WO2023221937A1 - Method for delivering self-replicating rna molecule - Google Patents

Method for delivering self-replicating rna molecule Download PDF

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Publication number
WO2023221937A1
WO2023221937A1 PCT/CN2023/094277 CN2023094277W WO2023221937A1 WO 2023221937 A1 WO2023221937 A1 WO 2023221937A1 CN 2023094277 W CN2023094277 W CN 2023094277W WO 2023221937 A1 WO2023221937 A1 WO 2023221937A1
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Prior art keywords
protein
composition
self
liposomes
replicating rna
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PCT/CN2023/094277
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French (fr)
Chinese (zh)
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吴可行
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上海行深生物科技有限公司
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Publication of WO2023221937A1 publication Critical patent/WO2023221937A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus

Definitions

  • the present invention relates to the biological field.
  • the present invention relates to methods of delivering self-replicating RNA molecules.
  • mRNA-based drugs or vaccines have become one of the hot topics of concern.
  • traditional mRNA is not very stable and degrades within days within cells, resulting in unsustainable protein expression levels. If used for long-term disease treatment, patients may be required to inject large amounts of mRNA, which may increase the toxic side effects of mRNA therapy.
  • RNA molecules can replicate themselves in the cytoplasm, so they can reach protein expression levels no lower than traditional mRNA at very low doses, and can express proteins for a long time within a certain period of time, thus showing more promise than mRNA. technical advantages.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
  • a composition in one aspect of the invention, includes: a nucleic acid molecule including a self-replicating RNA molecule; and a delivery vector carrying the nucleic acid molecule.
  • a delivery carrier is required to deliver them into cells, which will help achieve intracellular RNA self-replication, maintain long-term high protein levels, and better exert its effect.
  • the use of the composition of the present invention helps to achieve in vivo delivery of nucleic acid drugs, especially self-replicating RNA molecules.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes: the aforementioned composition.
  • the invention provides a method of delivering self-replicating RNA molecules.
  • the method includes: formulating a self-replicating RNA molecule into a composition as described above; and optionally, providing the composition to a subject to be treated.
  • Figure 1 shows a schematic structural diagram of a self-replicating RNA molecule (reRNA TM ) according to one embodiment of the present invention
  • Figure 2 shows a schematic structural diagram of a protein-RNA complex containing self-replicating RNA molecules according to one embodiment of the present invention
  • Figure 3 shows a schematic diagram of an LNP delivery system containing self-replicating RNA molecules according to one embodiment of the present invention
  • Figure 4 shows a schematic structural diagram of a protein delivery system containing self-replicating RNA molecules according to one embodiment of the present invention
  • Figure 5 shows a schematic diagram of the analysis of reRNA TM -GFP cell entry efficiency detection according to one embodiment of the present invention
  • Figure 6 shows a GFP fluorescence image of reRNA TM -GFP delivered to vero cells using LCMV-GP as a delivery protein according to one embodiment of the present invention
  • Figure 7 shows a GFP fluorescence image of reRNA TM -GFP delivered to vero cells using NDV-F/NDV-HN as a delivery protein according to one embodiment of the present invention
  • Figure 8 shows the GFP fluorescence image of reRNA TM -GFP delivered to vero cells using LNP as a delivery carrier according to one embodiment of the present invention.
  • self-replicating RNA molecule used in this article can also be called “self-amplifying RNA”. Compared with ordinary mRNA, the important difference between self-replicating RNA molecules is that it can use its own RNA sequence as a template to self-amplify. copy. According to embodiments of the present application, self-replicating RNA molecules can also perform translation and replication in the cytoplasm without entering the cell nucleus, thereby avoiding potential risks caused by integration with the genome. Usually, mRNA codes for the protein that needs to be expressed, and ribosomes in the cell are used to complete translation and protein production.
  • the self-replicating RNA molecule will carry a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase). After the RNA molecule is translated in the cytoplasm to produce RNA polymerase, it can use the self-replicating RNA molecules serve as templates to create more self-replicating RNA molecules.
  • RNA-dependent RNA polymerase RNA-dependent RNA polymerase
  • a composition in one aspect of the invention, includes: a nucleic acid molecule including a self-replicating RNA molecule; and a delivery vector carrying the nucleic acid molecule.
  • RNA molecules entering cells and stably existing in cells they are carried on delivery carriers.
  • the delivery carriers can carry self-replicating RNA molecules into cells, which helps achieve intracellular RNA self-replication and maintain long-lasting high Protein level, better performance.
  • the use of the composition of the present invention helps to achieve in vivo delivery of nucleic acid drugs, especially self-replicating RNA molecules.
  • the inventor found that since the self-replicating RNA molecule connects at least the sequence encoding RNA polymerase with the sequence expressing the target protein, the molecular weight of the entire mRNA molecule is much larger than that of traditional mRNA. Excessive molecular weight may cause Delivery efficiency, translation efficiency, and replication efficiency are significantly reduced. In order to improve these efficiencies, the inventors conducted in-depth research, hoping to find the shortest nucleic acid fragment that can function normally in self-replication and translation.
  • the self-replicating RNA molecule includes: a first RNA sequence encoding an N protein or a functional fragment thereof; a second RNA sequence encoding a P protein or a functional fragment thereof; Functional fragments; a third RNA sequence encoding L protein or a functional fragment thereof; and a target molecule coding region encoding at least one target molecule.
  • RNA molecules can realize self-replication and translation of RNA in animal cells, and as a powerful "engine", this core region can provide efficient transcription amplification and "kinetic energy" to start macromolecular proteins, and can further carry ""CargoZone" to copy or translate target molecules, which cover almost all protein drugs currently on the market.
  • the "cargo area" can be designed with different protein coding cassettes to enable the body to produce a variety of peptides, enzymes, antibodies, channel proteins, receptor proteins, etc. within cells, thereby achieving different prevention or treatment purposes, covering Oncology pipeline, vaccine pipeline, rare disease pipeline and forward-looking general product pipeline.
  • the novel self-replicating RNA molecule proposed in the present invention is named reRNA TM .
  • the term "functional fragment” used in this article refers to a part of the full-length sequence of a protein that can still perform functions related to the self-replication of RNA molecules. For example, it can be a truncated version of the full-length sequence or the entire protein sequence. Proteins that have been modified by substitution, mutation, or deletion of long amino acid sequences. According to the embodiments of the present application, the functional fragment of the N protein can be combined with an RNA molecule to protect the RNA from the influence of nucleases.
  • the functional fragment of the P protein can be combined with the N protein to position the L polymerase on the template, while also Able to act as RNA polymerase to transcribe and
  • the basic component of the replication complex and further the functional fragment of L protein can function as RNA polymerase and is related to the transcription and replication of RNA.
  • At least one of the N protein, the P protein, and the L protein is independently from a Rhabdoviridae virus.
  • the N protein, the P protein, and the L protein can be from the Indiana strain of vesicular stomatitis virus.
  • the N protein includes but is not limited to Uniprot IDs: P03521, P11212, Q77E03, Q8B0H4, and B7UCZ2 sequences;
  • the P protein includes But are not limited to sequences with Uniprot IDs: P04880, Q8B0H8, P04879, P03520, and B7UCZ3;
  • L protein includes but is not limited to sequences with Uniprot IDs: Q8B0H0, Q98776, Q8B0I0, Q8B0H5, and P03523.
  • the N protein, the P protein, and the L protein can be from the New Jersey strain of vesicular stomatitis virus.
  • the N protein includes but is not limited to sequences with Uniprot IDs: P04881, Q89034, S5TKS4, Q89036, and Q89037;
  • the P protein includes But are not limited to sequences with Uniprot IDs: P04877, Q89057, Q89052, Q89050, Q89049;
  • L protein includes but is not limited to sequences with Uniprot IDs: P16379, P16379, I7DDL0, Q8B545, S5TC82.
  • the N protein, the P protein, and the L protein can also be from other vesicular virus genera (such as Chandipura vesicular virus, Malabar vesicular virus) and rabies virus.
  • vesicular virus genera such as Chandipura vesicular virus, Malabar vesicular virus
  • rabies virus such as Chandipura vesicular virus, Malabar vesicular virus
  • the N protein has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology with it
  • the P protein has the amino acid sequence shown in SEQ ID NO: 2 or has an amino acid sequence with at least 80% homology with it.
  • the L protein has an amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology thereto.
  • the first RNA sequence has a nucleotide sequence as shown in SEQ ID NO: 4, and the second RNA sequence has a nucleotide sequence as shown in SEQ ID NO: 5, so The third RNA sequence has the nucleotide sequence shown in SEQ ID NO:6.
  • the delivery vehicle includes at least one of proteins, liposomes and exosomes.
  • Proteins can serve as delivery vehicles to carry nucleic acid molecules into cells to achieve intracellular delivery of genetic material.
  • Liposomes are widely used in drug delivery as nanoscale drug delivery carriers, which can self-assemble into virus-sized particles and can release mRNA from endosomes to the cytoplasm. At the same time, it can promote cellular uptake, improve endosomal escape, protect nucleic acid molecules from being recognized by TLRs, and avoid excessive activation of the innate immune system.
  • the LNP delivery system mainly determines the amount of nucleic acid packaged by the LNP potential and the nucleic acid potential. Therefore, an LNP delivery system can package one to multiple nucleic acid molecules.
  • Exosomes are 50-150nm-sized vesicles released from living cells into the extracellular microenvironment. Due to their small size and the fact that they are cell products themselves, drug delivery through exosomes can avoid phagocytosis or phagocytosis by macrophages. It degrades and can circulate in the body for a long time to maintain the effect. Among them, the ability of exosomes to cross the blood-brain barrier to deliver nucleic acid drugs to the central nervous system is a significant advantage of exosome-delivered drugs.
  • the protein includes non-human protein and human protein.
  • the present invention does not strictly limit the type of human protein, as long as it can be used as a delivery vector to deliver nucleic acid molecules into cells, including but not limited to the SNARE protein family.
  • the non-human protein includes a viral capsid protein. Compared with human proteins, the delivery effect of viral capsid proteins is better. Since viral capsid proteins do not contain viral genetic material, they are easy to infect and enter cells. They have strong cell targeting specificity and can achieve intracellular delivery of proteins and genetic materials. . And, virus Capsid proteins are easy to obtain, which improves the efficiency of preparing delivery vectors and reduces costs. As shown in Figure 4, viral protein delivery vectors are derived from the characteristics of the virus itself, so a protein delivery vector can only package one nucleic acid molecule.
  • the coat proteins of other viruses can also be used for the delivery of reRNA TM .
  • the virus includes at least one of poxvirus, rabies virus, flavivirus, measles virus, coronavirus, vesicular stomatitis virus, Newcastle disease virus and lymphocytic choriomeningitis virus.
  • the above-mentioned viruses all have capsid proteins and can be used as delivery vectors.
  • the protein is selected from the group consisting of vesicular stomatitis virus receptor, lymphocytic choriomeningitis virus coat protein and/or Newcastle disease virus coat protein.
  • the vesicular stomatitis virus receptor VSV-G has an amino acid sequence such as SEQ ID NO: 7.
  • sequence information of lymphocytic choriomeningitis virus coat protein LCMV-GP please refer to UniProtKB/Swiss-Prot:P09991; Newcastle disease virus coat proteins are NDV-F and NDV-HN respectively.
  • the liposome contains a targeting element.
  • the carried nucleic acid molecules can be targeted and delivered to specific tissues or organs.
  • the liposomes include permanently positively charged cationic liposomes (such as DOTAP, DOTMA), auxiliary liposomes (such as DSPC, DOPE), structural liposomes (such as cholesterol, cholesterol lipid), long-circulating liposomes (such as DMG-PEG2000) and ionizable cationic liposomes (such as DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP).
  • permanently positively charged cationic liposomes such as DOTAP, DOTMA
  • auxiliary liposomes such as DSPC, DOPE
  • structural liposomes such as cholesterol, cholesterol lipid
  • long-circulating liposomes such as DMG-PEG2000
  • ionizable cationic liposomes such as DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP.
  • the liposome is selected from DOTAP, DSPC, cholesterol and DMG-PEG2000.
  • the inventor found through extensive experimental research that the self-replicating RNA delivery efficiency of the above-mentioned liposomes is high.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes: the aforementioned composition.
  • the delivery vector in the composition can carry nucleic acid molecules containing self-replicating RNA molecules into cells. Self-replicating RNA molecules can replicate themselves in the cytoplasm, and the target molecule coding regions they carry can express small molecules, various Peptides, enzymes, antibodies, channel proteins and receptor proteins, etc., to achieve different preventive or therapeutic purposes.
  • the pharmaceutical composition further includes pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients may include buffers, solubilizers, etc., and the pharmaceutical composition may be prepared, for example, in ampoules of a single dose dosage form or, for example, multiple doses. Unit dosage forms in measuring containers.
  • Pharmaceutical compositions can also be prepared into solutions, suspensions, lyophilized agents, liquids, injections and long-acting preparations.
  • the invention provides a method of delivering self-replicating RNA molecules.
  • the method includes formulating a self-replicating RNA molecule into the composition as described above.
  • the delivery vector can effectively deliver self-replicating RNA molecules into cells.
  • the method further includes providing the composition to a subject to be treated.
  • composition is also applicable to the method of delivering self-replicating RNA molecules, and will not be described again here.
  • VSV virus vesicular stomatitis virus
  • LDLR low-density lipoprotein
  • SEQ ID NO: 7 The specific VSV-G (SEQ ID NO: 7) screened by the inventor has high affinity. , using VSV-G protein as a delivery protein, it has high cell delivery efficiency.
  • transfection reagent Lipo8000 TM to culture HEK 293T cells to transfect reRNA TM plasmid (the plasmid carries the reRNA TM sequence, which contains the mRNA sequence, SEQ ID NO: 4-6 sequence, and GFP sequence) and the helper plasmid ( The helper plasmid contains a separate plasmid containing SEQ ID NO: 4-6 sequences, a VSV-G plasmid, and a T7 RNA polymerase plasmid). After the transfection is completed, the cells are cultured and cultured in an incubator for 48 hours. The supernatant is collected and carried out. After purification, reRNA TM -GFP seeds were obtained.
  • the transfection reagent Lipo8000 TM to transfect the cultured HEK 293 cells with the helper plasmid (the helper plasmid carries the nucleic acid molecule encoding VSV-G). After the transfection is completed, add reRNA TM -GFP seeds and culture in the incubator. After 48 hours, the supernatant was collected and purified to obtain VSV-G wrapped reRNA TM -GFP.
  • LCMV virus The coat protein of lymphocytic choriomeningitis virus (LCMV virus for short) is LCMV-GP, and its receptor is ⁇ -dystrophin, which is a ubiquitous protein and a high-quality candidate for reRNA TM delivery protein.
  • LCMV-GP Preparing LCMV-GP as a reRNA TM -GFP product for delivery protein can also complete the delivery of reRNA TM .
  • Example 1 Refer to Example 1 for the steps of wrapping reRNA TM -GFP with LCMV-GP protein.
  • Newcastle disease virus (NDV virus for short) is also an enveloped virus. It has two coat proteins, namely NDV-F and NDV-HN. The two proteins work together to complete cell entry and lysosome escape, and realize RNA detoxification. Delivery, using both proteins as delivery proteins at the same time, can also complete the delivery of reRNA TM -GFP.
  • NDV virus Newcastle disease virus
  • the specific experiments are as follows:
  • Example 1 Refer to Example 1 for the steps of wrapping reRNA TM -GFP with LCMV-GP protein.
  • Table 1 shows the changes in particle size and potential of reRNA TM -GFP before and after LNP encapsulation. From the results, it can be seen that the potential after LNP encapsulation is around +20mV and the particle size is around 150nm, which is more conducive to the delivery of RNA.
  • the packaged reRNA TM -GFP can complete RNA delivery in vero cells.
  • Figure 8 shows the expression of GFP after LNP packaged reRNA TM -GFP was delivered into vero cells for 24 hours.
  • the amount of reRNA TM -GFP is 18ng, and the number of vero cells is 2 ⁇ 10 5 pcs. It can be seen that liposomes can achieve effective delivery of reRNA TM -GFP.
  • references to the terms "one embodiment,””someembodiments,””anexample,””specificexamples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

Abstract

Provided in the present invention are a method for delivering a self-replicating RNA molecule, and a composition. The composition comprises a nucleic acid molecule and a delivering vector, wherein the nucleic acid molecule comprises a self-replicating RNA molecule, and the delivering vector carries the nucleic acid molecule, and comprises at least one of a protein, a liposome, and an exosome.

Description

递送自复制RNA分子的方法Methods of delivering self-replicating RNA molecules 技术领域Technical field
本发明涉及生物领域。具体地，本发明涉及递送自复制RNA分子的方法。The present invention relates to the biological field. In particular, the present invention relates to methods of delivering self-replicating RNA molecules.
背景技术Background technique
近年来基于mRNA药物或mRNA疫苗的开发成为人们关注的热点之一。然而，传统mRNA不是很稳定，在细胞内几天内就会降解，导致不可持续的蛋白质表达水平。如果长期用于疾病治疗，可能需要患者注射大量的mRNA，这可能会增加mRNA治疗的毒副作用。In recent years, the development of mRNA-based drugs or vaccines has become one of the hot topics of concern. However, traditional mRNA is not very stable and degrades within days within cells, resulting in unsustainable protein expression levels. If used for long-term disease treatment, patients may be required to inject large amounts of mRNA, which may increase the toxic side effects of mRNA therapy.
自复制RNA分子可以在细胞质内进行自我复制，因此可以在很低的剂量下达到不低于传统mRNA的蛋白表达水平，并且可以在一定时间内长效表达蛋白质，从而展现出比mRNA更有前景的技术优势。Self-replicating RNA molecules can replicate themselves in the cytoplasm, so they can reach protein expression levels no lower than traditional mRNA at very low doses, and can express proteins for a long time within a certain period of time, thus showing more promise than mRNA. technical advantages.
然而，针对RNA分子，目前缺乏有效的递送手段，这极大地限制了自复制RNA分子技术的应用。However, there is currently a lack of effective delivery methods for RNA molecules, which greatly limits the application of self-replicating RNA molecule technology.
发明内容Contents of the invention
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
在本发明的一个方面，本发明提出了一种组合物。根据本发明的实施例，所述组合物包括：核酸分子，所述核酸分子包括自复制RNA分子；递送载体，所述递送载体携带所述核酸分子。In one aspect of the invention, a composition is provided. According to an embodiment of the present invention, the composition includes: a nucleic acid molecule including a self-replicating RNA molecule; and a delivery vector carrying the nucleic acid molecule.
为了实现自复制RNA分子进入细胞且稳定存在于细胞内，需要递送载体将其递送至细胞内，有助于实现细胞内RNA自复制，维持长效的高蛋白水平，更好地发挥作用效果。根据本发明的实施例，利用本发明的组合物有助于实现核酸类药物尤其是自复制RNA分子的体内递送。In order for self-replicating RNA molecules to enter cells and exist stably within cells, a delivery carrier is required to deliver them into cells, which will help achieve intracellular RNA self-replication, maintain long-term high protein levels, and better exert its effect. According to embodiments of the present invention, the use of the composition of the present invention helps to achieve in vivo delivery of nucleic acid drugs, especially self-replicating RNA molecules.
在本发明的另一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：前面所述的组合物。In another aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: the aforementioned composition.
在本发明的又一方面，本发明提出了一种递送自复制RNA分子的方法。根据本发明的实施例，所述方法包括：将自复制RNA分子配制为前面所述的组合物；和任选的，为待处理对象提供所述组合物。In yet another aspect of the invention, the invention provides a method of delivering self-replicating RNA molecules. According to an embodiment of the present invention, the method includes: formulating a self-replicating RNA molecule into a composition as described above; and optionally, providing the composition to a subject to be treated.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。 Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
附图说明Description of the drawings
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:
图1显示了根据本发明一个实施例的自复制RNA分子(reRNATM)的结构示意图；Figure 1 shows a schematic structural diagram of a self-replicating RNA molecule (reRNA ™ ) according to one embodiment of the present invention;
图2显示了根据本发明一个实施例的含有自复制RNA分子的蛋白质-RNA复合物的结构示意图；Figure 2 shows a schematic structural diagram of a protein-RNA complex containing self-replicating RNA molecules according to one embodiment of the present invention;
图3显示了根据本发明一个实施例的含自复制RNA分子的LNP递送***示意图；Figure 3 shows a schematic diagram of an LNP delivery system containing self-replicating RNA molecules according to one embodiment of the present invention;
图4显示了根据本发明一个实施例的含自复制RNA分子的蛋白质递送***结构示意图；Figure 4 shows a schematic structural diagram of a protein delivery system containing self-replicating RNA molecules according to one embodiment of the present invention;
图5显示了根据本发明一个实施例的reRNATM-GFP入胞效率检测的分析示意图；Figure 5 shows a schematic diagram of the analysis of reRNA TM -GFP cell entry efficiency detection according to one embodiment of the present invention;
图6显示了根据本发明一个实施例的以LCMV-GP作为递送蛋白递送reRNATM-GFP至vero细胞的GFP荧光图；Figure 6 shows a GFP fluorescence image of reRNA TM -GFP delivered to vero cells using LCMV-GP as a delivery protein according to one embodiment of the present invention;
图7显示了根据本发明一个实施例的以NDV-F/NDV-HN作为递送蛋白递送reRNATM-GFP至vero细胞的GFP荧光图；Figure 7 shows a GFP fluorescence image of reRNA TM -GFP delivered to vero cells using NDV-F/NDV-HN as a delivery protein according to one embodiment of the present invention;
图8显示了根据本发明一个实施例的以LNP作为递送载体递送reRNATM-GFP至vero细胞的GFP荧光图。Figure 8 shows the GFP fluorescence image of reRNA ™ -GFP delivered to vero cells using LNP as a delivery carrier according to one embodiment of the present invention.
具体实施方式Detailed ways
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。Embodiments of the present invention are described in detail below. The embodiments described below are illustrative and are only used to explain the present invention and are not to be construed as limitations of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
在本文中所使用的术语“自复制RNA分子”也可以称为“自我扩增RNA”，与普通mRNA相比，自复制RNA分子的重要的区别在于它可以使用自己的RNA序列作为模板进行自我复制。根据本申请的实施例，自复制RNA分子还可以在细胞质进行翻译和复制工作，不进入细胞核，可以避免与基因组发生整合所带来的潜在风险。通常mRNA编码需要表达的蛋白质，细胞内的核糖体用来完成翻译和蛋白质生产。根据本申请的实施例，自复制RNA分子会携带一个能够表达RNA聚合酶(RNA依赖RNA聚合酶)的序列，在该RNA分子在细胞质中通过翻译产生RNA聚合酶后，它可以用该自复制RNA分子作为模板来产生更多的自复制RNA分子。 The term "self-replicating RNA molecule" used in this article can also be called "self-amplifying RNA". Compared with ordinary mRNA, the important difference between self-replicating RNA molecules is that it can use its own RNA sequence as a template to self-amplify. copy. According to embodiments of the present application, self-replicating RNA molecules can also perform translation and replication in the cytoplasm without entering the cell nucleus, thereby avoiding potential risks caused by integration with the genome. Usually, mRNA codes for the protein that needs to be expressed, and ribosomes in the cell are used to complete translation and protein production. According to embodiments of the present application, the self-replicating RNA molecule will carry a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase). After the RNA molecule is translated in the cytoplasm to produce RNA polymerase, it can use the self-replicating RNA molecules serve as templates to create more self-replicating RNA molecules.
组合物combination
在本发明的一个方面，本发明提出了一种组合物。根据本发明的实施例，所述组合物包括：核酸分子，所述核酸分子包括自复制RNA分子；递送载体，所述递送载体携带所述核酸分子。In one aspect of the invention, a composition is provided. According to an embodiment of the present invention, the composition includes: a nucleic acid molecule including a self-replicating RNA molecule; and a delivery vector carrying the nucleic acid molecule.
为了实现自复制RNA分子进入细胞且稳定存在于细胞内，将其承载在递送载体上，递送载体可以携带自复制RNA分子进入细胞内，有助于实现细胞内RNA自复制，维持长效的高蛋白水平，更好地发挥作用效果。根据本发明的实施例，利用本发明的组合物有助于实现核酸类药物尤其是自复制RNA分子的体内递送。In order to achieve self-replicating RNA molecules entering cells and stably existing in cells, they are carried on delivery carriers. The delivery carriers can carry self-replicating RNA molecules into cells, which helps achieve intracellular RNA self-replication and maintain long-lasting high Protein level, better performance. According to embodiments of the present invention, the use of the composition of the present invention helps to achieve in vivo delivery of nucleic acid drugs, especially self-replicating RNA molecules.
发明人在研究过程中发现，由于自复制RNA分子将至少编码RNA聚合酶的序列与表达靶蛋白的序列连接起来，因此整个mRNA分子的分子量比传统mRNA的分子量大得多，分子量过大可能导致递送效率、翻译效率以及复制效率显著降低。为了对这些效率进行改善，发明人进行了深入研究，希望能够寻找到最短的核酸片段，可以正常发挥自我复制和翻译的功能。During the research process, the inventor found that since the self-replicating RNA molecule connects at least the sequence encoding RNA polymerase with the sequence expressing the target protein, the molecular weight of the entire mRNA molecule is much larger than that of traditional mRNA. Excessive molecular weight may cause Delivery efficiency, translation efficiency, and replication efficiency are significantly reduced. In order to improve these efficiencies, the inventors conducted in-depth research, hoping to find the shortest nucleic acid fragment that can function normally in self-replication and translation.
根据本发明的实施例，所述自复制RNA分子包括：第一RNA序列，所述第一RNA序列编码N蛋白或其功能片段；第二RNA序列，所述第二RNA序列编码P蛋白或其功能片段；第三RNA序列，所述第三RNA序列编码L蛋白或其功能片段；和靶分子编码区，所述靶分子编码区编码至少一个靶分子。According to an embodiment of the present invention, the self-replicating RNA molecule includes: a first RNA sequence encoding an N protein or a functional fragment thereof; a second RNA sequence encoding a P protein or a functional fragment thereof; Functional fragments; a third RNA sequence encoding L protein or a functional fragment thereof; and a target molecule coding region encoding at least one target molecule.
参见图1和图2，发明人通过对各种RNA病毒的RNA在细胞内的翻译及自我扩增机制进行了深入研究，发现通过采用编码来自弹状病毒的N蛋白，P蛋白及L蛋白的RNA分子作为核心区域，可以实现RNA在动物细胞内的自我复制和翻译，并且该核心区域作为强大的“引擎”，可以提供高效转录扩增和启动大分子蛋白的“动能”，能够进一步搭载“货物区”来复制或者翻译靶分子，这些靶分子几乎涵盖了目前市场上所有的蛋白质药物。根据本发明的实施例，“货物区”可设计不同的蛋白编码盒使机体在细胞内生产多种肽、酶、抗体、通道蛋白以及受体蛋白等，从而达到不同的预防或治疗目的，覆盖肿瘤管线、疫苗管线、罕见病管线及前瞻性通用型产品管线。本发明中将提出的新型自复制RNA分子命名为reRNATM。Referring to Figures 1 and 2, the inventor conducted in-depth research on the intracellular translation and self-amplification mechanisms of RNA of various RNA viruses and found that by using proteins encoding the N protein, P protein and L protein from rhabdoviruses, As a core region, RNA molecules can realize self-replication and translation of RNA in animal cells, and as a powerful "engine", this core region can provide efficient transcription amplification and "kinetic energy" to start macromolecular proteins, and can further carry ""CargoZone" to copy or translate target molecules, which cover almost all protein drugs currently on the market. According to embodiments of the present invention, the "cargo area" can be designed with different protein coding cassettes to enable the body to produce a variety of peptides, enzymes, antibodies, channel proteins, receptor proteins, etc. within cells, thereby achieving different prevention or treatment purposes, covering Oncology pipeline, vaccine pipeline, rare disease pipeline and forward-looking general product pipeline. The novel self-replicating RNA molecule proposed in the present invention is named reRNA TM .
在本文中所使用的术语“功能片段”是指蛋白质的全长序列的一部分，但仍能够发挥与RNA分子自我复制相关的功能，例如可以是全长序列的截断型的，也可以是蛋白质全长序列的氨基酸序列发生替换、突变或者删除等改变后的蛋白质。根据本申请的实施例，对于N蛋白的功能片段，可以结合RNA分子，保护RNA不受核酸酶的影响，对于P蛋白的功能片段，能够结合N蛋白，在模板上定位L聚合酶，同时也能够作为RNA聚合酶转录和 复制复合体的基本组成部分，进一步L蛋白的功能片段能够发挥RNA聚合酶的功能，与RNA的转录及复制有关。The term "functional fragment" used in this article refers to a part of the full-length sequence of a protein that can still perform functions related to the self-replication of RNA molecules. For example, it can be a truncated version of the full-length sequence or the entire protein sequence. Proteins that have been modified by substitution, mutation, or deletion of long amino acid sequences. According to the embodiments of the present application, the functional fragment of the N protein can be combined with an RNA molecule to protect the RNA from the influence of nucleases. The functional fragment of the P protein can be combined with the N protein to position the L polymerase on the template, while also Able to act as RNA polymerase to transcribe and The basic component of the replication complex, and further the functional fragment of L protein can function as RNA polymerase and is related to the transcription and replication of RNA.
根据本发明的实施例，所述N蛋白、所述P蛋白、所述L蛋白的至少之一分别独立地来自弹状病毒科病毒。According to an embodiment of the present invention, at least one of the N protein, the P protein, and the L protein is independently from a Rhabdoviridae virus.
所述N蛋白、所述P蛋白、所述L蛋白，可以来自水疱性口炎病毒印第安纳株，N蛋白包括但不限于Uniprot ID为：P03521、P11212、Q77E03、Q8B0H4、B7UCZ2的序列；P蛋白包括但不限于Uniprot ID为：P04880、Q8B0H8、P04879、P03520、B7UCZ3的序列；L蛋白包括但不限于Uniprot ID为：Q8B0H0、Q98776、Q8B0I0、Q8B0H5、P03523的序列。The N protein, the P protein, and the L protein can be from the Indiana strain of vesicular stomatitis virus. The N protein includes but is not limited to Uniprot IDs: P03521, P11212, Q77E03, Q8B0H4, and B7UCZ2 sequences; the P protein includes But are not limited to sequences with Uniprot IDs: P04880, Q8B0H8, P04879, P03520, and B7UCZ3; L protein includes but is not limited to sequences with Uniprot IDs: Q8B0H0, Q98776, Q8B0I0, Q8B0H5, and P03523.
所述N蛋白、所述P蛋白、所述L蛋白，可以来自水疱性口炎病毒新泽西株，N蛋白包括但不限于Uniprot ID为：P04881、Q89034、S5TKS4、Q89036、Q89037的序列；P蛋白包括但不限于Uniprot ID为：P04877、Q89057、Q89052、Q89050、Q89049的序列；L蛋白包括但不限于Uniprot ID为：P16379、P16379、I7DDL0、Q8B545、S5TC82的序列。The N protein, the P protein, and the L protein can be from the New Jersey strain of vesicular stomatitis virus. The N protein includes but is not limited to sequences with Uniprot IDs: P04881, Q89034, S5TKS4, Q89036, and Q89037; the P protein includes But are not limited to sequences with Uniprot IDs: P04877, Q89057, Q89052, Q89050, Q89049; L protein includes but is not limited to sequences with Uniprot IDs: P16379, P16379, I7DDL0, Q8B545, S5TC82.
所述N蛋白、所述P蛋白、所述L蛋白，还可以来自其他水泡性病毒属(如钱迪普拉水疱病毒、马拉巴水泡病毒)、狂犬病毒属。The N protein, the P protein, and the L protein can also be from other vesicular virus genera (such as Chandipura vesicular virus, Malabar vesicular virus) and rabies virus.
根据本发明的实施例，所述N蛋白具有SEQ ID NO：1所示氨基酸序列或与其具有至少80％同源性的氨基酸序列，所述P蛋白具有SEQ ID NO：2所示氨基酸序列或与其具有至少80％同源性的氨基酸序列，所述L蛋白具有SEQ ID NO：3所示氨基酸序列或与其具有至少80％同源性的氨基酸序列。

According to an embodiment of the present invention, the N protein has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology with it, and the P protein has the amino acid sequence shown in SEQ ID NO: 2 or has an amino acid sequence with at least 80% homology with it. An amino acid sequence having at least 80% homology, the L protein has an amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology thereto.

根据本申请的实施例，所述第一RNA序列具有如SEQ ID NO：4所示的核苷酸序列，所述第二RNA序列具有如SEQ ID NO：5所示的核苷酸序列，所述第三RNA序列具有如SEQ ID NO：6所示的核苷酸序列。




According to an embodiment of the present application, the first RNA sequence has a nucleotide sequence as shown in SEQ ID NO: 4, and the second RNA sequence has a nucleotide sequence as shown in SEQ ID NO: 5, so The third RNA sequence has the nucleotide sequence shown in SEQ ID NO:6.




根据本发明的实施例，所述递送载体包括蛋白质、脂质体和外泌体的至少之一。According to an embodiment of the present invention, the delivery vehicle includes at least one of proteins, liposomes and exosomes.
蛋白质可以作为递送载体携带核酸分子进入细胞，以实现遗传物质的细胞内递送。Proteins can serve as delivery vehicles to carry nucleic acid molecules into cells to achieve intracellular delivery of genetic material.
脂质体作为纳米级药物递送载体被广泛地应用于药物递送中，可以自组装成病毒大小的颗粒，并能够将mRNA从内质体释放到细胞质。同时，可以起到促进细胞摄取、提高核内体逃逸、保护核酸分子不被TLRs识别、避免先天免疫***的过度激活等作用。如图3所示，LNP递送***主要是由LNP电势及核酸电势决定包裹核酸的量，由此一个LNP递送***可以包裹一个到多个核酸分子。Liposomes are widely used in drug delivery as nanoscale drug delivery carriers, which can self-assemble into virus-sized particles and can release mRNA from endosomes to the cytoplasm. At the same time, it can promote cellular uptake, improve endosomal escape, protect nucleic acid molecules from being recognized by TLRs, and avoid excessive activation of the innate immune system. As shown in Figure 3, the LNP delivery system mainly determines the amount of nucleic acid packaged by the LNP potential and the nucleic acid potential. Therefore, an LNP delivery system can package one to multiple nucleic acid molecules.
外泌体是从活细胞释放到细胞外微环境的50-150nm大小的囊泡，由于外泌体体积小和其本身就是细胞产物，通过外泌体递送药物可以避免巨噬细胞的吞噬作用或降解，还可以在体内长时间循环，保持效果。其中，外泌体能够穿过血脑屏障以将核酸药物递送到中枢神经***是外泌体递送药物的一个显著优势。Exosomes are 50-150nm-sized vesicles released from living cells into the extracellular microenvironment. Due to their small size and the fact that they are cell products themselves, drug delivery through exosomes can avoid phagocytosis or phagocytosis by macrophages. It degrades and can circulate in the body for a long time to maintain the effect. Among them, the ability of exosomes to cross the blood-brain barrier to deliver nucleic acid drugs to the central nervous system is a significant advantage of exosome-delivered drugs.
根据本发明的实施例，所述蛋白质包括非人源蛋白和人源蛋白。本发明对于人源蛋白的类型不作严格限定，只要是能够作为递送载体将核酸分子递送到细胞内即可，包括但不限于SNARE蛋白家族。根据本发明的实施例，所述非人源蛋白包括病毒衣壳蛋白。相比于人源蛋白，病毒衣壳蛋白的递送效果更佳，由于病毒衣壳蛋白不具有病毒遗传物质，易感染进入细胞，靶向细胞特异性强，可以实现蛋白及遗传物质的细胞内递送。并且，病毒 衣壳蛋白容易获得，提高了制备递送载体的效率，降低成本。如图4所示，病毒蛋白递送载体是源自病毒自身的特性，所以一个蛋白质递送载体只能包裹一个核酸分子。According to embodiments of the present invention, the protein includes non-human protein and human protein. The present invention does not strictly limit the type of human protein, as long as it can be used as a delivery vector to deliver nucleic acid molecules into cells, including but not limited to the SNARE protein family. According to an embodiment of the invention, the non-human protein includes a viral capsid protein. Compared with human proteins, the delivery effect of viral capsid proteins is better. Since viral capsid proteins do not contain viral genetic material, they are easy to infect and enter cells. They have strong cell targeting specificity and can achieve intracellular delivery of proteins and genetic materials. . And, virus Capsid proteins are easy to obtain, which improves the efficiency of preparing delivery vectors and reduces costs. As shown in Figure 4, viral protein delivery vectors are derived from the characteristics of the virus itself, so a protein delivery vector can only package one nucleic acid molecule.
除了弹状病毒科的外壳蛋白因其相似的结构可以完成reRNATM的递送外，其他病毒的外壳蛋白也可以用于reRNATM的递送。根据本发明的实施例，所述病毒包括痘类病毒、狂犬病病毒、黄病毒、麻疹病毒、冠状病毒、水疱性口炎病毒、新城疫病毒和淋巴细胞脉络丛脑膜炎病毒的至少之一。上述病毒均具有衣壳蛋白，可以用作递送载体。In addition to the coat proteins of the Rhabdoviridae family that can complete the delivery of reRNA TM due to their similar structures, the coat proteins of other viruses can also be used for the delivery of reRNA TM . According to an embodiment of the present invention, the virus includes at least one of poxvirus, rabies virus, flavivirus, measles virus, coronavirus, vesicular stomatitis virus, Newcastle disease virus and lymphocytic choriomeningitis virus. The above-mentioned viruses all have capsid proteins and can be used as delivery vectors.
根据本发明的实施例，所述蛋白质选自水疱性口炎病毒受体、淋巴细胞性脉络丛脑膜炎病毒外壳蛋白和/或新城疫病毒外壳蛋白。具体地，水疱性口炎病毒受体VSV-G具有如SEQ ID NO：7的氨基酸序列。淋巴细胞性脉络丛脑膜炎病毒外壳蛋白LCMV-GP的具体序列信息参考UniProtKB/Swiss-Prot:P09991；新城疫病毒外壳蛋白分别为NDV-F和NDV-HN，具体序列信息参考UniProtKB/Swiss-Prot:Q9DLD4和UniProtKB/Swiss-Prot:Q91UL0。发明人经过大量实验研究发现，上述三类蛋白质的自复制RNA递送效率高。According to an embodiment of the present invention, the protein is selected from the group consisting of vesicular stomatitis virus receptor, lymphocytic choriomeningitis virus coat protein and/or Newcastle disease virus coat protein. Specifically, the vesicular stomatitis virus receptor VSV-G has an amino acid sequence such as SEQ ID NO: 7. For specific sequence information of lymphocytic choriomeningitis virus coat protein LCMV-GP, please refer to UniProtKB/Swiss-Prot:P09991; Newcastle disease virus coat proteins are NDV-F and NDV-HN respectively. For specific sequence information, please refer to UniProtKB/Swiss-Prot :Q9DLD4 and UniProtKB/Swiss-Prot:Q91UL0. The inventor found through extensive experimental research that the self-replicating RNA delivery efficiency of the above three types of proteins is high.
根据本发明的实施例，所述脂质体上含有靶向元件。由此，可以将携带的核酸分子靶向递送至特定组织或器官。According to an embodiment of the invention, the liposome contains a targeting element. Thus, the carried nucleic acid molecules can be targeted and delivered to specific tissues or organs.
根据本发明的实施例，所述脂质体包括带永久正电荷的阳离子脂质体(如DOTAP、DOTMA)、辅助脂质体(如DSPC、DOPE)、结构脂质体、(如胆固醇、胆固醇脂)、长循环脂质体(如DMG-PEG2000)和可电离的阳离子脂质体(如DLin-MC3-DMA、DLin-KC2-DMA、DLin-DMA、DODMA、DODAP)的至少之一。According to embodiments of the present invention, the liposomes include permanently positively charged cationic liposomes (such as DOTAP, DOTMA), auxiliary liposomes (such as DSPC, DOPE), structural liposomes (such as cholesterol, cholesterol lipid), long-circulating liposomes (such as DMG-PEG2000) and ionizable cationic liposomes (such as DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP).
根据本发明的实施例，所述脂质体选自DOTAP、DSPC、胆固醇和DMG-PEG2000。发明人经过大量实验研究发现，上述脂质体的自复制RNA递送效率高。According to an embodiment of the present invention, the liposome is selected from DOTAP, DSPC, cholesterol and DMG-PEG2000. The inventor found through extensive experimental research that the self-replicating RNA delivery efficiency of the above-mentioned liposomes is high.
药物组合物pharmaceutical composition
在本发明的另一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：前面所述组合物。如前所述，组合物中递送载体可以携带包含自复制RNA分子的核酸分子进入细胞内，自复制RNA分子可以在细胞质内进行自我复制，其搭载的靶分子编码区可以表达小分子、多种肽、酶、抗体、通道蛋白以及受体蛋白等，从而达到不同的预防或治疗目的。In another aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: the aforementioned composition. As mentioned above, the delivery vector in the composition can carry nucleic acid molecules containing self-replicating RNA molecules into cells. Self-replicating RNA molecules can replicate themselves in the cytoplasm, and the target molecule coding regions they carry can express small molecules, various Peptides, enzymes, antibodies, channel proteins and receptor proteins, etc., to achieve different preventive or therapeutic purposes.
根据本发明的实施例，药物组合物进一步包括药学上可接受的辅料。本发明对于辅料的种类不做严格限定，可以根据情况灵活选择。对于注射制剂，药物上可接受的辅料可以包括缓冲剂、增溶剂等，药物组合物可以被制备成例如一次剂量的剂型的安瓿或例如多剂 量容器的单元型剂型。药物组合物还可以被制备成溶液、悬浮液、冻干剂、水剂、针剂和长效制剂。According to embodiments of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients. The present invention does not strictly limit the types of auxiliary materials, and can be flexibly selected according to the situation. For injectable preparations, pharmaceutically acceptable excipients may include buffers, solubilizers, etc., and the pharmaceutical composition may be prepared, for example, in ampoules of a single dose dosage form or, for example, multiple doses. Unit dosage forms in measuring containers. Pharmaceutical compositions can also be prepared into solutions, suspensions, lyophilized agents, liquids, injections and long-acting preparations.
需要说明的是，前面针对组合物所描述的特征和优点，同样适用于该药物组合物，在此不再赘述。It should be noted that the features and advantages described above for the composition are also applicable to the pharmaceutical composition and will not be repeated here.
递送自复制RNA分子的方法Methods of delivering self-replicating RNA molecules
在本发明的又一方面，本发明提出了一种递送自复制RNA分子的方法。根据本发明的实施例，所述方法包括：将自复制RNA分子配制为前面所述的组合物。由此，递送载体可以有效地递送自复制RNA分子进入细胞内。In yet another aspect of the invention, the invention provides a method of delivering self-replicating RNA molecules. According to an embodiment of the invention, the method includes formulating a self-replicating RNA molecule into the composition as described above. Thus, the delivery vector can effectively deliver self-replicating RNA molecules into cells.
根据本发明的实施例，所述方法进一步包括：为待处理对象提供所述组合物。According to an embodiment of the present invention, the method further includes providing the composition to a subject to be treated.
需要说明的是，前面针对组合物所描述的特征和优点，同样适用于该递送自复制RNA分子的方法，在此不再赘述。It should be noted that the features and advantages described above for the composition are also applicable to the method of delivering self-replicating RNA molecules, and will not be described again here.
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The solutions of the present invention will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
实施例1Example 1
水疱性口炎病毒(简称VSV病毒)的受体是低密度脂蛋白(LDLR)，具有广泛的组织分布，发明人筛选出的特定的VSV-G(SEQ ID NO：7)具有较高的亲和力，以此VSV-G蛋白作为递送蛋白，具有很高的细胞递送效率。The receptor of vesicular stomatitis virus (referred to as VSV virus) is low-density lipoprotein (LDLR), which has wide tissue distribution. The specific VSV-G (SEQ ID NO: 7) screened by the inventor has high affinity. , using VSV-G protein as a delivery protein, it has high cell delivery efficiency.
具体实验如下：The specific experiments are as follows:
1、将经培养的HEK 293T细胞用转染试剂Lipo8000TM进行reRNATM质粒(质粒上携带有reRNATM序列，该序列包含mRNA序列、SEQ ID NO：4-6序列、GFP序列)及辅助质粒(辅助质粒包含分别包括SEQ ID NO：4-6序列的单独质粒、VSV-G质粒、T7RNA聚合酶质粒)的转染，转染完毕后，培养细胞，培养箱中培养48小时，收取上清进行纯化，得到reRNATM-GFP种子。1. Use transfection reagent Lipo8000 TM to culture HEK 293T cells to transfect reRNA TM plasmid (the plasmid carries the reRNA TM sequence, which contains the mRNA sequence, SEQ ID NO: 4-6 sequence, and GFP sequence) and the helper plasmid ( The helper plasmid contains a separate plasmid containing SEQ ID NO: 4-6 sequences, a VSV-G plasmid, and a T7 RNA polymerase plasmid). After the transfection is completed, the cells are cultured and cultured in an incubator for 48 hours. The supernatant is collected and carried out. After purification, reRNA TM -GFP seeds were obtained.
2、将经培养的HEK 293细胞用转染试剂Lipo8000TM进行辅助质粒(辅助质粒上携带编码VSV-G的核酸分子)的转染，转染完毕，加入reRNATM-GFP种子，培养箱中培养48小时，收取上清进行纯化，得到VSV-G包裹的reRNATM-GFP。 2. Use the transfection reagent Lipo8000 TM to transfect the cultured HEK 293 cells with the helper plasmid (the helper plasmid carries the nucleic acid molecule encoding VSV-G). After the transfection is completed, add reRNA TM -GFP seeds and culture in the incubator. After 48 hours, the supernatant was collected and purified to obtain VSV-G wrapped reRNA TM -GFP.
在6孔板上铺2×106个293细胞，使用含1％FBS的培养基培养细胞，分别将VSV-G包裹的reRNATM-GFP以1.4pg、14pg和140pg的添加量加入到2ml细胞液中，16小时后对培养的细胞进行流式检测。Spread 2 × 10 6 293 cells on a 6-well plate, culture the cells in culture medium containing 1% FBS, and add VSV-G-coated reRNA TM -GFP to 2 ml cells at 1.4pg, 14pg and 140pg. solution, and flow cytometry was performed on the cultured cells 16 hours later.
结果如图5所示，reRNATM-GFP添加量为1.4pg时，16小时细胞的阳性率可达96.5％；reRNATM-GFP添加量为14pg和140pg时，16小时细胞的阳性率可达98.2％。培养时间延长，可以一直维持这种表达，可见VSV-G蛋白具有很高的递送效率。
The results are shown in Figure 5. When reRNA TM -GFP was added at 1.4pg, the positive rate of cells at 16 hours could reach 96.5%; when reRNA TM -GFP was added at 14pg and 140pg, the positive rate of cells at 16 hours could reach 98.2 %. This expression can be maintained with prolonged culture time, indicating that VSV-G protein has a high delivery efficiency.
实施例2Example 2
淋巴细胞性脉络丛脑膜炎病毒(简称LCMV病毒)的外壳蛋白为LCMV-GP，其受体为α-肌营养不良蛋白，是一种普遍存在的蛋白质，也是reRNATM递送蛋白的优质备选。制备LCMV-GP作为递送蛋白的reRNATM-GFP产品，也可完成reRNATM的递送，具体实验如下：The coat protein of lymphocytic choriomeningitis virus (LCMV virus for short) is LCMV-GP, and its receptor is α-dystrophin, which is a ubiquitous protein and a high-quality candidate for reRNA TM delivery protein. Preparing LCMV-GP as a reRNA TM -GFP product for delivery protein can also complete the delivery of reRNA TM . The specific experiments are as follows:
1、LCMV-GP蛋白包裹reRNATM-GFP的步骤参考实施例1。1. Refer to Example 1 for the steps of wrapping reRNA TM -GFP with LCMV-GP protein.
2、在6孔板上铺2×106个vero细胞，使用含1％FBS的培养基培养细胞，加入以LCMV-GP蛋白为递送蛋白的reRNATM-GFP(同实施例1)，观察vreo形态及GFP荧光。2. Spread 2×10 6 vero cells on a 6-well plate, culture the cells in a medium containing 1% FBS, add reRNA TM -GFP with LCMV-GP protein as the delivery protein (same as Example 1), and observe vreo Morphology and GFP fluorescence.
结果如图6所示，可以看出，reRNATM-GFP被有效地递送至vero细胞中。The results are shown in Figure 6. It can be seen that reRNA ™ -GFP was effectively delivered into vero cells.
实施例3Example 3
新城疫病毒(简称NDV病毒)也是一种有囊膜的病毒，其外壳蛋白有两个，分别为NDV-F和NDV-HN，两个蛋白共同完成入胞及溶酶体逃逸，实现RNA的递送，将两个蛋白同时作为递送蛋白，也可完成reRNATM-GFP的递送，具体实验如下：Newcastle disease virus (NDV virus for short) is also an enveloped virus. It has two coat proteins, namely NDV-F and NDV-HN. The two proteins work together to complete cell entry and lysosome escape, and realize RNA detoxification. Delivery, using both proteins as delivery proteins at the same time, can also complete the delivery of reRNA TM -GFP. The specific experiments are as follows:
1、LCMV-GP蛋白包裹reRNATM-GFP的步骤参考实施例1。 1. Refer to Example 1 for the steps of wrapping reRNA TM -GFP with LCMV-GP protein.
2、在6孔板上铺2×106个vero细胞，使用含1％FBS的培养基培养细胞，加入以NDV-F蛋白和NDV-HN蛋白为递送蛋白的reRNATM-GFP(同实施例1)，观察vero形态及GFP荧光。2. Spread 2×10 6 vero cells on a 6-well plate, culture the cells in a medium containing 1% FBS, and add reRNA TM -GFP with NDV-F protein and NDV-HN protein as delivery proteins (same as in the Example 1), observe vero morphology and GFP fluorescence.
如图7所示，以新城疫病毒F蛋白和HN蛋白为递送蛋白，可以实现reRNATM-GFP的有效递送。As shown in Figure 7, using Newcastle disease virus F protein and HN protein as delivery proteins, effective delivery of reRNA TM -GFP can be achieved.
实施例4Example 4
裸露的reRNATM-GFP不能进入胞内，以LNP作为递送载体可以有效地实现递送reRNATM。具体实验如下：Naked reRNA TM -GFP cannot enter the cell, and using LNP as a delivery carrier can effectively deliver reRNA TM . The specific experiments are as follows:
1、将LNP溶液(DOTAP：DSPC：胆固醇：DMG-PEG2000＝44:16:37.5:2.5v/v)与reRNATM-GFP种子(制备方法参考实施例1)通过微流控芯片进行混合。混合完成后混入PBS缓冲液中稀释3倍，浓缩纯化，过滤除菌，得到LNP包裹的reRNATM-GFP。1. Mix the LNP solution (DOTAP:DSPC:cholesterol:DMG-PEG2000=44:16:37.5:2.5v/v) and reRNA TM -GFP seeds (refer to Example 1 for the preparation method) through a microfluidic chip. After the mixing is completed, mix it into PBS buffer and dilute it 3 times, concentrate and purify, and filter and sterilize to obtain LNP-coated reRNA TM -GFP.
2、在6孔板上铺2×106个vero细胞，使用含1％FBS的培养基培养细胞，加入LNP包裹的reRNATM-GFP，观察vero形态及GFP荧光。2. Plate 2×10 6 vero cells on a 6-well plate, culture the cells in a medium containing 1% FBS, add LNP-coated reRNA TM -GFP, and observe the vero morphology and GFP fluorescence.
表1为LNP包裹前后reRNATM-GFP粒径及电势的变化，从结果可以看出LNP包裹之后的电势在+20mV左右，粒径在150nm左右，比较利于RNA的递送。Table 1 shows the changes in particle size and potential of reRNA TM -GFP before and after LNP encapsulation. From the results, it can be seen that the potential after LNP encapsulation is around +20mV and the particle size is around 150nm, which is more conducive to the delivery of RNA.
表1 LNP包裹前后reRNATM-GFP电势及粒径的变化
Table 1 Changes in potential and particle size of reRNA TM -GFP before and after LNP encapsulation
包裹后的reRNATM-GFP可以完成在vero细胞内的RNA递送，图8为LNP包裹reRNATM-GFP递送入vero细胞24小时之后的GFP表达情况，reRNATM-GFP量为18ng，vero细胞数量为2×105个。可以看出，脂质体可以实现reRNATM-GFP的有效递送。The packaged reRNA TM -GFP can complete RNA delivery in vero cells. Figure 8 shows the expression of GFP after LNP packaged reRNA TM -GFP was delivered into vero cells for 24 hours. The amount of reRNA TM -GFP is 18ng, and the number of vero cells is 2×10 5 pcs. It can be seen that liposomes can achieve effective delivery of reRNA ™ -GFP.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。 In the description of this specification, reference to the terms "one embodiment,""someembodiments,""anexample,""specificexamples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。 Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.

Claims (13)

  1. 一种组合物，其特征在于，包括：A composition, characterized in that it includes:
    核酸分子，所述核酸分子包括自复制RNA分子；Nucleic acid molecules, including self-replicating RNA molecules;
    递送载体，所述递送载体携带所述核酸分子。A delivery vector carrying the nucleic acid molecule.
  2. 根据权利要求1所述的组合物，其特征在于，所述自复制RNA分子包括：The composition of claim 1, wherein the self-replicating RNA molecule includes:
    第一RNA序列，所述第一RNA序列编码N蛋白或其功能片段；A first RNA sequence encoding N protein or a functional fragment thereof;
    第二RNA序列，所述第二RNA序列编码P蛋白或其功能片段；a second RNA sequence encoding P protein or a functional fragment thereof;
    第三RNA序列，所述第三RNA序列编码L蛋白或其功能片段；和a third RNA sequence encoding the L protein or a functional fragment thereof; and
    靶分子编码区，所述靶分子编码区编码至少一个靶分子。A target molecule coding region encoding at least one target molecule.
  3. 根据权利要求2所述的组合物，其特征在于，所述N蛋白、所述P蛋白、所述L蛋白的至少之一分别独立地来自弹状病毒科病毒。The composition according to claim 2, characterized in that at least one of the N protein, the P protein, and the L protein are each independently from a Rhabdoviridae virus.
  4. 根据权利要求2所述的组合物，其特征在于，所述N蛋白具有SEQ ID NO：1所示氨基酸序列或与其具有至少80％同源性的氨基酸序列，所述P蛋白具有SEQ ID NO：2所示氨基酸序列或与其具有至少80％同源性的氨基酸序列，所述L蛋白具有SEQ ID NO：3所示氨基酸序列或与其具有至少80％同源性的氨基酸序列。The composition according to claim 2, wherein the N protein has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology with it, and the P protein has SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology thereto, the L protein has an amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology thereto.
  5. 根据权利要求1所述的组合物，其特征在于，所述递送载体包括蛋白质、脂质体和外泌体的至少之一。The composition according to claim 1, wherein the delivery vehicle includes at least one of proteins, liposomes and exosomes.
  6. 根据权利要求5所述的组合物，其特征在于，所述蛋白质包括非人源蛋白和人源蛋白；The composition according to claim 5, wherein the protein includes non-human protein and human protein;
    所述非人源蛋白包括病毒衣壳蛋白；The non-human protein includes viral capsid protein;
    所述人源蛋白包括SNARE蛋白家族。The human proteins include the SNARE protein family.
  7. 根据权利要求6所述的组合物，其特征在于，所述病毒包括痘类病毒、狂犬病病毒、黄病毒、麻疹病毒、冠状病毒、水疱性口炎病毒、新城疫病毒和淋巴细胞脉络丛脑膜炎病毒的至少之一。The composition of claim 6, wherein the viruses include poxviruses, rabies viruses, flaviviruses, measles viruses, coronaviruses, vesicular stomatitis virus, Newcastle disease virus and lymphocytic choriomeningitis At least one of the viruses.
  8. 根据权利要求5所述的组合物，其特征在于，所述蛋白质选自水疱性口炎病毒受体、淋巴细胞性脉络丛脑膜炎病毒外壳蛋白和/或新城疫病毒外壳蛋白。The composition according to claim 5, wherein the protein is selected from the group consisting of vesicular stomatitis virus receptor, lymphocytic choriomeningitis virus coat protein and/or Newcastle disease virus coat protein.
  9. 根据权利要求5所述的组合物，其特征在于，所述脂质体包括带永久正电荷的阳离子脂质体、辅助脂质体、结构脂质体、长循环脂质体和可电离的阳离子脂质体的至少之一。The composition according to claim 5, wherein the liposomes comprise permanently positively charged cationic liposomes, auxiliary liposomes, structured liposomes, long circulating liposomes and ionizable cationic liposomes. At least one of the liposomes.
  10. 根据权利要求5所述的组合物，其特征在于，所述脂质体选自DOTAP、DSPC、胆固醇和DMG-PEG2000。 The composition of claim 5, wherein the liposome is selected from the group consisting of DOTAP, DSPC, cholesterol and DMG-PEG2000.
  11. 一种药物组合物，其特征在于，包括：权利要求1～10任一项所述的组合物。A pharmaceutical composition, characterized by comprising: the composition according to any one of claims 1 to 10.
  12. 一种递送自复制RNA分子的方法，其特征在于，包括：A method of delivering self-replicating RNA molecules, characterized by comprising:
    将自复制RNA分子配制为权利要求1～10任一项所述的组合物。The self-replicating RNA molecule is formulated into the composition of any one of claims 1-10.
  13. 根据权利要求12所述的方法，其特征在于，所述方法进一步包括：为待处理对象提供所述组合物。 The method of claim 12, further comprising providing the composition to a subject to be treated.
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