking the rRanp molecule as a template. It can be understood by those skilled in the art that it is possible to construct a new rR3GE with similar functions of rRanp by editing and recombining these sequences as templates:

[0102]

[0103] Example 6

[0104] According to the analysis and test of the sequence, structural characteristics and functional groups of the rRanp molecule by the inventors, the common characteristics of rR3GE are summarized. The sequence, structure and functional groups of the rR3GE molecule that may have the function of enhancing gene expression are further discussed and tested, and the induction of the sequence, structure and function of rR3GE by the inventors is verified. The influence of RN3 activity and low RI affinity on the enhancement of rR3GE gene expression, and the degradation activity of mRNA (single-stranded RNA) are required to be avoided.

[0105] The catalytic triad (His12, Lys41 and His119) of RNase A is the key site for the high activity of RNase A nuclease. It is strictly conserved in all variants of Ranp (His10, Lys31 and His97) and Amph (His15, Lys42 and His107). The nucleolytic activity of Ranp and Amph-1 is significantly lower than that of RNase A and most of the homologues. The variant of RNase A in which Lys41 is replaced by an arginine residue was shown to have about 2% of the enzymatic hydrolytic activity of wild-type RNase A. The molecular basis of this difference has not been fully elucidated. The most obvious factor is the atypical conformation of the catalytic residues, Lys31 and His97 in Ranp and the equivalent Lys42 and His107 in Amph-1. These residues, in comparison with the highly active members of the RNase A superfamily, have fewer hydrogen bonds with adjacent residues, and the substrate binding of both enzymes is impaired.

[0106] In this embodiment, the inventors constructed a group of recombinant gene enhanced mRNA containing different RNase A hydrolytic activity and RI affinity according to the characteristics of rR3GE, and the construct is shown in Figure 10 .

[0107] In this embodiment, the mRNA-LNP described above is prepared. 7-week-old female Balb / c mice are divided into six groups, 3 mice in each group. 5 μg is administered by intramuscular injection, and in vivo IVIS imaging analysis is performed at 0.25, 1 to 9 days after administration. The results are shown in Figure 11 .

[0108] The experimental results are shown in Table 1. Under intramuscular administration, rRanp, rRanpK32R, rAmph1, rBS-RNase and other recombinant genes with RN3 hydrolytic activity and low RI affinity have similar gene expression enhancement functions of rR3GE, while the expression level of protein expressed in the liver within 1 day after administration of rRNaseI is slightly increased, but the expression duration of the gene is not increased. The K32R mutation reduces the RNase A hydrolytic enzyme activity (RN1) of rRanpK32R, and further improves the expression level of the co-transfected gene based on rRanp, indicating that the RNase A hydrolytic activity has a limited promotion effect on the protein expression level of the co-transfected mRNA within a short time, and has no obvious help for improving the expression duration of the co-transfected gene.

[0109] Table 1: Comparison of activity characteristics and gene expression enhancement of different rR3GE genes

[0110]

[0111] *: Dimeric BS-RNase Kd Value is 2E-06; monomeric BS-RNase K d Value is 1E-12;**: RNase III activity based on rRanp

[0112] ***: Rutkoski, T. J. and R. T. Raines (2008). "Evasion of ribonuclease inhibitor as a determinant of ribonuclease cytotoxicity." Curr Pharm Biotechnol 9(3): 185-189.

[0113] Conclusion: RN3 activity and low RI affinity are required for the enhanced expression of rR3GE. After co-transfecting cells with RNase I, the over-expression of RNase I in a short time degrades various RNAs in the cytoplasm, and down-regulates the normal metabolic level of cells. The co-transfected mRNA protein expression is assisted and enhanced, but the expression time of the target gene is not increased. By mutating RN1 of rR3GE, the expression level of the co-transfected gene can be further improved. Example 7

[0114] A549 cells are p53 wild type, H1299 cells are p53 deletion type, and H322 cells are p53 mutant heterozygous type. To evaluate the effect of rRanp on the tracer gene in vitro cells, 2 x 10 5A549, H322, and H1299 cells were seeded at 1,000 cells / mL into 6-well plates and incubated at 37°C in 5% CO2 overnight. pcDNA3.1 was mixed with pCMV-eGFP, pCMV-BP-Rnase, pCMV-Bax, pCMV-p53, pCMV-rMQD-Ranp, and pCMV-rMSD-Ranp plasmid DNA at a 1:9 weight ratio, encapsulated with Dotap:Lecithin (molar ratio 20:9) liposomes, and then diluted with culture medium containing 10% FBS to 1 μg or 5 μg DNA per ml of medium. The culture medium was replaced with the DNA-liposome-containing medium, and the cells were incubated in the incubator. After 2 days of transfection, the cells were replaced with culture medium containing different concentrations of G418, H1299 (400 μg / ml), H322 (100 μg / ml), and A549 (100 μg / ml). When the cell density reached more than 80%, the cells were fixed with 10% formalin and stained with Giemsa solution, photographed, and counted. Cell toxicity was determined by the percentage of remaining viable cells after exposure to the test gene expression relative to eGFP-negative control cells (% cell toxicity = 100 - [(experimental group mean / eGFP group mean) x 100]), where the experimental group was one of the five genes tested, and the eGFP group was the negative control. Unpaired one-tailed Student's t-tests were used to determine statistical significance. t - Statistical evaluation of observed differences was tested and significance was established at the P < 0.05 confidence level.

[0115] The colony formation inhibition rate of rRanp recombinant genes in cultured cells was as shown in Table 1. Figure 12 As shown in Table 1, the experiments showed that the Bax and p53 genes had different colony formation inhibition rates in the three cultured cell lines. The Bax gene significantly inhibited the formation of colonies in A549 cells and H322 cells; the p53 gene inhibited the formation of colonies in H1299 cells. The rMQD-Ranp and rMSD-Ranp genes had no inhibitory effect on the formation of colonies in the three cells, Figure 12 At the same time, the BP-Rnase recombinant gene also showed no significant colony growth inhibition ability.

[0116] Conclusion: Based on the colony formation inhibition experiments on different p53 mutant phenotype cell lines, the rMQD-Ranp, rMSD-Ranp, and BP-Rnase recombinant proteins showed no significant cytotoxicity. Example 8

[0117] To evaluate the effect of rR3GE on the preservation stability of mRNA-LNP and verify whether it can prolong the preservation time of mRNA, the inventors conducted a comparative experiment on the expression intensity in tracer mice under the storage conditions of 4°C and -20°C. The experiment used mRNA of pFluc purified by oligo dT column and pFluc-rRanp mRNA. Microfluidic chip preparation was performed with LNP. The prepared mRNA-nLNP was stored in 4°C refrigerator and -20°C refrigerator, respectively. After 0W, 1W, 2W, 4W, 8W, one of each was taken from 4°C and -20°C, respectively, diluted with 400 uL 1XPBS (1:2 dilution, mRNA concentration 0.1667 ug / uL) for particle size determination and mRNA encapsulation rate determination. At the same time, mouse intramuscular administration of tracer protein expression experiment was conducted, and IVIS live imaging detection was performed at 0.25, 1, 2 days after administration.

[0118] The results show that after 4 weeks of storage in a 4°C refrigerator, the total amount of tracer protein expression of pFluc-LNP decreases significantly, while the gene expression level of pFluc-rRanp-LNP does not change significantly, Figure 13 After two weeks of storage in a -20°C refrigerator, the gene expression levels of pFluc-LNP and pFluc-rRanp-LNP change significantly. rRanp can promote the stability of mRNA-LNP. After 4 weeks of storage in a -20°C refrigerator, the protein expression level delivered by pFluc-LNP decreases significantly, while the protein expression level of pFluc-rRanp-LNP does not decrease significantly.

[0119] Conclusion: The rRanp gene fragment significantly prolongs the storage time of mRNA-rR3GE-LNP preparation under the conditions of 4°C or -20°C, and prolongs the storage time by at least one time.

[0120] The present application is a synthetic recombinant gene fragment, rR3GE, which encodes a recombinant polypeptide with nuclease III activity. When co-transfected with a pharmacodynamic mRNA, it can inhibit the innate immune response of host cells and mRNA degradation, thereby improving the expression efficiency and expression time of pharmacodynamic proteins. rR3GE can be integrated into mRNA without changing the physicochemical properties of mRNA, so it is suitable for all delivery methods and administration methods for delivering mRNA, without changing the biological function and pharmacodynamics of mRNA.

[0121] The present application has been illustrated by various specific embodiments. However, those skilled in the art can understand that the present application is not limited to the specific embodiments, and each technical feature mentioned in the specification can be combined with each other without departing from the spirit and scope of the present application. Such modifications and variations are within the scope of the present application.

Claims

1. Use of a recombinant gene for the preparation of a medicament for increasing the level of protein expression and the length of expression of co-transfected mRNAs, characterized in that, The amino acid sequence of the polypeptide encoded by the recombinant gene is shown in SEQ ID NO. 1, SEQ ID NO. 3-6, SEQ ID NO.

8.

2. A method for increasing the expression level of an exogenous target RNA in an organism using a recombinant gene, characterized in that: The amino acid sequence of the polypeptide encoded by the recombinant gene is shown in SEQ ID NO. 1, SEQ ID NO. 3-8. The method is to recombine the recombinant gene and the target protein gene into the same mRNA, and to generate independent rR3GE polypeptide and target protein in cells simultaneously through self-cleavage polypeptide or IRES, and the method is not for therapeutic purposes.

3. The method of claim 2, wherein: The nucleotide sequence of the recombinant gene is added to the RNA expression vector for eukaryotic expression, and protein expression is carried out in the form of independent transfection or co-transfection, so as to improve the expression level and expression time of the co-transfection target gene in the body and cells.

4. The method of claim 2, wherein: The introduction of the recombinant gene is combined with nucleotide modification.

5. An mRNA preparation made from a recombinant gene encoding the polypeptide sequence of claim 1, characterized by: The preparation is an injection, and the preparation is to recombine the recombinant gene and the target protein gene into the same mRNA, and to generate independent recombinant gene polypeptide and target protein in cells simultaneously through self-cleavage polypeptide or IRES.

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