MRNA (messenger ribonucleic acid) medicine for treating propionemia and application thereof

Abstract

The invention relates to an mRNA (messenger ribonucleic acid) medicine for treating propionemia and application of the mRNA medicine. According to the invention, an optimal candidate mRNA molecule-PUMCH-MR-01 is selected by carrying out mRNA sequence optimization design on a propionyl-coenzyme A carboxylase alpha or beta (PCCA or PCCB) subunit amino acid sequence and carrying out in-vitro expression screening verification. Then, the protein expression level of PUMCH-MR-01 is evaluated in a wild type mouse body, and pharmacodynamic verification is carried out on cynomolgus monkeys. The result shows that the PUMCH-MR-01 can efficiently express the PCC enzyme protein, shows a certain drug effect trend in non-human primates, and can be used for effectively treating propionemia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an mRNA drug for treating propionic acidemia and a preparation method thereof, as well as an application of the mRNA drug in treating propionic acidemia. Background Art

[0002] Rare diseases generally refer to rare diseases and rare diseases. Compared with common diseases and frequently occurring diseases, they are a general term for a large class of different diseases scattered in various disease systems. The "Research Report on the Definition of Rare Diseases in China 2021" defines rare diseases as "diseases with a neonatal incidence rate of less than 1 / 10,000, a prevalence rate of less than 1 / 10,000, and a number of patients less than 140,000; diseases that meet any one of these conditions are rare diseases." At present, there are 207 rare diseases in my country that have been included in the rare disease catalog jointly issued by the National Health Commission and other five ministries. Studies have shown that more than 80% of rare diseases are caused by genetic factors, and 50% occur at birth or in childhood. Less than 5% of rare diseases worldwide have drugs that can be treated. The lack of drugs is a major problem facing patients with rare diseases around the world. Even if there are drugs to treat them, patients often face high treatment costs, and many people give up treatment because they cannot afford the expenses.

[0003] Propionic acidemia (PA), also known as propionyl-CoA carboxylase deficiency, is an autosomal recessive organic acidemia. PA is caused by defects in the PCCA or PCCB genes encoding the mitochondrial multimeric enzyme propionyl-CoA carboxylase (PCC). PCC deficiency can lead to abnormal conversion of propionyl-CoA to methylmalonyl-CoA in the body, abnormal accumulation of propionic acid and its related metabolites, leading to organic acidemia and a series of biochemical abnormalities, symptoms of damage to the nervous system and other organs. The overall prevalence of PA among different ethnic groups abroad is 1 / 100,000 to 100 / 100,000, and in my country it is 0.6 / 100,000 to 0.7 / 100,000.

[0004] For the treatment of propionic acidemia, in the acute phase, acidosis, hypoglycemia and electrolyte imbalance are corrected mainly by rehydration, natural protein intake is restricted and sufficient calories are supplied. At the same time, intravenous drip of L-carnitine is required to promote organic acid excretion, neomycin or metronidazole is used to inhibit intestinal bacteria to reduce propionic acid production, and carbamylglutamate can reduce blood ammonia and improve metabolic stability during the acute decompensation period. Long-term treatment is mainly to limit natural protein diet to ensure sufficient protein and energy intake. It is recommended to feed with special formula nutritional powder to reduce propionic acid production, but because it contains essential amino acids, it cannot be used as the only protein source. A small amount of natural protein is also required. Clinically, the ratio of special milk powder to natural protein is adjusted from 1:3 according to blood amino acid levels and metabolite concentrations, and hunger is avoided to inhibit tissue catabolism. A few patients who are still in poor condition after good dietary control may consider liver transplantation.

[0005] mRNA therapy technology uses a specific delivery system to deliver specific fragments of mRNA into human cells, and then uses the body's own cells to translate mRNA into protein. In addition to infectious diseases, mRNA vaccine technology has also shown good prospects in cancer immunotherapy, gene editing and protein replacement therapy. Protein replacement therapy mainly restores the function of a certain protein that is missing or mutated in the body through the body's encoding of normal target proteins. Compared with recombinant proteases, mRNA drugs have relatively low immunogenicity. mRNA can be delivered through lipid nanoparticles to treat metabolic diseases caused by protein deficiency. mRNA is only expressed in the cytoplasm, and while effectively expressing the target protein, it does not cause the risk of gene mutation caused by inserting DNA, and is safer.

[0006] Moderna's team used mRNA technology to develop an intravenous drug, mRNA-3927, to treat propionic acidemia. mRNA-3927 uses lipid nanoparticles to deliver two mRNAs encoding PCCA and PCCB into liver cells, which are translated and assembled into active propionyl-CoA carboxylase in the cytoplasm, thereby restoring the liver's propionic acid metabolism pathway to normal. Summary of the invention

[0007] 1. Technical issues to be resolved

[0008] There are no reports on propionic acidemia mRNA in my country. The present invention is based on the mRNA nucleic acid drug conversion application platform for rare diseases, constructs the mRNA fragment of propionyl-CoA carboxylase, uses a lipid nanoparticle (LNP) delivery system for intravenous injection, and utilizes the body's own cells to translate mRNA into propionyl-CoA carboxylase, thereby alleviating the patient's metabolic abnormalities and can be used to effectively treat propionic acidemia.

[0009] (II) Technical solution

[0010] In order to achieve the above-mentioned object, as one aspect of the present invention, a propionyl-CoA carboxylase alpha subunit (PCCA) mRNA molecule is provided, wherein the mRNA comprises a CDS coding region encoding PCCA, and the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 1-3 or a nucleotide sequence having codon degeneracy and at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO: 1-3.

[0011] As another aspect of the present invention, a propionyl-CoA carboxylase β subunit (PCCB) mRNA molecule is provided, wherein the mRNA comprises a CDS coding region encoding PCCB, and the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO:4-6 or a nucleotide sequence having codon degeneracy and at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO:4-6.

[0012] As another aspect of the present invention, a plasmid encoding the above-mentioned PCCA mRNA molecule or PCCB mRNA molecule is provided.

[0013] As another aspect of the present invention, a host cell is provided, which comprises the above-mentioned mRNA molecule or plasmid.

[0014] As another aspect of the present invention, an mRNA pharmaceutical composition is provided, wherein the pharmaceutical composition comprises the above-mentioned PCCA mRNA molecule and / or PCCB mRNA molecule.

[0015] As another aspect of the present invention, an mRNA drug for treating propionic acidemia is provided, wherein the drug comprises a pharmaceutical composition and a drug delivery carrier.

[0016] As another aspect of the present invention, a method for preparing the above-mentioned mRNA drug for treating propionic acidemia is provided, comprising the following steps:

[0017] (1) Dissolving a cationic lipid, an auxiliary phospholipid, cholesterol and a pegylated phospholipid in an organic solvent, ethanol, to obtain a mixture A of lipid nanoparticles; wherein the cationic lipid is DLin-MC3-DMA, the auxiliary phospholipid is DSPC, and the pegylated phospholipid is DMG-PEG2000.

[0018] (2) diluting the mRNA with a sodium acetate solution to obtain a mixture B;

[0019] (3) Mixture A and mixture B are mixed by a microfluidic device to prepare a lipid nanoparticle composition, and dialyzed in PBS.

[0020] As another aspect of the present invention, provided is the use of the above-mentioned pharmaceutical composition, mRNA drug or the mRNA drug prepared according to the above-mentioned preparation method in the preparation of a drug for preventing, treating or improving propionic acidemia and / or alleviating metabolic disorders or metabolic disorder symptoms caused by PCC deficiency, wherein the use comprises administering an effective amount of the drug to a subject.

[0021] In one embodiment, the mRNA molecule further comprises a 5′UTR and a 3′UTR, wherein the 5′UTR preferably comprises a nucleotide sequence as shown in SEQ ID NO: 7, and the 3′UTR preferably comprises a nucleotide sequence as shown in SEQ ID NO: 8. The mRNA molecule further comprises a flagtag and Poly(A), wherein the flag tag is preferably Flag tag x2, and the Poly(A) is preferably Poly(A100).

[0022] In one embodiment, the above mRNA may be modified to improve the stability and translation efficiency of the mRNA. The modification of mRNA includes: N6-methyladenosine (m6A), 5-methylcytosine (m5C), 1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), 4-acetylcytosine (ac4C), etc. Preferably, the modification of mRNA is to replace all uridine (U) in the CDS sequence with N1-methyl-pseudouridine (N1-Methyl-Pseudouridine, m1Ψ) bases.

[0023] In one embodiment, the drug delivery carrier is selected from liposome nanoparticles (LNP), cationic lipid complexes, polymer nanoparticles, protamine nanoparticles, polypeptide nanoparticles, polypeptide nanoparticles, viral vectors, inorganic nanoparticles, cell penetrating peptides or self-assembling RNA-lipid complexes. The LNP comprises cationic lipids, auxiliary phospholipids, cholesterol and PEGylated phospholipids.

[0024] (III) Beneficial effects

[0025] The present invention provides an mRNA drug for treating propionic acidemia and its application. Compared with the prior art, it has the following beneficial effects:

[0026] The present invention optimizes the mRNA sequence design of the amino acid sequence of the propionyl-CoA carboxylase α or β (PCCA or PCCB) subunit, and selects the best candidate mRNA molecule, PUMCH-MR-01, by in vitro expression screening and verification. Then, the protein expression level of PUMCH-MR-01 was evaluated in wild-type mice, and pharmacodynamics was verified in crab-eating monkeys. The results showed that PUMCH-MR-01 can efficiently express PCC enzyme protein, and show a certain pharmacodynamic trend in non-human primates, and can be used to effectively treat propionic acidemia.

[0027] (IV) Terms and Definitions

[0028] In this application, including the appended claims, the singular forms "a," "an," and "the" are often used for convenience. However, it should be understood that these singular forms include plural referents unless otherwise stated.

[0029] As used herein, "nucleotide" refers to a nucleoside containing a phosphate group. Nucleotides are generally molecules composed of three parts: a five-carbon sugar (ribose or deoxyribose), a phosphate group, and a nitrogenous base (purine or pyrimidine).

[0030] "mRNA (messenger RNA)" as described herein refers to a single-stranded nucleic acid molecule that is transcribed from a DNA template, carries genetic information, and directs protein synthesis on ribosomes in the cytoplasm. The structure of mRNA generally includes a 5' cap structure (5' cap), a 5' untranslated region (5'-UTR), a coding region (open reading frame, ORF), a 3' untranslated region (3'-UTR) and / or a polyadenylic acid tail (Poly-A tail).

[0031] The "CDS coding region" described herein refers to the portion of the mRNA molecule that is actually translated into protein. The CDS starts with the start codon (usually AUG) and ends with the stop codon (such as UAA, UAG or UGA). It is located between the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR). The CDS consists of exons and contains all the nucleotide sequences that encode proteins.

[0032] The "codon degeneracy" described herein refers to the phenomenon that multiple different codons in the genetic code can encode the same amino acid.

[0033] "Identity" as used herein refers to the degree of similarity between two or more biomacromolecule sequences (such as DNA, RNA or protein sequences). When comparing two sequences, sequence identity is quantified by determining the ratio of sequences with the same components at the same position.

[0034] "Modification" as used herein refers to a change in the state or structure of a molecule. Modification can be performed in a variety of ways, including chemical, structural and functional modifications. For example, an mRNA molecule can be modified by introducing non-natural nucleosides and / or nucleotides, such as modifications of natural ribonucleotides A, U, G and C.

[0035] "Drug delivery carrier" as described herein refers to a system for safely and effectively delivering mRNA drugs to target cells or tissues. The types of drug delivery carriers generally include liposome nanoparticles (LNP), cationic lipid complexes, polymer nanoparticles, protamine nanoparticles, polypeptide nanoparticles, polypeptide nanoparticles, viral vectors, inorganic nanoparticles, cell penetrating peptides or self-assembling RNA-lipid complexes.

[0036] The "liposome nanoparticle (LNP)" described in this article is a nanoscale delivery system composed of lipid molecules and is widely used in drug and gene delivery. LNPs are composed of multiple lipid bilayers that can encapsulate and protect drug molecules such as mRNA, siRNA, DNA, etc. Lipid nanoparticles (LNPs) are generally composed of four main types of lipids: ionizable cationic lipids, phospholipids, cholesterol or its derivatives, and polyethylene glycol (PEG)-lipids. The size of liposome nanoparticles (LNPs) is generally between 50 and 200 nanometers. The size of LNPs can be controlled by adjusting its composition and preparation method to optimize its delivery efficiency and biodistribution. The U.S. Food and Drug Administration (FDA) has approved it for liver siRNA delivery and the delivery of antigen mRNA for the new crown vaccine mRNA-1273 and BNT162b21mRNA. FDA-approved LNPs contain four basic components: cationic / ionizable lipids, cholesterol, auxiliary lipids, and polyethylene glycol (PEG) lipids. The LNPs of international pharmaceutical companies Alnylam, Moderna and Pfizer / BioNTech / Acuitas are composed of four parts: 1) cationic / ionizable lipids DLin-MC3-DMA (Alnylam), SM-102 (Moderna) or ALC-0315 (Pfizer / BioNTech / Acuitas), 2) cholesterol, 3) polyethylene glycol (PEG) lipids PEG-2000-C-DMG (Alnylam), PEG-2000-DMG (Moderna) or ALC-0159 (Pfizer / BioNTech / Acuitas), and 4) 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC).

[0037] The "mRNA drugs" described in this article refer to drugs developed based on messenger RNA (mRNA) technology. Its working principle is to introduce mRNA encoding a specific protein into human cells, so that the cells themselves produce the functional protein, thereby exerting their effects. The structure of mRNA drugs mainly includes the following key parts: mRNA sequence, delivery vector and excipients such as stabilizers and buffers.

[0038] As used herein, an "effective amount" refers to a dose of a drug that produces a desired or beneficial effect, such as the results achieved in clinical treatment. Therefore, the "effective amount" may vary depending on the context of the specific application. For example, in the treatment of a disease, an effective amount of a drug refers to a dose that achieves the desired therapeutic effect compared to not using the drug.

[0039] "Administering" as described herein includes any route of administration, e.g., oral, parenteral, intramuscular, transdermal, intravenous, interarterial, nasal, vaginal, sublingual, subungual, etc. Administering can also include prescribing a drug to be delivered to a subject, e.g., according to a specific dosing regimen, or filling a prescription for a drug to be delivered to a subject, e.g., according to a specific dosing regimen.

[0040] As used herein, "treating" includes the following actions: (i) preventing a particular disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition; (ii) curing, treating or inhibiting the disease, i.e., arresting its development; or (iii) ameliorating the disease by reducing or eliminating symptoms, ailments and / or by causing regression of the disease.

[0041] The "host cell" described herein refers to a cell that can accept exogenous genes (such as recombinant DNA molecules) and provide them with the conditions required for a series of physiological processes such as replication, transcription, and translation, so that the exogenous genes can be expressed therein and produce corresponding products (such as proteins). Host cells include prokaryotic cells or eukaryotic cells. Examples of suitable host cells in the present invention include, but are not limited to, bacteria, yeast cells, insect cells, animal cells, and mammalian cells.

[0042] The "plasmid" mentioned in this article refers to a DNA fragment extracted from a virus, plasmid or higher biological cell, which can be inserted into foreign DNA for cloning and / or expression, can be stably maintained in the organism, contains a replication origin, a selective marker or reporter gene and a multiple cloning site, etc., and covers a variety of forms such as linear DNA fragments, plasmid vectors, and viral vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 The technical roadmap for PUMCH-MR-01, the mRNA drug for propionic acidemia.

[0045] Figure 2 This is a schematic diagram of the structure of the PCC protein mRNA in Example 1.

[0046] Figure 3 Prepare the DNA electrophoresis quality control profile for PCR in Example 2.

[0047] Figure 4 Prepare the mRNA electrophoresis quality control profile for the IVT in Example 2.

[0048] Figure 5 This is the Western blot expression spectrum of the PCCA protein in Example 3.

[0049] Figure 6 This is the Western blot expression spectrum of the PCCB protein in Example 3.

[0050] Figure 7 is the expression level of PCCA protein in the cell and supernatant in Example 3.

[0051] Figure 8 is the expression level of PCCB protein in the cell and supernatant in Example 3.

[0052] Fig. 9 The liver protein expression of PUMCH-MR-01 in Example 5 in Balb / C mice after a single dose of 0.1 mg / kg.

[0053] Fig.10 The pharmacodynamic effects of repeated administration of different doses of PUMCH-MR-01 in cynomolgus monkeys in Example 6 are shown.

[0054] Fig.11 This is the toxicity reaction of repeated administration of different doses of PUMCH-MR-01 in cynomolgus monkeys in Example 8. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] In order to better understand the technical solution, the technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0057] Example 1: Sequence optimization design of mRNA

[0058] The sequence characteristics of mRNA have an important impact on its stability, expressibility, and even the preparation process. At present, the research on mRNA sequence design is mainly carried out from multiple dimensions such as codon frequency, degeneracy and rarity, and sequence free energy. The advantages of mRNA sequence characteristics will be reflected in the expression of protein. Therefore, the above-mentioned important optimization parameters are combined to screen candidate sequences by protein expression differences.

[0059] The present invention adopts a self-developed deep learning-based mRNA sequence design method to comprehensively evaluate parameters such as codon frequency, degeneracy and rarity; GC content and Gibbs free energy of the sequence; and secondary structure of the sequence, and designs corresponding mRNA sequences for PCC protein sequences. The protein is expressed by in vitro transfection of HEK293T cell lines, and the expression level and duration of the protein are compared to evaluate and select the sequences.

[0060] The schematic diagram of the designed PCC protein mRNA structure is shown in Figure 2 The mRNA sequences of PCCA and PCCB are shown in Table 1. Among them, the UTR uses a combination of 5'UTR (5'-CBT3) and 3'UTR (3'-CBT4). The selected Tag tag is located at the C-terminus, and Flagtag x2 is selected. PolyA (A100) and m1ψ (N1-Methylpseudouridine) base modifications are selected.

[0061] Based on the target amino acid sequence (PCCA), three PCCA mRNA CDS coding region sequences (SEQ ID NO: 1-3) were designed. The full-length mRNA structure includes: 5'UTR (SEQ ID NO: 7), CDS, flag tag (SEQ ID NO: 11), 3'UTR (SEQ ID NO: 8) and Poly (A100) (SEQ ID NO: 12).

[0062] Based on the target amino acid sequence (PCCB), the CDS coding regions of three PCCB mRNA sequences (SEQ ID NO: 4-6) were designed. The full-length mRNA structure includes: 5'UTR (SEQ ID NO: 7), CDS, flag tag (SEQ ID NO: 11), 3'UTR (SEQ ID NO: 8) and Poly (A100) (SEQ ID NO: 12).

[0063] CYN230052-A2187, CYN230052-A2512, and CYN230052-A2753 sequences are optimized PCCA sequences, and CYN230052-B2261, CYN230052-B2390, and CYN230052-B2745 sequences are optimized PCCB sequences. The CDS of CYN230052-AC sequence is replaced by the Moderna PCCA sequence, and the CDS of CYN230052-BC sequence is replaced by the Moderna PCCB sequence, while the 5′UTR, 3′UTR, flag tag, and Poly(A100) sequences remain unchanged.

[0064] Table 1 Designed mRNA and corresponding sequences

[0065]

[0066]

[0067] Example 2: Preparation of mRNA

[0068] 1. PCR preparation of DNA

[0069] PCR preparation of DNA mainly includes two main processes: PCR linearization of plasmid DNA template and subsequent purification of linear DNA template.

[0070] In the stage of linearizing the plasmid DNA template by PCR, the reaction system must be configured first, including 25 μL of 2x Reaction Buffer, 1 μL of dNTP Mix, 1 μL of DNA Polymerase, 3 μL of plasmid DNA, 2 μL each of CBT3-F and CBT4-R, and 16 μL of RNase-free Water, with a total volume of 50 μL. Then set the PCR program, pre-denaturation at 95°C for 3 minutes, only 1 cycle; denaturation temperature at 95°C for 15 seconds, a total of 35 cycles; annealing at 60°C for 15 seconds, also for 35 cycles; extension stage at 72°C per kb per minute according to the size of the plasmid DNA, also for 35 cycles; finally, thorough extension at 72°C for 5 minutes, only 1 cycle, and then kept at 4°C until the next step.

[0071] After completing the PCR reaction, proceed to the step of purifying the linear DNA template. First, add an appropriate amount of DNA binding buffer to the PCR reaction solution and mix thoroughly. Then transfer the mixed sample to the purification centrifuge column, place it in a centrifuge and centrifuge it at 10,000g for 30 seconds, and then discard the waste liquid. After that, wash it twice with washing buffer, transfer the purification centrifuge column to a new centrifuge tube, add nuclease-free water and let it stand at room temperature for 3 minutes. Then put the sample column into the centrifuge again and centrifuge it at 10,000g for 30 seconds. The resulting solution is the linear DNA product. Finally, it is necessary to test the concentration and purity of the DNA, and perform quality control on the DNA template by agarose gel electrophoresis to ensure that the prepared linear DNA meets the requirements of subsequent experiments.

[0072] The experimental results showed that the length of the PCCA sequence was 2496 bp, and the length of the PCCB sequence was 1929 bp. The DNA bands in each lane were clear, without any mixed bands, with normal concentration and qualified purity, indicating that the prepared DNA met the requirements of subsequent experiments (see Figure 3 ).

[0073] 2. Preparation of mRNA by in vitro transcription (IVT)

[0074] Using linearized DNA as a template, in vitro transcription (IVT) was performed with the help of T7 promoter to prepare mRNA, which was then purified and quality controlled through a series of operations.

[0075] First, an IVT reaction was performed using the NEB HiScribe T7 in vitro transcription kit. Preparation included thawing and mixing 100 mM NTPs (ATP, GTP, CTP, and UTP nucleotides or their chemically modified counterparts) to make a 2.5X NTP mix. A 50 μL IVT reaction system contained approximately 2.5 μL DNA template, 10X IVT reaction buffer, NTP mix, and 5 μL T7 RNA polymerase mix. The reaction mixture was incubated at 37°C for 2 hours, then diluted with 700 μL nuclease-free water, and then 10X DNase I buffer and 20 μL RNase-free DNase I were added, and incubated at 37°C for 15 minutes to degrade the DNA template.

[0076] Then, mRNA purification was performed. 2 volumes of binding buffer and 3 volumes of anhydrous ethanol were added to the solution after the IVT reaction and mixed thoroughly; the sample was transferred to the purification column, centrifuged at 13000g for 1 min and the waste liquid was discarded; 500 μL of washing buffer was added, centrifuged at 13000g for 1 min and the waste liquid was discarded, and this step was repeated twice; the purification column was transferred to a new centrifuge tube, the product was eluted with nuclease-free water, and after standing at room temperature for 5 min, it was centrifuged at 13000g for 1 min to obtain the mRNA product. The mRNA concentration was then detected, and the mRNA was quality controlled by agarose gel electrophoresis.

[0077] In addition, the NEB enzymatic method was used for Cap 1GAG capping. The mRNA was denatured at 65°C for 5 minutes and then rapidly cooled to release the secondary conformation. 100 μg of denatured mRNA, 10 μL of 1X capping buffer, 5 μL of 10 mM GTP, 5 μL of 4 mMSAM, 5 μL of 10 U / mL vaccinia virus capping enzyme, and 5 μL of mRNA cap 2'-O-methyltransferase (50 U / mL) were mixed, and the final volume was adjusted to 100 μL with nuclease-free water, and incubated at 37°C for 1 hour. The capped mRNA after the reaction was purified using an RNeasy column, eluted with nuclease-free water, and quantified using a NanoDrop spectrophotometer. Finally, the purity of the mRNA was confirmed by denaturing urea polyacrylamide gel electrophoresis.

[0078] The experimental results showed that the length of the PCCA sequence was 2496 bp and the length of the PCCB sequence was 1929 bp. The RNA bands in each lane were clear, without any mixed bands, with normal concentration and qualified purity, indicating that the prepared mRNA met the requirements of subsequent experiments (see Figure 4 ).

[0079] Example 3: Screening of mRNA expression

[0080] First, prepare HEK293T cells, plate 7*10^4 cells per well in a 24-well plate, add 500μL culture medium, and culture for 24 hours before transfection. During the transfection process, use RNAiMAX transfection reagent to encapsulate mRNA, transfect 1μg mRNA per well, and set up 3 replicate wells / sample.

[0081] After transfection, the cells were cultured for 48 hours, and then 500 μL of culture supernatant and 100 μL of cell lysate samples (treated with 100 μL of lysis buffer) were collected. The cell body concentration was then determined using the BCA method.

[0082] Then, a Western blot experiment was performed, with 2 μg of cell body sample and 10 μL of supernatant sample. Then, antibody incubation was performed, and after the incubation was completed, the protein was developed with ECL colorimetric solution and exposed to image on a developer.

[0083] Finally, by analyzing the imaging results, the protein expression level of the screened mRNA after transfection in HEK293T was evaluated according to the Western blot results, so as to obtain the influence of the CDS design sequence on protein expression and then determine the final sequence.

[0084] Western blot results showed that the size of PCCA protein was about 78.98KDa, and the size of PCCB protein was about 58.22KDa. The target bands were detected in the cell body and supernatant, and the internal reference was detected normally (see Figure 5 , 6 ). PCCA and PCCB were detected in the cell and supernatant. All optimized sequences were able to express PCCA and PCCB proteins normally. The expression level of PCCA in both the supernatant and the cell body was the highest in the CYN230052-A2187 sequence (see Figure 7 ); PCCB had the highest expression level of CYN230052-B2745 sequence in both the supernatant and the cell body (see Figure 8 ). The sequences with the highest expression levels were selected respectively, and CYN230052-A2187 and CYN230052-B2745 were combined into candidate molecules for preparing lipid nanoparticles.

[0085] Example 4: Preparation of lipid nanoparticles (LNP)

[0086] PUMCH-MR-01 is an mRNA lipid nanoparticle (mRNA-LNP) product with lipid nanoparticles (LNP) as drug delivery carriers, in which the LNP preparation contains four main ingredients, namely DLin-MC3-DMA, cholesterol, distearoylphosphatidylcholine (DSPC) and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000).

[0087] The mRNA stock solution (inorganic phase) and lipid excipients (organic phase) of CYN230052-A2187 and CYN230052-B2745 are mixed with microfluidic dynamic precision technology to quickly prepare nanolipid particles encapsulating mRNA under precise control of the flow rate. After solution dilution, ultrafiltration concentration, solvent replacement, sterilization filtration and filling processes, the finished drug that meets the quality standards can be obtained.

[0088] LNP-mRNA preparation and detection process:

[0089] First, lipid mixture A was prepared, and DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. Then, mRNA was processed to prepare mixture B, and 200 μg of mRNA stock solution of CYN230052-A2187 and CYN230052-B2745 was diluted with sodium acetate solution. Then A and B were mixed by microfluidic equipment, with parameters of flow rate 12 ml / min, water phase / ethanol phase 3 / 1, room temperature, and PBS dialyzed after preparation. After detection, the size and zeta potential of LNP were measured by nanoparticle size analyzer, and the mRNA encapsulation rate was measured by RiboGreen experiment. The free RNA was measured first, and then the structure was broken to measure the total amount and calculate the difference. This process provides a method and data basis for related research.

[0090] The quality standards of the drug mainly include the appearance of a slightly milky liquid with no visible particles, sequence identification consistent with the theoretical sequence, RNA concentration between 1.80 mg / mL and 2.20 mg / mL, RNA integrity greater than 70%, LNP encapsulation efficiency greater than 70%, LNP particle size in the range of 50 nm to 180 nm and PDI less than 0.3, preparation pH in the range of 7.0 to 8.0, and endotoxin less than 20.0 EU / mL.

[0091] The experimental results show that the prepared PUMCH-MR-01 has uniform and stable morphology and is not easy to aggregate, and the drug encapsulation efficiency meets the requirements.

[0092] Example 5: Protein expression level of PUMCH-MR-01 in mice after single dose administration

[0093] Wild-type Balb / C mice were given a single dose of 0.1 mg / kg PUMCH-MR-01 by tail vein injection. The mice were euthanized on days 1, 2, 4, 7, 10, and 14 after administration, and liver tissues were collected by dissection. The liver tissues were homogenized and the expression of PCCA and PCCB proteins was detected.

[0094] The experimental results showed that PCCA protein began to express on the first day after administration and reached its peak expression level on the second day after administration, then gradually decayed until it basically stopped expressing on the 14th day; PCCB protein showed almost the same expression level and expression characteristics as PCCA protein, also reaching its peak expression level on the second day, which was more than 3 times that of the first day, and basically stopped expressing on the 14th day (see Fig. 9 ). In summary, PUMCH-MR-01 can efficiently express the target protein in mice and can sustain the expression for 10 days.

[0095] Example 6: Pharmacodynamics of PUMCH-MR-01 in cynomolgus monkeys at different doses and multiple administrations

[0096] Low and high doses (1.0, 3.0 mg / kg) or a vehicle control group were given to cynomolgus monkeys by intravenous infusion, and the administration was repeated once a week for 4 weeks, with 1 male and 1 female in each group, aged 1-1.5 years. Serum samples were collected at the preset sampling points, and then the concentration of biomarkers of propionic acidemia was quantified by mass spectrometry to analyze the concentration changes of the markers.

[0097] The experimental results showed that compared with the solvent control group, 3-hydroxypropionate (3HP) in the test group showed a downward trend, indicating that PUMCH-MR-01 produced a certain drug effect. However, there were large differences between individual animals, and an increase was even observed in the low-dose group (see Fig.10 ).

[0098] This study was conducted in healthy young monkeys, and no consistency in biomarker changes was observed. The above research results are consistent with the results reported in the Phase 1 / 2 clinical trial of mRNA-3927, which is currently being used to treat propionic acidemia. In this clinical trial, different doses of mRNA-3927 were used to treat patients with propionic acidemia, and there was no consistency in the concentration changes of biomarkers such as 3HP, 2MC (2-methylcitrate), and C3 (propionylcarnitine) after treatment.

[0099] Example 7: Pharmacokinetics of PUMCH-MR-01

[0100] After PUMCH-MR-01 is injected into the body, it will be quickly cleared from the blood within 24 hours. Consistent with existing literature reports, the liver is the main target organ of mRNA-LNP. In addition, PUMCH-MR-01 is also distributed throughout the body, and low concentrations of mRNA can be detected in organs other than the kidneys, including the brain, heart, lungs, eyes, and intestines. After blood clearance, proximal lymph nodes, distal lymph nodes, and spleen are the main distribution organs of mRNA, and the highest exposure is reached within 2-24 hours after administration. Lipids are mainly effectively and rapidly metabolized and cleared through bile and kidney pathways. The accumulation of lipids in tissues does not exceed 168 hours.

[0101] Example 8: Toxicology Experiment of PUMCH-MR-01

[0102] A 4-week toxicology study of repeated doses via intravenous infusion in cynomolgus monkeys.

[0103] In the PUMCH-MR-01 intravenous infusion repeated dose toxicity test, the low-dose group was 1.0 mg / kg and the high-dose group was 3.0 mg / kg. The drug was administered once a week and repeated for 4 weeks, for a total of 4 times. A solvent control group was also set up, and the experimental treatment method was the same as that of the experimental group. During the experiment, clinical observations and records were made on the crab-eating macaques, including body weight, body temperature, diet, and local reactions; blood samples were collected for routine blood tests, blood biochemical liver function tests, and cytokine tests; at the end of the experiment, the crab-eating macaques were dissected to observe the pathological conditions of the main organs, and pathological examinations were performed on the main tissues.

[0104] The experimental results showed that no animal deaths occurred during the repeated administration test. In clinical observation, no obvious abnormalities were found in the animals' food intake, body weight, body temperature and infusion site. No obvious abnormalities were found in the blood routine examination indicators (such as: RBC, HGB, RET, WBC, NEUT, LYMP, MONO, PLT, etc.) and blood biochemical indicators (such as: GGT, TP, ALP, GLOB, etc.) (see Fig.11 ). However, in the high-dose group, ALT and AST showed transient reactions after the fourth dose. There were no obvious abnormalities in coagulation indicators (PT, FIB, APTT), urine test indicators (UBIL, UGL, UPRO, USG, UPH, etc.), immunological indicators (C3, C4) and cytokine indicators (IL-6, TNFα). Histopathological examinations of the heart, liver, brain, kidneys, spleen, eyeballs, lymph nodes and sexual organs showed no toxic and adverse reactions related to the test article. In summary, in this repeated dosing study, crab-eating monkeys tolerated PUMCH-MR-01 well, and no adverse reactions related to the test article were observed.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A propionyl-CoA carboxylase alpha subunit (PCCA) mRNA molecule, characterized in that: The mRNA comprises a CDS coding region encoding PCCA, wherein the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 1-3 or a nucleotide sequence having codon degeneracy and at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1-3.

2. A propionyl-CoA carboxylase β subunit (PCCB) mRNA molecule, characterized in that: The mRNA comprises a CDS coding region encoding PCCB, wherein the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 4-6 or a nucleotide sequence having codon degeneracy and at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 4-6.

3. The PCCA mRNA molecule according to claim 1 or the PCCB mRNA molecule according to claim 2, characterized in that: The mRNA molecule further includes a 5′UTR and a 3′UTR, wherein the 5′UTR is a nucleotide sequence shown in SEQ ID NO:7, and the 3′UTR is a nucleotide sequence shown in SEQ ID NO:

8.

4. The PCCA mRNA molecule according to claim 1 or the PCCB mRNA molecule according to claim 2, characterized in that: The mRNA is modified by one or more of N6-methyladenosine (m6A), 5-methylcytosine (m5C), 1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ) or 4-acetylcytosine (ac4C).

5. A plasmid encoding the PCCA mRNA molecule of claim 1 or the PCCB mRNA molecule of claim 2.

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

7. An mRNA pharmaceutical composition, characterized in that: The mRNA pharmaceutical composition comprises the PCCA mRNA molecule of claim 1 and / or the PCCB mRNA molecule of claim 2.

8. An mRNA drug for treating propionic acidemia, characterized in that: The drug comprises the pharmaceutical composition of claim 7 and a drug delivery carrier, wherein the drug delivery carrier is selected from liposome nanoparticles (LNP), cationic lipid complexes, polymer nanoparticles, protamine nanoparticles, polypeptide nanoparticles, polypeptide nanoparticles, viral vectors, inorganic nanoparticles, cell penetrating peptides or self-assembled RNA-lipid complexes.

9. The method for preparing the mRNA drug for treating propionic acidemia according to claim 8, characterized in that: The preparation method comprises the following steps: (1) dissolving a cationic lipid, an auxiliary phospholipid, cholesterol and a pegylated phospholipid in an organic solvent ethanol at a certain molar ratio to obtain a mixture A of lipid nanoparticles; wherein the cationic lipid is DLin-MC3-DMA, the auxiliary phospholipid is DSPC, and the pegylated phospholipid is DMG-PEG2000; (2) diluting the mRNA with a sodium acetate solution to obtain a mixture B; (3) Mixture A and mixture B are mixed by a microfluidic device to prepare a lipid nanoparticle composition, and dialyzed in PBS.

10. Use of the pharmaceutical composition according to claim 7, the mRNA drug according to claim 8 or the mRNA drug prepared by the preparation method according to claim 9 in the preparation of a drug for preventing, treating or improving propionic acidemia and / or alleviating the symptoms of metabolic disorders or metabolic disorders caused by PCC deficiency, characterized in that: The use comprises administering an effective amount of the drug to a subject.

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