CN118166004B - MRNA for encoding human PCCA or PCCB protein and use thereof - Google Patents
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Abstract
本公开涉及生物技术领域，具体涉及用于编码人PCCA或PCCB蛋白的mRNA及其用途，该mRNA分子包含编码人PCCA或PCCB蛋白的CDS，编码人PCCA蛋白的CDS包含选自SEQ ID NO:2‑6之一的核苷酸序列或与其具有至少80%同一性的核苷酸序列，编码人PCCB蛋白的CDS包含选自SEQ ID NO:8‑14之一的核苷酸序列或与其具有至少80%同一性的核苷酸序列。本公开提供了一种全新的RNA分子设计方案，通过合理优化功能区域的核酸序列，实现了更高的mRNA表达量，且组装成的PCC酶具有更高的酶活性和更长的半衰期，使得该RNA分子在针对丙酸血症的基因治疗领域具有广泛的应用前景。
The present disclosure relates to the field of biotechnology, and specifically to mRNA for encoding human PCCA or PCCB protein and its use, wherein the mRNA molecule comprises a CDS encoding human PCCA or PCCB protein, the CDS encoding human PCCA protein comprises a nucleotide sequence selected from one of SEQ ID NO:2-6 or a nucleotide sequence having at least 80% identity therewith, and the CDS encoding human PCCB protein comprises a nucleotide sequence selected from one of SEQ ID NO:8-14 or a nucleotide sequence having at least 80% identity therewith. The present disclosure provides a new RNA molecule design scheme, which achieves a higher mRNA expression amount by rationally optimizing the nucleic acid sequence of the functional region, and the assembled PCC enzyme has a higher enzyme activity and a longer half-life, so that the RNA molecule has a wide range of application prospects in the field of gene therapy for propionic acidemia.
Description
Technical Field
The present disclosure relates to the field of biotechnology, in particular to an mRNA for encoding human PCCA or PCCB proteins and uses thereof.
Background
The acidemia (propionic academia, PA) is an autosomal recessive genetic disease, which is a rare organic acidemia, and the defects of propionyl coenzyme a carboxylase (propionyl-CoA carboxylase, PCC) cause the metabolic disorder of branched chain amino acids and odd chain fatty acids, so that propionic acid and bypass metabolites accumulate in the body, and a series of biochemical metabolic disorders and systemic damage of brain, heart, liver, bone marrow and the like are caused, so that the acidulous or lethal effect is caused. The current lack of specific treatment of propionic acidemia is mainly life-supporting, symptomatic treatment in the acute phase, including limiting protein intake, intravenous fluid infusion and correcting acidosis, hypoglycemia and electrolyte disorders, and if necessary, peritoneal dialysis or hemodialysis. The long-term treatment mainly comprises limiting the intake of valine, isoleucine, threonine and methionine and is assisted by medicines such as levocarnitine, metronidazole, carbamylglutamic acid and the like. For patients with poor therapeutic effects via combination of diet and drug, and with repeated occurrence of severe metabolic decompensation or concomitant cardiomyopathy, liver transplantation can be considered.
The PA pathogenic genes are PCCA (propionyl-CoA carboxylase subunit alpha) and PCCB (propionyl-CoA carboxylase subunit beta), respectively, which are the two subunits that make up the PCC enzyme. The PCCA or PCCB mutation causes deficiency of propionyl-CoA carboxylase, which results in blockage of propionyl-CoA conversion to methylmalonyl-CoA, and further causes abnormal increase of metabolites such as propionic acid and precursor propionyl CoA, methylcitric acid and propionylglycine in the body, and causes damage to the body. Because of the need for PCC enzymes to localize to mitochondria, there is currently no approved therapy (including enzyme replacement therapies) for the underlying cause of PA. Some medicines are in clinical development stage, wherein the development of small molecular medicines does not realize etiology treatment, but only can supplement circulating substances and improve symptoms. mRNA has wide application prospect as a drug molecule and unique advantages in the treatment field. Compared to traditional DNA therapies, mRNA drugs do not introduce integration of the exogenous genome into the host genome, thus reducing possible safety risks. In addition, mRNA can also provide immediate and regulated protein expression, making it an ideal choice for treating diseases caused by specific gene deletions or mutations.
MRNA-3927 developed by Moderna is the only mRNA therapy currently being developed clinically and used for treating the hyperuricemia by encapsulating two mRNAs through lipid nanoparticles, encoding two protein subunits PCCA and PCCB respectively, thereby synthesizing PCCase. mRNA-3927 sequences are disclosed in patent WO2023287751A 1. The U.S. Food and Drug Administration (FDA) has granted mRNA-3927 a fast-channel qualification. mRNA-3927 is now in clinical phase I/II, and the existing clinical results show signs of early treatment effects, such as a significant decrease in the incidence of metabolic disorder events in the treated patients, indicating that mRNA technology can treat acrylic acid by replacing intracellular proteins, providing a potential solution for the patient population facing treatment selection scarcity, and showing great application prospects. However, the complexity of mRNA nucleic acid sequence design, and the low in vivo and in vitro stability of mRNA drugs, are difficulties that mRNA drug development needs to address. mRNA sequences screened from naturally occurring genomes may have certain limitations in terms of transcriptional regulatory mechanisms and translational efficiencies that fail to meet the demands for high levels of gene expression. In order to increase the expression level of the protein encoded by the mRNA, the sequences need to be optimized and screened. In addition, a large amount of mRNA degrading enzymes exist in the body, and simultaneously different mRNA sequences are in the same cell and the degradation speed of the same mRNA sequence in different cells is greatly different, so that in vitro, mRNA drugs are easy to lose efficacy due to degradation, and the instability is unfavorable for the storage and transportation of the mRNA drugs. Therefore, mRNA sequences with stable structure and high translation efficiency are designed and screened, and play an important role in the research and development of mRNA drugs.
Disclosure of Invention
In order to solve the technical problems, the present disclosure provides an mRNA for encoding human PCCA or PCCB protein and use thereof, the mRNA has stable structure, is not easily degraded, has high translation efficiency, shows higher mRNA expression in vitro cell experiments, and the assembled PCC enzyme has higher enzyme activity and longer half-life, and the mRNA has good patent drug potential.
In one aspect, the present disclosure provides an mRNA molecule, wherein the mRNA molecule comprises a coding region (CDS) encoding a human PCCA or PCCB protein, the CDS encoding a human PCCA protein comprising a nucleotide sequence selected from one of SEQ ID NOs 2-6 or having at least 80% identity thereto, and the CDS encoding a human PCCB protein comprising a nucleotide sequence selected from one of SEQ ID NOs 8-14 or having at least 80% identity thereto.
In some embodiments, the above mRNA molecules further comprise a5 'untranslated region (5' UTR) and/or a 3 'untranslated region (3' UTR), the 5'UTR comprising a nucleotide sequence selected from the group consisting of one of SEQ ID NOS: 16-18, or a complement thereof, or a nucleotide sequence having at least 80% identity to the nucleotide sequence of one of SEQ ID NOS: 16-18, or a complement thereof, the 3' UTR comprising a nucleotide sequence selected from the group consisting of one of SEQ ID NOS: 19-22, or a complement thereof, or a nucleotide sequence having at least 80% identity to the nucleotide sequence of one of SEQ ID NOS: 19-22, or a complement thereof;
Optionally, the mRNA molecule further comprises a 5 'Cap structure and/or a poly-A region, preferably the poly-A region comprises 20-500 adenine nucleotides, the 5' Cap structure being selected from the group consisting of Cap0 Cap structure, cap1 Cap structure, and Cap2 Cap structure.
In some embodiments, (1) the mRNA molecules described above are mRNA molecules encoding human PCCA protein comprising, in 5 'to 3' order, the 5'UTR shown in SEQ ID NO. 16, the CDS shown in SEQ ID NO. 2 and the 3' UTR shown in SEQ ID NO. 19, or,
(2) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises, in 5 'to 3' order, a 5'UTR shown in SEQ ID NO. 17, a CDS shown in SEQ ID NO. 2 and a 3' UTR shown in SEQ ID NO. 20, or,
(3) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises in 5 'to 3' order the 5'UTR shown in SEQ ID NO. 17, the CDS shown in SEQ ID NO. 3 and the 3' UTR shown in SEQ ID NO. 20, or,
(4) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises in 5 'to 3' order the 5'UTR shown in SEQ ID NO. 17, the CDS shown in SEQ ID NO. 4 and the 3' UTR shown in SEQ ID NO. 20, or,
(5) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises, in 5 'to 3' order, a 5'UTR shown in SEQ ID NO:17, a CDS shown in SEQ ID NO:5 and a 3' UTR shown in SEQ ID NO:20, or,
(6) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises, in 5 'to 3' order, a 5'UTR shown in SEQ ID NO:17, a CDS shown in SEQ ID NO:6 and a 3' UTR shown in SEQ ID NO:20, or,
(7) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises in 5 'to 3' order the 5'UTR shown in SEQ ID NO. 17, the CDS shown in SEQ ID NO. 2 and the 3' UTR shown in SEQ ID NO. 21, or,
(8) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprise in 5 'to 3' order the 5'UTR shown in SEQ ID NO. 18, the CDS shown in SEQ ID NO. 8 and the 3' UTR shown in SEQ ID NO. 22, or,
(9) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 16, CDS shown in SEQ ID NO. 9 and 3' UTR shown in SEQ ID NO. 19 in 5 'to 3' order, or,
(10) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 8 and 3' UTR shown in SEQ ID NO. 20 in the order of 5 'to 3',
(11) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 10 and 3' UTR shown in SEQ ID NO. 20 in 5 'to 3' order, or,
(12) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 11 and 3' UTR shown in SEQ ID NO. 20 in the order of 5 'to 3',
(13) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 12 and 3' UTR shown in SEQ ID NO. 20 in 5 'to 3' order, or,
(14) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 13 and 3' UTR shown in SEQ ID NO. 20 in the order of 5 'to 3',
(15) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 14 and 3' UTR shown in SEQ ID NO. 20 in 5 'to 3' order, or,
(16) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprise 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 8 and 3' UTR shown in SEQ ID NO. 21 in the order of 5 'to 3'.
In some embodiments, the mRNA molecules described above comprise at least one chemical modification, preferably at least one chemical modification selected from pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine or 5-methoxy-uridine and 2' -O-methyl-uridine.
In another aspect, the present disclosure provides a nucleic acid composition, wherein the nucleic acid composition comprises at least one mRNA molecule encoding a human PCCA protein, and at least one mRNA molecule encoding a human PCCB protein;
Preferably, the nucleic acid composition comprises one mRNA molecule encoding a human PCCA protein and one mRNA molecule encoding a human PCCB protein, wherein the mRNA molecule encoding a human PCCA protein and the mRNA molecule encoding a human PCCB protein are the mRNA molecules of any of the above;
More preferably, the nucleic acid composition comprises (1) the mRNA molecule encoding human PCCA protein and (9) the mRNA molecule encoding human PCCB protein, or,
The nucleic acid composition comprising (1) the mRNA molecule encoding a human PCCA protein and (8) the mRNA molecule encoding a human PCCB protein, or,
The nucleic acid composition comprising (2) the mRNA molecule encoding a human PCCA protein and (10) the mRNA molecule encoding a human PCCB protein, or,
The nucleic acid composition comprising (3) the mRNA molecule encoding human PCCA protein and (11) the mRNA molecule encoding human PCCB protein, or,
The nucleic acid composition comprising (4) the mRNA molecule encoding a human PCCA protein and (12) the mRNA molecule encoding a human PCCB protein, or,
The nucleic acid composition comprising (5) the mRNA molecule encoding human PCCA protein and (13) the mRNA molecule encoding human PCCB protein, or,
The nucleic acid composition comprising (6) the mRNA molecule encoding a human PCCA protein and (14) the mRNA molecule encoding a human PCCB protein, or,
The nucleic acid composition comprising (1) the mRNA molecule encoding human PCCA protein and (15) the mRNA molecule encoding human PCCB protein, or,
The nucleic acid composition comprises (7) the mRNA molecule encoding the human PCCA protein and (16) the mRNA molecule encoding the human PCCB protein.
In another aspect, the present disclosure provides a vector encoding any one of the mRNA molecules described above.
In another aspect, the present disclosure provides a host cell comprising an mRNA molecule, a nucleic acid composition, or a vector of any one of the above.
In another aspect, the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises any one of (1) - (4) and a pharmaceutically acceptable carrier therefor:
(1) An mRNA molecule of any one of the above;
(2) The nucleic acid composition of any of the above;
(3) The vector of any one of the above, or,
(4) The host cell of any one of the above.
In another aspect, the present disclosure provides a lipid nanoparticle comprising an mRNA molecule or nucleic acid composition of any of the above.
In another aspect, the present disclosure provides the use of any one of the mRNA molecules, nucleic acid compositions, vectors, host cells, pharmaceutical compositions or lipid nanoparticles described above in the manufacture of a medicament for preventing, treating or ameliorating a hyperkeratosis and/or alleviating a metabolic disorder or symptoms of a metabolic disorder caused by a defect in PCC.
The beneficial effects of the present disclosure are at least as follows:
compared with the mRNA of the prior art, the mRNA sequence of the present disclosure shows higher mRNA expression in vitro cell experiments, and the assembled PCC enzyme has higher enzyme activity and longer half-life.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the specification and together with the description, serve to explain the principles of the specification.
FIG. 1 shows the mRNA molecule sequence of PCCA-10;
FIG. 2 shows the results of PCCenzyme activity assays after transfection of mRNA encoding human PCCA and PCCB proteins into HepG2-PCCA KO cells.
Detailed Description
I. Definition and description
In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present disclosure, definitions and explanations of related terms are provided below. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions, or biological systems, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.
As used herein, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device. References to "a plurality" in this disclosure refer to two or more. "and/or" describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate that there are three cases of a alone, a and B together, and B alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.
As used herein, the terms "nucleic acid," "nucleotide," and "polynucleotide" are used interchangeably to refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and polymers thereof in single-stranded, double-stranded, or multi-stranded form. The term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and/or pyrimidine bases or other natural, chemical, biochemical, non-natural, synthetic or derivatized nucleotide bases. In some embodiments, the nucleic acid may comprise a mixture of DNA, RNA, and the like. The term also encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. "nucleic acid" is used interchangeably with "gene," "DNA," and "mRNA" encoded by a gene.
As used herein, the term "mRNA (messenger RNA)" refers to any polynucleotide encoding a polypeptide or protein of interest and capable of in vitro, in vivo, in situ, or ex vivo translation to produce the encoded polypeptide of interest.
As used herein, the term "CDS (Coding sequence, coding region or Coding sequence)" is a sequence that encodes a polypeptide or protein product that, when expressed, produces a polypeptide or protein.
As used herein, the term "identity" refers to the ratio of mismatched nucleotides to the total number of nucleotides in a homologous region in percent. For example, a 20 base oligonucleotide that hybridizes to a region (site) of homology in a target genome that has two mismatches is said to have 90% identity to that region.
As used herein, the term "5'utr" generally refers to a sequence of an mRNA molecule from the 5' end to the translation initiation codon that is capable of recruiting a ribosomal complex and initiating translation of the mRNA. The 5'UTR comprises the 5' UTR region structure on the mRNA or the structure corresponds to the coding sequence on the DNA template. The 5' utr regulates post-transcriptional modification, translation initiation complex formation, stability, and the like by interacting with transcription factors, ribosomes, and other transcriptional regulatory proteins. The sequence design and optimization of this region is critical to improving the efficiency of post-transcriptional modification and protein expression. As used herein, the terms "5'utr structure", "5' utr sequence", and "5'utr element", etc. are used interchangeably, and reference in this disclosure to a 5' utr sequence has a nucleotide sequence selected from the group consisting of a nucleotide sequence selected from one of SEQ ID NOs 16-18 or a complement thereof, or a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from one of SEQ ID NOs 16-18 or a complement thereof.
As used herein, the term "3' utr" refers to a sequence between a stop codon of a polypeptide coding sequence and a poly (a) sequence in an mRNA. The 3' utr may regulate translation of mRNA by interacting with mRNA binding proteins, mirnas, and the like. The 3'UTR comprises the 3' UTR region structure on the mRNA or the structure corresponds to the coding sequence on the DNA template. It is closely related to post-transcriptional modification and mRNA stability. The sequence and structural features of the 3' utr may affect mRNA stability, ribosome scanning, formation of translation termination complexes, etc., thereby affecting the expression level of the protein as used herein, the terms "3' utr structure", "3' utr sequence", and "3' utr element" are used interchangeably, and the 3' utr has a length of 3-500 nucleotides, 5-150 nucleotides, 10-100 nucleotides, 15-90 nucleotides, or 20-70 nucleotides. The 3' UTR elements referred to in the present disclosure comprise a nucleotide sequence selected from one of SEQ ID NOS: 19-22 or a complement thereof, or a nucleotide sequence having at least 80% identity to a nucleotide sequence of one of SEQ ID NOS: 19-22 or a complement thereof.
As used herein, the term "complementary sequence" refers to the formation of hydrogen bonds between one nucleic acid and another nucleic acid sequence by conventional Watson-Crick (Watson-Crick) or other non-conventional types.
As used herein, the term "poly-A region" or "poly-A sequence" is intended to include the structure of the poly-A region on an mRNA or the structure corresponding to a coding sequence on a DNA template. The addition of poly-A sequences aids in the stability and transport of mRNA, prevents its degradation, and plays an important role in post-transcriptional modification. The poly (poly-A) region sequence may be a continuous strand of pure adenine nucleotides or may contain non-adenine nucleotides. In any form, a sequence is considered a poly (A) sequence so long as it is functionally equivalent to a conventional poly (A) sequence, i.e., is capable of providing a biological function similar to that of a conventional poly (A) sequence, such as affecting mRNA stability, translation efficiency, ribosome binding, etc. This includes, but is not limited to, known variants such as the human growth hormone (hGH) poly (A) sequence and the monkey virus 40 (SV 40) poly (A) sequence, which variants may differ in nucleotide composition but are functionally identified as equivalent to the conventional poly (A) sequence. In the present disclosure, the poly-A region sequence comprises 20-500 adenine nucleotides, e.g., 25, 50, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, or 500 adenine nucleotides.
As used herein, the term "5 'cap structure" is meant to include 5' cap structures found on natural mRNA and analogs thereof. The 5 'cap structure on natural mRNA is the attachment of the nail-methylated guanylic acid to the 5' terminal nucleotide of the RNA via pyrophosphate, forming a 5',5' -triphosphate linkage (5 ',5' -triphosphate linkage). The 5' Cap structure is typically of three types (m 7G5' ppp5' Np, m7G5' ppp5' NmpNp, m7G5' ppp5' NmpNmpNp), designated Cap0, cap1 and Cap2, respectively. Cap0 refers to the ribose unmethylation of the terminal nucleotide, cap1 refers to the ribose methylation of the terminal one nucleotide, and Cap2 refers to the ribose homomethylation of the terminal two nucleotides. Methods for capping mRNA molecules are known in the art. The 5 'cap structure of the aforementioned mRNA molecules may be added by enzymatic reaction after the mRNA molecules have been obtained by chemical synthesis or in vitro transcription (e.g., by commercial kits comprising vaccinia capping enzyme and mRNA cap structure 2' -O-methyltransferase). However, mRNA with a cap structure can also be produced by incorporating a nucleotide analogue with a cap structure as the first nucleotide directly into the transcript during in vitro transcription.
As used herein, the term "chemically modified" or "chemically modified" refers to modification of one or more of the position, pattern, percentage or population of adenosine (a), guanosine (G), uridine (U) or cytidine (C) ribonucleosides or deoxyribonucleosides. In this context, these terms are not intended to refer to ribonucleotide modifications in the naturally occurring 5' cap structure.
As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid or gel fillers that are suitable for use in humans or animals, and must be of sufficient purity and low enough toxicity. "compatibility" means that the components of the pharmaceutical composition and the active ingredients of the drug and the combination thereof are blended with each other without significantly reducing the efficacy. In the present invention, the aforementioned pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers, or combinations thereof. The amount of pharmaceutically acceptable carrier in the pharmaceutical composition can be determined experimentally based on the activity of the carrier and the desired characteristics of the formulation, such as stability and/or minimal oxidation.
As used herein, the term "lipid nanoparticle" refers to a particle having a nanoscale size that contains a lipid component.
As used herein, the term "ionizable cationic lipid" refers to a lipid molecule capable of being positively charged under physiological pH conditions. Such as amino lipids, as ionizable cationic lipids.
As used herein, the term "neutral lipid" refers to a lipid molecule that is uncharged at a particular pH condition, such as physiological pH conditions. Examples of neutral lipids include, but are not limited to, one or more of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine (DOPE), 1, 2-dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylethanolamine (POPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), preferably DSPC and/or DOPE.
As used herein, the term "structural lipid" refers to a lipid that enhances nanoparticle stability by filling in the interstices between the lipids, commonly known as steroids. The steroid is a compound having a cyclopenta-dihydrophenanthrene carbon skeleton, and in a preferred embodiment, the steroid is selected from one or more of cholesterol, sitosterol, stigmasterol, saponariol, brassicasterol, ergosterol, lycorine, ursolic acid, alpha-tocopherol, stigmasterol, avenanthrol, ergocalcitol or campesterol, preferably cholesterol and/or beta-sitosterol, more preferably cholesterol.
As used herein, the term "polymer lipid" refers to a molecule that contains a polymer moiety and a lipid moiety. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid. Other lipids capable of reducing aggregation, such as products of coupling compounds having uncharged, hydrophilic, steric-blocking moieties to lipids, may also be used. In a preferred embodiment, the pegylated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Alternatively, the pegylated lipid comprises a PEG moiety of about 1000Da to about 20kDa, preferably about 1000Da to about 5000 Da. Optionally, the pegylated lipid is selected from one or more of DMPE-PEG1000、DPPE-PEG1000、DSPE-PEG1000、DOPE-PEG1000、DMG-PEG2000、Ceramide-PEG2000、DMPE-PEG2000、DPPE-PEG2000、DSPE-PEG2000、Azido-PEG2000、DSPE-PEG2000-Mannose、Ceramide-PEG5000、DSPE-PEG5000, preferably DMG-PEG2000.
As used herein, the term "vector" refers to a piece of DNA extracted from a virus, plasmid, or cell of a higher organism into which a foreign DNA fragment may be inserted or has been inserted for cloning and/or expression purposes. In certain embodiments, the carrier may be stably maintained in the organism. Vectors may contain, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, and/or a Multiple Cloning Site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial Artificial Chromosomes (BACs), yeast Artificial Chromosomes (YACs), and the like.
As used herein, the terms "cell" and "host cell" are used interchangeably herein to refer to a cell that expresses or is capable of expressing a sequence to be expressed. The host cells of the invention express polynucleotides encoding polypeptides or RNAs that have a variety of uses, including biotechnology, molecular biology and clinical applications. 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.
Detailed description of the preferred embodiments
In one aspect, the present disclosure provides an mRNA molecule, wherein the mRNA molecule comprises a CDS encoding a human PCCA or PCCB protein comprising a nucleotide sequence selected from one of SEQ ID NOs 2-6 or having at least 80% identity thereto, and wherein the CDS encoding a human PCCB protein comprises a nucleotide sequence selected from one of SEQ ID NOs 8-14 or having at least 80% identity thereto.
In some embodiments, the mRNA molecules described above further comprise a 5'UTR comprising a nucleotide sequence selected from one of SEQ ID NOS: 16-18 or a complement thereof, or a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from one of SEQ ID NOS: 16-18 or a complement thereof, and/or a 3' UTR comprising a nucleotide sequence selected from one of SEQ ID NOS: 19-22, or a complement thereof, or a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from one of SEQ ID NOS: 19-22, or a complement thereof;
Optionally, the mRNA molecule further comprises a 5 'Cap structure and/or a poly-A region, preferably the poly-A region comprises 20-500 adenine nucleotides, the 5' Cap structure being selected from the group consisting of Cap0 Cap structure, cap1 Cap structure, and Cap2 Cap structure.
In some alternative embodiments, the 3'utr sequence further comprises a 3' utr sequence or a complement thereof into which an additional sequence is inserted, preferably the additional sequence is a miRNA binding site, more preferably the additional sequence is selected from the group consisting of a full-length microRNA reverse complement sequence or a reverse complement of a seed sequence thereof, preferably the full-length microRNA reverse complement sequence is 19-25 nt, preferably the seed sequence has a reverse complement length of 7-8nt, preferably the 3'utr sequence further comprises a 3' utr sequence into which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional sequences, or a complement thereof, are inserted.
In some alternative embodiments, the poly-A sequence comprises 25, 50, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450 or 500 adenine nucleotides (A), preferably 120 adenine nucleotides, particularly the poly-A region sequence set forth in SEQ ID NO. 15.
In some alternative embodiments, the 5' Cap structure described above is preferably a Cap1 Cap structure.
In some embodiments, (1) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in 5 'to 3' order, the 5'UTR shown in SEQ ID NO. 16, the CDS shown in SEQ ID NO. 2 and the 3' UTR shown in SEQ ID NO. 19, or,
(2) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises, in 5 'to 3' order, a 5'UTR shown in SEQ ID NO. 17, a CDS shown in SEQ ID NO. 2 and a 3' UTR shown in SEQ ID NO. 20, or,
(3) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises in 5 'to 3' order the 5'UTR shown in SEQ ID NO. 17, the CDS shown in SEQ ID NO. 3 and the 3' UTR shown in SEQ ID NO. 20, or,
(4) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises in 5 'to 3' order the 5'UTR shown in SEQ ID NO. 17, the CDS shown in SEQ ID NO. 4 and the 3' UTR shown in SEQ ID NO. 20, or,
(5) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises, in 5 'to 3' order, a 5'UTR shown in SEQ ID NO:17, a CDS shown in SEQ ID NO:5 and a 3' UTR shown in SEQ ID NO:20, or,
(6) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises, in 5 'to 3' order, a 5'UTR shown in SEQ ID NO:17, a CDS shown in SEQ ID NO:6 and a 3' UTR shown in SEQ ID NO:20, or,
(7) The mRNA molecules are mRNA molecules encoding human PCCA protein, which comprises in 5 'to 3' order the 5'UTR shown in SEQ ID NO. 17, the CDS shown in SEQ ID NO. 2 and the 3' UTR shown in SEQ ID NO. 21, or,
(8) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprise in 5 'to 3' order the 5'UTR shown in SEQ ID NO. 18, the CDS shown in SEQ ID NO. 8 and the 3' UTR shown in SEQ ID NO. 22, or,
(9) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 16, CDS shown in SEQ ID NO. 9 and 3' UTR shown in SEQ ID NO. 19 in 5 'to 3' order, or,
(10) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 8 and 3' UTR shown in SEQ ID NO. 20 in the order of 5 'to 3',
(11) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 10 and 3' UTR shown in SEQ ID NO. 20 in 5 'to 3' order, or,
(12) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 11 and 3' UTR shown in SEQ ID NO. 20 in the order of 5 'to 3',
(13) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 12 and 3' UTR shown in SEQ ID NO. 20 in 5 'to 3' order, or,
(14) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 13 and 3' UTR shown in SEQ ID NO. 20 in the order of 5 'to 3',
(15) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprises 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 14 and 3' UTR shown in SEQ ID NO. 20 in 5 'to 3' order, or,
(16) The mRNA molecules are mRNA molecules encoding human PCCB protein, which comprise 5'UTR shown in SEQ ID NO. 17, CDS shown in SEQ ID NO. 8 and 3' UTR shown in SEQ ID NO. 21 in the order of 5 'to 3'.
In some embodiments, (I-1) the mRNA molecules described above are mRNA molecules encoding human PCCA protein comprising, in 5 'to 3' order, cap1 Cap structure, 5'UTR shown in SEQ ID NO. 16, CDS shown in SEQ ID NO. 2, 3' UTR shown in SEQ ID NO. 19 and poly-A region shown in SEQ ID NO. 15, or,
(I-2) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in 5 'to 3' order, cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:2, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-3) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in 5 'to 3' order, cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:3, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-4) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in 5 'to 3' order, cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:4, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-5) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:5, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-6) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in 5 'to 3' order, cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:6, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-7) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:2, 3' UTR shown in SEQ ID NO:21 and poly-A region shown in SEQ ID NO:15, or,
(I-8) the mRNA molecule is an mRNA molecule encoding a human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:18, CDS shown in SEQ ID NO:8, 3' UTR shown in SEQ ID NO:22 and poly-A region shown in SEQ ID NO:15, or,
(I-9) the mRNA molecule is an mRNA molecule encoding a human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:16, CDS shown in SEQ ID NO:9, 3' UTR shown in SEQ ID NO:19 and poly-A region shown in SEQ ID NO:15, or,
(I-10) the mRNA molecule is an mRNA molecule encoding a human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:8, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-11) the mRNA molecule is an mRNA molecule encoding a human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:10, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-12) the mRNA molecule is an mRNA molecule encoding a human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:11, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-13) the mRNA molecule is an mRNA molecule encoding a human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:12, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-14) the mRNA molecule is an mRNA molecule encoding a human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:13, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-15) the mRNA molecule is an mRNA molecule encoding a human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:14, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, or,
(I-16) the mRNA molecule is an mRNA molecule encoding human PCCB protein, which comprises, in the order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:8, 3' UTR shown in SEQ ID NO:21 and poly-A region shown in SEQ ID NO: 15.
In some embodiments, the mRNA molecules described above comprise at least one chemical modification selected from at least one of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytidine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine or 5-methoxy-uridine and 2' -O-methyl-uridine, preferably N1-methyl-pseudouridine.
In some embodiments, uracil in the mRNA molecules has a chemical modification, in some embodiments, the chemical modification is at the 5-position of uracil, preferably, the chemical modification is N1-methyl pseudouridine, more preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of uracil in the mRNA molecules has a chemical modification, which is N1-methyl pseudouridine.
In some embodiments, the mRNA molecules described above are mRNA molecules encoding human PCCA protein comprising, in 5' to 3' order, a 5' UTR, CDS encoding human PCCA protein and a 3' UTR, wherein 100% of the uracil in the mRNA molecule is N1-methyl pseudouridine, preferably, the mRNA molecules described above comprise, in 5' to 3' order, a 5' cap structure, a 5' UTR, CDS encoding human PCCA protein, a 3' UTR and a poly-A region, wherein 100% of the uracil in the mRNA molecule is N1-methyl pseudouridine.
In some embodiments, the mRNA molecules described above are mRNA molecules encoding human PCCB protein comprising in 5' to 3' order a 5' UTR, CDS encoding human PCCB protein and a 3' UTR, wherein 100% of the uracil in the mRNA molecule is N1-methyl pseudouridine, preferably the mRNA molecules described above comprise in 5' to 3' order a 5' cap structure, a 5' UTR, CDS encoding human PCCB protein, a 3' UTR and a poly-A region, wherein 100% of the uracil in the mRNA molecule is N1-methyl pseudouridine.
In some embodiments, (II-1) the mRNA molecules described above are mRNA molecules encoding human PCCA protein comprising, in 5 'to 3' order, cap1 Cap structure, 5'UTR shown in SEQ ID NO:16, CDS shown in SEQ ID NO:2, 3' UTR shown in SEQ ID NO:19 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecules is N1-methyl pseudouridine, or,
(II-2) the mRNA molecule is mRNA molecule 9 encoding human PCCA protein, which comprises, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:2, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule is N1-methyl pseudouridine, or,
(II-3) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:3, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-4) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:4, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-5) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:5, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-6) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:6, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-7) the mRNA molecule is an mRNA molecule encoding human PCCA protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:2, 3' UTR shown in SEQ ID NO:21 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-8) the mRNA molecule is an mRNA molecule encoding human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:18, CDS shown in SEQ ID NO:8, 3' UTR shown in SEQ ID NO:22 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-9) the mRNA molecule is an mRNA molecule encoding human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:16, CDS shown in SEQ ID NO:9, 3' UTR shown in SEQ ID NO:19 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-10) the mRNA molecule is an mRNA molecule encoding human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:8, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-11) the mRNA molecule is an mRNA molecule encoding human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:10, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-12) the mRNA molecule is an mRNA molecule encoding human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:11, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-13) the mRNA molecule is an mRNA molecule encoding human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:12, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-14) the mRNA molecule is an mRNA molecule encoding human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:13, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-15) the mRNA molecule is an mRNA molecule encoding human PCCB protein comprising, in order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:14, 3' UTR shown in SEQ ID NO:20 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule being N1-methyl pseudouridine, or,
(II-16) the mRNA molecule is an mRNA molecule encoding human PCCB protein, which comprises, in the order of 5 'to 3', cap1 Cap structure, 5'UTR shown in SEQ ID NO:17, CDS shown in SEQ ID NO:8, 3' UTR shown in SEQ ID NO:21 and poly-A region shown in SEQ ID NO:15, 100% uracil in the mRNA molecule is N1-methyl pseudouridine.
In another aspect, the present disclosure provides a nucleic acid composition, wherein the nucleic acid composition comprises at least one mRNA molecule encoding a human PCCA protein, and at least one mRNA molecule encoding a human PCCB protein;
Preferably, the nucleic acid composition comprises one mRNA molecule encoding a human PCCA protein and one mRNA molecule encoding a human PCCB protein, wherein the mRNA molecule encoding a human PCCA protein and the mRNA molecule encoding a human PCCB protein are the mRNA molecules of any of the above;
More preferably, the nucleic acid composition comprises the mRNA molecule encoding human PCCA protein as described in the above (1) and the mRNA molecule encoding human PCCB protein as described in the above (9), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (1) and the mRNA molecule encoding human PCCB protein as described in the above (8), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (2) and the mRNA molecule encoding human PCCB protein as described in the above (10), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (3) and the mRNA molecule encoding human PCCB protein as described in the above (11), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (4) and the mRNA molecule encoding human PCCB protein as described in the above (12), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (5) and the mRNA molecule encoding human PCCB protein as described in the above (13), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (6) and the mRNA molecule encoding human PCCB protein as described in the above (14), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (1) and the mRNA molecule encoding human PCCB protein as described in the above (15), or,
The nucleic acid composition comprises the mRNA molecule encoding human PCCA protein as described in the above (7) and the mRNA molecule encoding human PCCB protein as described in the above (16).
More preferably, the nucleic acid composition comprises the mRNA molecule encoding human PCCA protein as described in the above (I-1) and the mRNA molecule encoding human PCCB protein as described in the above (I-9), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (I-1) and the mRNA molecule encoding human PCCB protein as described in the above (I-8), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (I-2) and the mRNA molecule encoding human PCCB protein as described in the above (I-10), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (I-3) and the mRNA molecule encoding human PCCB protein as described in the above (I-11), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (I-4) and the mRNA molecule encoding human PCCB protein as described in the above (I-12), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (I-5) and the mRNA molecule encoding human PCCB protein as described in the above (I-13), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (I-6) and the mRNA molecule encoding human PCCB protein as described in the above (I-14), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (I-1) and the mRNA molecule encoding human PCCB protein as described in the above (I-15), or,
The nucleic acid composition comprises the mRNA molecule encoding human PCCA protein as described in the above (I-7) and the mRNA molecule encoding human PCCB protein as described in the above (I-16).
More preferably, the nucleic acid composition comprises the mRNA molecule encoding human PCCA protein as described in the above (II-1) and the mRNA molecule encoding human PCCB protein as described in the above (II-9), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (II-1) and the mRNA molecule encoding human PCCB protein as described in the above (II-8), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (II-2) and the mRNA molecule encoding human PCCB protein as described in the above (II-10), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (II-3) and the mRNA molecule encoding human PCCB protein as described in the above (II-11), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (II-4) and the mRNA molecule encoding human PCCB protein as described in the above (II-12), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (II-5) and the mRNA molecule encoding human PCCB protein as described in the above (II-13), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (II-6) and the mRNA molecule encoding human PCCB protein as described in the above (II-14), or,
The nucleic acid composition comprising the mRNA molecule encoding human PCCA protein as described in the above (II-1) and the mRNA molecule encoding human PCCB protein as described in the above (II-15), or,
The nucleic acid composition comprises the mRNA molecule encoding human PCCA protein as described in the above (II-7) and the mRNA molecule encoding human PCCB protein as described in the above (II-16).
In some preferred embodiments, the molar ratio of the above-described mRNA molecule encoding human PCCA protein to the mRNA molecule encoding human PCCB protein is from 1:0.1 to 10.
In some preferred embodiments, the molar ratio of the mRNA molecule encoding the human PCCA protein to the mRNA molecule encoding the human PCCB protein is selected from 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, preferably the molar ratio of the mRNA molecule encoding the human PCCA protein to the mRNA molecule encoding the human PCCB protein is 1:1.
In another aspect, the present disclosure provides a vector encoding any one of the mRNA molecules described above.
In another aspect, the present disclosure provides a host cell comprising an mRNA molecule, a nucleic acid composition, or a vector of any one of the above.
In another aspect, the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises any one of (1) - (4) and a pharmaceutically acceptable carrier therefor:
(1) An mRNA molecule of any one of the above;
(2) The nucleic acid composition of any of the above;
(3) A vector according to any one of the above, or
(4) The host cell of any one of the above.
In another aspect, the present disclosure provides a lipid nanoparticle comprising an mRNA molecule or nucleic acid composition of any of the above.
In some preferred embodiments, the lipid nanoparticle further comprises one or more lipid moieties selected from the group consisting of ionizable cationic lipids, structural lipids, neutral lipids, or polymeric lipids.
In another aspect, the present disclosure provides the use of any one of the mRNA molecules, nucleic acid compositions, vectors, host cells, pharmaceutical compositions or lipid nanoparticles described above in the manufacture of a medicament for preventing, treating or ameliorating a hyperkeratosis and/or alleviating a metabolic disorder or symptoms of a metabolic disorder caused by a defect in PCC.
In some preferred embodiments, the metabolic disorder or symptoms of a metabolic disorder resulting from a deficiency in PCC described above include electrolyte disorders, dehydration, hypoglycemia, rash, muscle cramps, brain damage, coma, vision loss, osteoporosis, epilepsy, or stroke.
For purposes of clarity and conciseness of description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include some embodiments having a combination of all or some of the features described.
Examples
Example 1 mRNA sequences encoding PCCA and PCCB proteins
Aiming at wild type human PCCA and PCCB proteins (the amino acid sequence of the wild type human PCCA protein is shown as SEQ ID NO:1, the amino acid sequence of the wild type human PCCB protein is shown as SEQ ID NO: 7), through different optimization algorithms such as CDSfold, ribotree, optimization is carried out in terms of stability, translation efficiency and the like, and a plurality of mRNA sequences are designed. The mRNA molecules are obtained by the steps of gene synthesis, in vitro transcription template preparation, plasmid linearization, IVT mRNA synthesis, purification quality inspection and the like and are used for in vitro cell experiments. The final mRNA sequence is shown in Table 1, and the total uracil in the mRNA sequence is N1-methyl pseudouridine, for example, the mRNA sequence of PCCA-10 is shown in FIG. 1, and the total uracil in the mRNA sequence of PCCA-10 is N1-methyl pseudouridine. Among them, the present application uses PCCA-06 and PCCB-06 as controls, which are derived from mRNA-3927 sequences disclosed in WO2023287751A 1.
TABLE 1 mRNA molecular sequences encoding human PCCA and PCCB proteins
Example 2 detection of protein expression and enzymatic Activity after transfection of mRNA encoding human PCCA and PCCB proteins into HepG2
The mRNA obtained in example 1 was used in an in vitro cell experiment to test the protein expression amounts and PCCb enzyme activity levels of human PCCA and PCCB.
The specific experimental procedure for determining the protein expression levels and PCCb enzyme activity levels of PCCA and PCCB was as follows:
Human hepatoma cells HepG2 (product number: SCSP-510, purchased from cell bank of China academy of sciences) were cultured in DMEM high-sugar medium containing 10% fetal bovine serum at 37℃under 5% CO2 to a cell density of 90% or more. 24h before transfection, hepG2 cells were counted by a cell counter (brand: bodabo; model: JSY-FL-049) and plated in 24-well plates at a density of 1.5X105 cells/well with 500. Mu.L of medium per well.
MRNA (see tables 1 and 2 for mRNA information) was transfected 24h later using Lipofectamine MessengerMAX (brand: sieimer, cat# LMRNA; lot # 2646532) in combination with Opti-MEM (brand: sieimer, cat# 31985070; lot # 2508952) and co-transfected 500ng of mRNA (i.e., PCCA 284 ng, PCCB 214 ng) at a molar ratio of 1:1. The negative control ctrl group was not transfected with mRNA. The single well transfection system was as follows:
25 mu L of Opti-MEM+1.5 mu L Lipofectamine MessengerMAX, and standing for 5min at room temperature;
25 mu L of Opti-MEM+500ng mRNA, and standing for 5min at room temperature;
A+B stands for 20min at room temperature.
The mixed transfection solution was added slowly in an amount of 50. Mu.L to 450. Mu.L of opti-MEM, and after thoroughly mixing, the mixture was dropped into the culture plate, and after 4 hours, the transfection medium was removed, and 1mL of complete medium was added to continue the culture.
Samples of 48H, 96H and 144H after transfection were collected, 1 well was lysed by adding 500. Mu.L of lysis solution (RIPA (high): PMSF: cocktail=100:1:4), and protein expression was measured using a BCA kit (brand: bioTek; model: SYNERGY H1) using PCCA and PCCB ELISA kits (PCCA ELISA KIT (brand: abbexa Ltd; brand: abx585094; lot: E2212484W)), PCCB ELISA kit (brand: wuhan feen; brand: EH0800; lot: H0800I 072P)) after quantifying the total protein concentration using a BCA kit (brand: biyun day; brand: P0009; lot: 092822221121), and using an enzyme-labeled instrument (brand: bioTek; model: SYNERGY H1), see Table 2. Simultaneously, samples of 48h and 144h were collected, 100. Mu.L of lysate (RIPA (high): PMSF: cocktail=100:1:4) was added to each well, and after 30min of ice lysis, the supernatant was centrifuged at 12000rpm for 15min, and the supernatant was collected to a total protein concentration of 0.5mg/mL and then assayed for enzyme activity by HPLC, as shown in Table 3. The mRNA sequences of the present disclosure have significantly higher levels of protein expression and enzyme activity than mRNA-3927 (PCCA-06+PCCB-06).
TABLE 2 PCCA and PCCB protein ELISA assay results
TABLE 3 PCC enzyme Activity assay
Example 3 detection of enzymatic Activity after transfection of mRNA encoding human PCCA and PCCB proteins into HepG2-PCCA KO cells
Construction of PCCA knockout HepG2 cell line (HepG 2-PCCA KO, shanghai-derived well biosystems), and measurement of the enzyme activity level after cotransverse of PCCA and PCCB mRNA in the absence of PCC background enzyme activity in HepG2-PCCA KO cells.
MRNA used was PCCA-06, PCCA-10, PCCA-31, PCCB-06, PCCB-31, PCCB-38.
The specific experimental process is as follows:
HepG2-PCCA KO cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum at 37℃under 5% CO2 to a cell density of 90% or more. 24h before transfection, after counting with a cell counter (brand: bodabo; model: JSY-FL-049), the cells were plated in 24-well plates at a density of 1.5X105 cells/well, 500. Mu.L of medium per well.
After 24h, mRNAPCCA, PCCB was co-transfected with mRNA 500ng (i.e., PCCA 284 ng, PCCB 214 ng) at a molar ratio of 1:1 using Lipofectamine MessengerMAX complex Opti-MEM. The negative control ctrl group was not transfected with mRNA. The single well transfection system was as follows:
25 mu L of Opti-MEM+1.5 mu L Lipofectamine MessengerMAX, and standing for 5min at room temperature;
25 mu L of Opti-MEM+500ng mRNA, and standing for 5min at room temperature;
A+B stands for 20min at room temperature.
After mixing, a total of 50. Mu.L of the transfection solution was slowly added to 450. Mu.L of opti-MEM, and after thoroughly mixing, the mixture was dropped into the culture plate, and after 4 hours, the transfection medium was removed, and 1mL of complete medium (FBS concentration 15%) was added to continue the culture.
Samples of 2d, 4d, 7d, 9d, 11d post transfection were harvested, 100. Mu.L of lysate (RIPA (high): PMSF: cocktail=100:1:4) was added to each well, centrifuged at 12000rpm for 15min after 30min on ice lysis, enzyme activity was detected using HPLC after quantifying total protein concentration to 0.5mg/mL, AUC and half-life were calculated by Phoenix software analysis, and PCC enzyme activity generated by comparison of different mRNA combinations was measured and shown in FIGS. 2, 4 and 5.
TABLE 4 PCC enzyme Activity assay
TABLE 5 PCC enzyme AUC and T1/2 calculation
The results in FIGS. 2, 4 and 5 show that expression of PCC enzyme activity was detected after transfection of PCCA and PCCB mRNA in the event of loss of PCC background enzyme activity in HepG2-PCCA KO cells. The mRNA sequence combinations of the present disclosure, PCCA-10+PCCB-31, PCCA-10+PCCB-38, PCCA-31+PCCB-31, have significantly higher enzyme activity levels at each time point tested and have a higher AUC than mRNA-3927 (PCCA-06+PCCB-06). Wherein PCCA-10+PCCB-38 also has a significantly longer half-life.
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