WO2023221938A1 - Protein-encapsulated self-replicating rna and preparation method therefor - Google Patents
Protein-encapsulated self-replicating rna and preparation method therefor Download PDFInfo
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- WO2023221938A1 WO2023221938A1 PCT/CN2023/094280 CN2023094280W WO2023221938A1 WO 2023221938 A1 WO2023221938 A1 WO 2023221938A1 CN 2023094280 W CN2023094280 W CN 2023094280W WO 2023221938 A1 WO2023221938 A1 WO 2023221938A1
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Definitions
- the present invention relates to the biological field. Specifically, the present invention relates to protein-coated self-replicating RNA and its preparation method.
- 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.
- the invention provides a recombinant cell.
- the recombinant cell includes: a first nucleic acid molecule including a self-replicating RNA molecule; and a second nucleic acid molecule encoding a delivery carrier protein.
- the self-replicating RNA molecule enters the cell and exists stably in the cell, it is carried on a delivery carrier, and the delivery carrier can carry the self-replicating RNA molecule into the cell.
- the delivery carrier protein encoded and expressed by the second nucleic acid molecule can self-assemble in the cell to package the self-replicating RNA molecules, the delivery carriers wrapped with self-replicating RNA molecules produced by this cell line can achieve in vivo delivery of self-replicating RNA molecules, which helps to achieve intracellular RNA self-replication, maintain long-lasting high protein levels, and function better. Effect.
- the invention provides a method for preparing the aforementioned recombinant cells.
- the method includes: causing the cell to carry a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule being as defined in the recombinant cell as described above.
- the invention provides a method for preparing a self-replicating RNA molecule composition.
- the self-replicating RNA molecule includes self-replicating RNA and a delivery carrier protein, and the method includes: cultivating the aforementioned recombinant cells; and obtaining the self-replicating RNA molecule composition.
- 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 the production of reRNA TM using a helper plasmid as a source of coat protein according to one embodiment of the present invention
- Figure 4 shows a picture of reRNA TM -GFP production cells using the VSV-G auxiliary plasmid as an auxiliary material according to one embodiment of the present invention
- Figure 5 shows a picture of reRNA TM -GFP production cells using LCMV-GP auxiliary plasmid as an auxiliary material according to one embodiment of the present invention
- Figure 6 shows a schematic diagram of the production of reRNA TM in coat protein stably transduced cell lines according to one embodiment of the present invention
- Figure 7 shows a picture of the 293 cell line and the VSV-G stably transduced 293 cell line reRNA TM -GFP production cells 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.
- self-replicating RNA The molecule will carry a sequence that expresses RNA polymerase (RNA-dependent RNA polymerase), which can use the self-replicating RNA molecule as a template to produce more after the RNA molecule is translated in the cytoplasm to produce RNA polymerase.
- RNA-dependent RNA polymerase RNA-dependent RNA polymerase
- the invention provides a recombinant cell.
- the recombinant cell includes: a first nucleic acid molecule including a self-replicating RNA molecule; and a second nucleic acid molecule encoding a delivery carrier protein.
- the self-replicating RNA molecule enters the cell and exists stably in the cell, it is carried on a delivery carrier, and the delivery carrier can carry the self-replicating RNA molecule into the cell.
- the delivery carrier protein encoded and expressed by the second nucleic acid molecule can self-assemble in the cell to package the self-replicating RNA molecules, the delivery carriers wrapped with self-replicating RNA molecules produced by this cell line can achieve in vivo delivery of self-replicating RNA molecules, which helps to achieve intracellular RNA self-replication, maintain long-lasting high protein levels, and function better. Effect.
- 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 molecules proposed in the present invention will be named is 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.
- 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 It can serve as a basic component of the RNA polymerase transcription and replication complex. Further, the functional fragment of L protein can exert the function of 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 self-replicating core sequence includes:
- RNA sequence encoding P protein or a functional fragment thereof, the second RNA sequence having a nucleotide sequence as shown in SEQ ID NO: 5;
- a third RNA sequence encoding L protein or a functional fragment thereof, and the third RNA sequence has a nucleotide sequence as shown in SEQ ID NO: 6.
- 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 human proteins including 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. . Moreover, viral capsid proteins are easy to obtain, which improves the efficiency of preparing delivery vectors and reduces costs.
- 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 that can self-assemble in cells to wrap the first nucleic acid molecule and obtain a delivery vector carrying the first nucleic acid molecule.
- the delivery carrier protein is selected from the group consisting of vesicular stomatitis virus receptor and/or lymphocytic choriomeningitis virus coat protein.
- the vesicular stomatitis virus receptor VSV-G has an amino acid sequence such as SEQ ID NO: 7, and the specific sequence information of the lymphocytic choriomeningitis virus coat protein LCMV-GP is referred to UniProtKB/Swiss-Prot: P09991.
- the inventor found through extensive experimental research that the self-replicating RNA delivery efficiency of the above two types of proteins is high.
- the first nucleic acid molecule and the second nucleic acid molecule exist independently in the recombinant cell in a free form or in a form integrated into the genome.
- the first nucleic acid molecule and the second nucleic acid molecule independently exist in the recombinant cell in the form of plasmids.
- the first nucleic acid molecule and the second nucleic acid molecule are present on the same plasmid.
- the first nucleic acid molecule and the second nucleic acid molecule can exist in the recombinant cell in three ways.
- the first way is that both exist in a free form, for example, they are constructed on a plasmid and transferred into the recombinant cell, in the form of a free plasmid. Form exists.
- the first nucleic acid molecule and the second nucleic acid molecule can be constructed on the same plasmid, or they can be constructed on two different plasmids. When constructed on the same plasmid, they can be expressed separately under the same or different promoters. .
- the second way is that both of them exist in the cell in the form of integration into the cell genome, thereby achieving the purpose of long-term stable expression of the first nucleic acid molecule and the second nucleic acid molecule.
- the third way is that one of the two nucleic acid molecules exists in a free form and the other exists in an integrated genome form.
- the first nucleic acid molecule exists in a free form and the second nucleic acid molecule exists in an integrated genome form, so that Can express proteins efficiently.
- the invention provides a method for preparing recombinant cells.
- the method includes: causing the cell to carry a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule being as defined in the recombinant cell as described above.
- the second nucleic acid molecule can express the delivery carrier protein and self-assemble, thus wrapping the first nucleic acid molecule to obtain a composition containing the delivery carrier protein and the first nucleic acid molecule it carries, which helps to deliver the third nucleic acid molecule.
- the purpose of a nucleic acid molecule is not limited to carry a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule being as defined in the recombinant cell as described above.
- the second nucleic acid molecule can express the delivery carrier protein and self-assemble, thus wrapping the first nucleic acid molecule to obtain a composition
- the method includes introducing at least one of the first nucleic acid molecule and the second nucleic acid molecule into the cell in the form of a plasmid.
- plasmids can exist in cells in free form or to integrate into the cell's genome.
- the introduction is performed by transfecting the plasmid.
- the transfection methods include calcium phosphate transfection, lipofectamine transfection or electroporation transfection.
- plasmids can be quickly transferred into cells.
- the second nucleic acid molecule is provided in the form of lentivirus or CRISPR/Cas9. This facilitates the integration of the second nucleic acid molecule into the genome of the cell and achieves the purpose of long-term stable expression of the delivery vector protein.
- the cells are selected from HEK 293 cell lines, Vero cell lines, CHO-K1 cells or NIH-3T3 cells.
- a stably transduced cell line can be constructed to achieve stable expression.
- the invention provides a method for preparing a self-replicating RNA molecule composition.
- the self-replicating RNA molecule includes self-replicating RNA and a delivery carrier protein, and the method includes: cultivating the aforementioned recombinant cells; and obtaining the self-replicating RNA molecule composition.
- the recombinant cells contain a first nucleic acid molecule including a self-replicating RNA molecule and a second nucleic acid molecule encoding a delivery carrier protein.
- the second nucleic acid molecule expresses the delivery carrier protein, and the delivery carrier protein self-assembles. Wrapping the first nucleic acid molecule to obtain a composition containing a delivery carrier protein and the first nucleic acid molecule it carries helps achieve the purpose of delivering the first nucleic acid molecule.
- the present invention does not strictly limit the method of obtaining the self-replicating RNA molecule composition from the recombinant cells, as long as the separation purpose can be achieved, and the specific method can be flexibly selected according to actual needs. For example, centrifugal separation of the recombinant cells in the culture medium obtained, disrupting the recombinant cells, etc.
- the reRNA TM protein delivery system requires excipients to be added to the production system during production.
- the production model is shown in Figure 3.
- ReRNA TM and plasmids are used as raw materials and put into cells to produce reRNA TM drug stock solution.
- Vesicular stomatitis virus vesicular stomatitis virus
- the receptor of VSV virus is low-density lipoprotein (LDLR), which has wide tissue distribution.
- LDLR low-density lipoprotein
- the specific VSV-G (SEQ ID NO: 7) screened by the inventor has higher affinity, so VSV-G protein as a delivery protein.
- 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, a VSV-G plasmid, and a T7 RNA polymerase plasmid each including the SEQ ID NO: 4-6 sequence. The amino acid sequence of VSV-G is as shown in SEQ ID NO: 7). After the transfection is completed , culture the cells in an incubator for 24-48 hours, collect the supernatant for purification, and obtain reRNA TM -GFP seeds.
- 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 -GFP2 seeds and culture in the incubator. After 48 hours, the supernatant was collected and purified to obtain VSV-G wrapped reRNA TM -GFP.
- 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 virus lymphocytic choriomeningitis virus
- ⁇ -dystrophin a ubiquitous protein and a high-quality candidate for reRNA TM delivery protein.
- reRNA TM -GFP and LCMV-GP plasmids were used as raw materials and put into cells to produce reRNA TM drug stock solution.
- coat protein-coated reRNA TM drugs are prepared using coat protein stably transduced cell lines. The specific steps are as follows:
- resuscitate the cells add reRNA TM -GFP seeds after culture (refer to Example 1 for the preparation method), continue culturing for 48 hours after feeding, and collect the supernatant as the harvest liquid for purification.
- the non-stably transduced cell line is HEK 293 cells that do not contain VSV-G protein. Only reRNA TM -GFP seeds are fed into the cells. After 48 hours, only a few cells have fluorescent expression of GFP ( Figure 7). The production capacity is extremely low. However, because the stably transduced cell line contains VSV-G protein, it can effectively deliver reRNA TM -GFP into the cells. The proportion of cells that can be used as production cells is greatly increased, almost 100%, and the production capacity is also considerable (5.94 ⁇ g /ml).
- references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” 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
The present invention provides a protein-encapsulated self-replicating RNA and a preparation method therefor. The protein-encapsulated self-replicating RNA is obtained by culturing recombinant cells. A recombinant cell comprises: a first nucleic acid molecule, the first nucleic acid molecule comprising a self-replicating RNA molecule; and a second nucleic acid molecule, the second nucleic acid molecule encoding a delivery vector protein.
Description
本发明涉及生物领域，具体地，本发明涉及蛋白包裹自复制RNA及其制备方法。The present invention relates to the biological field. Specifically, the present invention relates to protein-coated self-replicating RNA and its preparation method.
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，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞包括：第一核酸分子，所述第一核酸分子包括自复制RNA分子；第二核酸分子，所述第二核酸分子编码递送载体蛋白。In one aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes: a first nucleic acid molecule including a self-replicating RNA molecule; and a second nucleic acid molecule encoding a delivery carrier protein.
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。In order to realize that the self-replicating RNA molecule enters the cell and exists stably in the cell, it is carried on a delivery carrier, and the delivery carrier can carry the self-replicating RNA molecule into the cell.
通过将包括自复制RNA分子的第一核酸分子和编码递送载体蛋白的第二核酸分子以重组细胞的形式提供，由第二核酸分子编码表达的递送载体蛋白能够在细胞中自组装以包裹自复制RNA分子，以此细胞株生产的包裹自复制RNA分子的递送载体可以实现自复制RNA分子的体内递送，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。By providing a first nucleic acid molecule including a self-replicating RNA molecule and a second nucleic acid molecule encoding a delivery carrier protein in the form of a recombinant cell, the delivery carrier protein encoded and expressed by the second nucleic acid molecule can self-assemble in the cell to package the self-replicating RNA molecules, the delivery carriers wrapped with self-replicating RNA molecules produced by this cell line can achieve in vivo delivery of self-replicating RNA molecules, which helps to achieve intracellular RNA self-replication, maintain long-lasting high protein levels, and function better. Effect.
在本发明的另一方面，本发明提出了一种制备前面所述重组细胞的方法。根据本发明的实施例，所述方法包括：使细胞携带第一核酸分子和第二核酸分子，所述第一核酸分子和第二核酸分子是如前面所述重组细胞中所限定的。In another aspect of the invention, the invention provides a method for preparing the aforementioned recombinant cells. According to an embodiment of the present invention, the method includes: causing the cell to carry a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule being as defined in the recombinant cell as described above.
在本发明的又一方面，本发明提出了一种制备自复制RNA分子组合物的方法。根据本
发明的实施例，所述自复制RNA分子包括自复制RNA和递送载体蛋白，所述方法包括：培养前面所述的重组细胞；和获取所述自复制RNA分子组合物。In yet another aspect of the invention, the invention provides a method for preparing a self-replicating RNA molecule composition. According to this In an embodiment of the invention, the self-replicating RNA molecule includes self-replicating RNA and a delivery carrier protein, and the method includes: cultivating the aforementioned recombinant cells; and obtaining the self-replicating RNA molecule composition.
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.
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辅助质粒作为外壳蛋白来源的reRNATM生产示意图；Figure 3 shows a schematic diagram of the production of reRNA TM using a helper plasmid as a source of coat protein according to one embodiment of the present invention;
图4显示了根据本发明一个实施例的VSV-G辅助质粒作为辅料reRNATM-GFP生产细胞图片；Figure 4 shows a picture of reRNA TM -GFP production cells using the VSV-G auxiliary plasmid as an auxiliary material according to one embodiment of the present invention;
图5显示了根据本发明一个实施例的LCMV-GP辅助质粒作为辅料reRNATM-GFP生产细胞图片；Figure 5 shows a picture of reRNA TM -GFP production cells using LCMV-GP auxiliary plasmid as an auxiliary material according to one embodiment of the present invention;
图6显示了根据本发明一个实施例的外壳蛋白稳转细胞株reRNATM生产示意图；Figure 6 shows a schematic diagram of the production of reRNA TM in coat protein stably transduced cell lines according to one embodiment of the present invention;
图7显示了根据本发明一个实施例的293细胞株和VSV-G稳转293细胞株reRNATM-GFP生产细胞图片。Figure 7 shows a picture of the 293 cell line and the VSV-G stably transduced 293 cell line reRNA TM -GFP production cells according to one embodiment of the present invention.
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聚合酶产生后，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, self-replicating RNA The molecule will carry a sequence that expresses RNA polymerase (RNA-dependent RNA polymerase), which can use the self-replicating RNA molecule as a template to produce more after the RNA molecule is translated in the cytoplasm to produce RNA polymerase. Self-replicating RNA molecules.
重组细胞recombinant cells
在本发明的一个方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞包括：第一核酸分子，所述第一核酸分子包括自复制RNA分子；第二核酸分子，所述第二核酸分子编码递送载体蛋白。In one aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes: a first nucleic acid molecule including a self-replicating RNA molecule; and a second nucleic acid molecule encoding a delivery carrier protein.
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。In order to realize that the self-replicating RNA molecule enters the cell and exists stably in the cell, it is carried on a delivery carrier, and the delivery carrier can carry the self-replicating RNA molecule into the cell.
通过将包括自复制RNA分子的第一核酸分子和编码递送载体蛋白的第二核酸分子以重组细胞的形式提供，由第二核酸分子编码表达的递送载体蛋白能够在细胞中自组装以包裹自复制RNA分子，以此细胞株生产的包裹自复制RNA分子的递送载体可以实现自复制RNA分子的体内递送，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。By providing a first nucleic acid molecule including a self-replicating RNA molecule and a second nucleic acid molecule encoding a delivery carrier protein in the form of a recombinant cell, the delivery carrier protein encoded and expressed by the second nucleic acid molecule can self-assemble in the cell to package the self-replicating RNA molecules, the delivery carriers wrapped with self-replicating RNA molecules produced by this cell line can achieve in vivo delivery of self-replicating RNA molecules, which helps to achieve intracellular RNA self-replication, maintain long-lasting high protein levels, and function better. Effect.
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 molecules proposed in the present invention will be named is reRNA ™ .
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 It can serve as a basic component of the RNA polymerase transcription and replication complex. Further, the functional fragment of L protein can exert the function of 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.
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，所述自复制核心序列包括：According to an embodiment of the present application, the self-replicating core sequence includes:
第一RNA序列，所述第一RNA序列编码N蛋白或其功能片段，所述第一RNA序列具有如SEQ ID NO：4所示的核苷酸序列；A first RNA sequence encoding N protein or a functional fragment thereof, the first RNA sequence having a nucleotide sequence as shown in SEQ ID NO: 4;
第二RNA序列，所述第二RNA序列编码P蛋白或其功能片段，所述第二RNA序列具有如SEQ ID NO：5所示的核苷酸序列；a second RNA sequence encoding P protein or a functional fragment thereof, the second RNA sequence having a nucleotide sequence as shown in SEQ ID NO: 5;
第三RNA序列，所述第三RNA序列编码L蛋白或其功能片段，所述第三RNA序列具有如SEQ ID NO：6所示的核苷酸序列。
A third RNA sequence encoding L protein or a functional fragment thereof, and the third RNA sequence has a nucleotide sequence as shown in SEQ ID NO: 6.
A third RNA sequence encoding L protein or a functional fragment thereof, and the third RNA sequence has a nucleotide sequence as shown in SEQ ID NO: 6.
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，包括但不限于人源蛋白包括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。
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 human proteins including 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. . Moreover, viral capsid proteins are easy to obtain, which improves the efficiency of preparing delivery vectors and reduces costs.
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、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，可以在细胞内发生自组装，从而包裹第一核酸分子，获得携带第一核酸分子的递送载体。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 that can self-assemble in cells to wrap the first nucleic acid molecule and obtain a delivery vector carrying the first nucleic acid molecule.
根据本发明的实施例，所述递送载体蛋白选自水疱性口炎病毒受体和/或淋巴细胞性脉络丛脑膜炎病毒外壳蛋白。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具体序列信息参考UniProtKB/Swiss-Prot：P09991。发明人经过大量实验研究发现，上述两类蛋白质的自复制RNA递送效率高。According to an embodiment of the present invention, the delivery carrier protein is selected from the group consisting of vesicular stomatitis virus receptor and/or lymphocytic choriomeningitis virus coat protein. Specifically, the vesicular stomatitis virus receptor VSV-G has an amino acid sequence such as SEQ ID NO: 7, and the specific sequence information of the lymphocytic choriomeningitis virus coat protein LCMV-GP is referred to UniProtKB/Swiss-Prot: P09991. The inventor found through extensive experimental research that the self-replicating RNA delivery efficiency of the above two types of proteins is high.
根据本发明的实施例，所述第一核酸分子和所述第二核酸分子分别独立地以游离形式或者整合至基因组的形式存在于所述重组细胞中。According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule exist independently in the recombinant cell in a free form or in a form integrated into the genome.
根据本发明的实施例，所述第一核酸分子和所述第二核酸分子分别独立地以质粒的形式存在于所述重组细胞中。According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule independently exist in the recombinant cell in the form of plasmids.
根据本发明的实施例，所述第一核酸分子和所述第二核酸分子存在于同一质粒上。According to an embodiment of the invention, the first nucleic acid molecule and the second nucleic acid molecule are present on the same plasmid.
第一核酸分子和第二核酸分子可以以三种方式存在于重组细胞中，第一种方式是两者均以游离形式存在，例如是构建在质粒上并转入重组细胞中，以游离质粒的形式存在。具体地，第一核酸分子和第二核酸分子既可以构建在同一质粒上，也可以分别构建在两种不同的质粒上，当构建于同一质粒上，可以在相同或者不同的启动子下分别表达。第二种方式是两者均以整合于细胞基因组的形式存在于细胞中，由此，可以实现长期稳定表达第一核酸分子和第二核酸分子的目的。第三种方式是两种核酸分子中的一种以游离形式存在，另一种以整合基因组的形式存在，例如，第一核酸分子以游离形式存在，第二核酸分子以整合基因组形式存在，从而可以高效表达蛋白。The first nucleic acid molecule and the second nucleic acid molecule can exist in the recombinant cell in three ways. The first way is that both exist in a free form, for example, they are constructed on a plasmid and transferred into the recombinant cell, in the form of a free plasmid. Form exists. Specifically, the first nucleic acid molecule and the second nucleic acid molecule can be constructed on the same plasmid, or they can be constructed on two different plasmids. When constructed on the same plasmid, they can be expressed separately under the same or different promoters. . The second way is that both of them exist in the cell in the form of integration into the cell genome, thereby achieving the purpose of long-term stable expression of the first nucleic acid molecule and the second nucleic acid molecule. The third way is that one of the two nucleic acid molecules exists in a free form and the other exists in an integrated genome form. For example, the first nucleic acid molecule exists in a free form and the second nucleic acid molecule exists in an integrated genome form, so that Can express proteins efficiently.
制备重组细胞的方法Methods for preparing recombinant cells
在本发明的另一方面，本发明提出了一种制备重组细胞的方法。根据本发明的实施例，所述方法包括：使细胞携带第一核酸分子和第二核酸分子，所述第一核酸分子和第二核酸分子是如前面所述重组细胞中所限定的。在细胞内，第二核酸分子可以表达递送载体蛋白，并发生自组装，从而包裹第一核酸分子，获得含有递送载体蛋白及其所携带的第一核酸分子的组合物，有助于实现递送第一核酸分子的目的。In another aspect of the invention, the invention provides a method for preparing recombinant cells. According to an embodiment of the present invention, the method includes: causing the cell to carry a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule being as defined in the recombinant cell as described above. In the cell, the second nucleic acid molecule can express the delivery carrier protein and self-assemble, thus wrapping the first nucleic acid molecule to obtain a composition containing the delivery carrier protein and the first nucleic acid molecule it carries, which helps to deliver the third nucleic acid molecule. The purpose of a nucleic acid molecule.
根据本发明的实施例，所述方法包括：将所述第一核酸分子和所述第二核酸分子的至少之一以质粒形式引入至所述细胞中。由此，质粒既可以以游离形式存在于细胞中，又可
以整合到细胞的基因组上。According to an embodiment of the invention, the method includes introducing at least one of the first nucleic acid molecule and the second nucleic acid molecule into the cell in the form of a plasmid. Thus, plasmids can exist in cells in free form or to integrate into the cell's genome.
根据本发明的实施例，所述引入是通过将所述质粒采用转染方式进行的。具体地，转染方式包括磷酸钙转染、脂质体转染或电穿孔转染。由此，可以快速将质粒转入到细胞中。According to an embodiment of the present invention, the introduction is performed by transfecting the plasmid. Specifically, the transfection methods include calcium phosphate transfection, lipofectamine transfection or electroporation transfection. As a result, plasmids can be quickly transferred into cells.
根据本发明的实施例，所述第二核酸分子是以慢病毒或CRISPR/Cas9的形式提供。由此，以便于使第二核酸分子整合到细胞的基因组上，实现长期稳定表达递送载体蛋白的目的。According to an embodiment of the present invention, the second nucleic acid molecule is provided in the form of lentivirus or CRISPR/Cas9. This facilitates the integration of the second nucleic acid molecule into the genome of the cell and achieves the purpose of long-term stable expression of the delivery vector protein.
根据本发明的实施例，所述细胞选自HEK 293细胞系、Vero细胞系、CHO-K1细胞或NIH-3T3细胞。由此，可以构建稳转细胞株，实现稳定表达的目的。According to embodiments of the present invention, the cells are selected from HEK 293 cell lines, Vero cell lines, CHO-K1 cells or NIH-3T3 cells. Thus, a stably transduced cell line can be constructed to achieve stable expression.
本领域技术人员能够理解的是，前面针对重组细胞所描述的特征和优点，同样适用于该制备重组细胞的方法，在此不再赘述。Those skilled in the art can understand that the characteristics and advantages described above for recombinant cells are also applicable to the method of preparing recombinant cells, and will not be described again here.
制备自复制RNA分子组合物的方法Methods for preparing self-replicating RNA molecule compositions
在本发明的又一方面，本发明提出了一种制备自复制RNA分子组合物的方法。根据本发明的实施例，所述自复制RNA分子包括自复制RNA和递送载体蛋白，所述方法包括：培养前面所述的重组细胞；和获取所述自复制RNA分子组合物。In yet another aspect of the invention, the invention provides a method for preparing a self-replicating RNA molecule composition. According to an embodiment of the present invention, the self-replicating RNA molecule includes self-replicating RNA and a delivery carrier protein, and the method includes: cultivating the aforementioned recombinant cells; and obtaining the self-replicating RNA molecule composition.
如前所述，重组细胞中含有包含自复制RNA分子的第一核酸分子和编码递送载体蛋白的第二核酸分子，通过对重组细胞培养，第二核酸分子表达递送载体蛋白，递送载体蛋白自组装以包裹第一核酸分子，获得含有递送载体蛋白及其所携带的第一核酸分子的组合物，有助于实现递送第一核酸分子的目的。As mentioned above, the recombinant cells contain a first nucleic acid molecule including a self-replicating RNA molecule and a second nucleic acid molecule encoding a delivery carrier protein. By culturing the recombinant cells, the second nucleic acid molecule expresses the delivery carrier protein, and the delivery carrier protein self-assembles. Wrapping the first nucleic acid molecule to obtain a composition containing a delivery carrier protein and the first nucleic acid molecule it carries helps achieve the purpose of delivering the first nucleic acid molecule.
本发明对于如何从重组细胞中获取自复制RNA分子组合物的方式不作严格限定，只要能够实现分离目的即可，具体可以根据实际需要灵活选择。例如，离心分离培养所得培养液中的重组细胞，对重组细胞进行破碎等等。The present invention does not strictly limit the method of obtaining the self-replicating RNA molecule composition from the recombinant cells, as long as the separation purpose can be achieved, and the specific method can be flexibly selected according to actual needs. For example, centrifugal separation of the recombinant cells in the culture medium obtained, disrupting the recombinant cells, etc.
本领域技术人员能够理解的是，前面针对重组细胞所描述的特征和优点，同样适用于该制备自复制RNA分子组合物的方法，在此不再赘述。Those skilled in the art can understand that the characteristics and advantages described above for recombinant cells are also applicable to the method of preparing a self-replicating RNA molecule composition, 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
reRNATM的蛋白递送系统需要在生产时将辅料加入生产体系，生产模式如图3所示，以reRNATM及质粒为原料投入到细胞内，即可产生reRNATM药物原液。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。The reRNA TM protein delivery system requires excipients to be added to the production system during production. The production model is shown in Figure 3. ReRNA TM and plasmids are used as raw materials and put into cells to produce reRNA TM drug stock solution. Vesicular stomatitis virus (vesicular stomatitis virus) The receptor of 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 higher affinity, so VSV-G protein as a delivery protein.
具体制备步骤为：The specific preparation steps are:
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，VSV-G的氨基酸序列如SEQ ID NO：7所示)的转染，转染完毕后，培养细胞，培养箱中培养24-48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, a VSV-G plasmid, and a T7 RNA polymerase plasmid each including the SEQ ID NO: 4-6 sequence. The amino acid sequence of VSV-G is as shown in SEQ ID NO: 7). After the transfection is completed , culture the cells in an incubator for 24-48 hours, collect the supernatant for purification, and obtain reRNA TM -GFP seeds.
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-GFP2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 -GFP2 seeds and culture in the incubator. After 48 hours, the supernatant was collected and purified to obtain VSV-G wrapped reRNA TM -GFP.
如图4所示，当仅投料reRNATM-GFP作为种子时，细胞培养48小时之后，只有很少的细胞会有GFP的荧光表达，而且几乎没有产能。当用VSV-G质粒作为辅料时，可以有效地递送reRNATM-GFP进入细胞，细胞内的GFP明显可见，相同的投料下，可以作为reRNATM-GFP生产细胞的比例大幅提高，并且产能也很可观(4.83μg/ml)。
As shown in Figure 4, when only reRNA TM -GFP is fed as a seed, after 48 hours of cell culture, only a few cells have fluorescent expression of GFP, and there is almost no production capacity. When VSV-G plasmid is used as an auxiliary material, reRNA TM -GFP can be effectively delivered into the cells, and the GFP in the cells is clearly visible. With the same input, the proportion of cells that can be used as reRNA TM -GFP production cells is greatly increased, and the production capacity is also very high. Considerable (4.83μg/ml).
As shown in Figure 4, when only reRNA TM -GFP is fed as a seed, after 48 hours of cell culture, only a few cells have fluorescent expression of GFP, and there is almost no production capacity. When VSV-G plasmid is used as an auxiliary material, reRNA TM -GFP can be effectively delivered into the cells, and the GFP in the cells is clearly visible. With the same input, the proportion of cells that can be used as reRNA TM -GFP production cells is greatly increased, and the production capacity is also very high. Considerable (4.83μg/ml).
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。参考实施例1的方法，以reRNATM-GFP及LCMV-GP质粒为原料投入到细胞内，生产reRNATM药物原液。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. Referring to the method of Example 1, reRNA TM -GFP and LCMV-GP plasmids were used as raw materials and put into cells to produce reRNA TM drug stock solution.
当仅投料reRNATM-GFP作为种子，48小时之后，只有很少的细胞会有GFP的荧光表达(图5)，而且几乎没有产能。当用LCMV-GP质粒作为辅料时，可以有效地递送reRNATM-GFP进入细胞，细胞内的GFP明显可见，相同的投料下，可以作为reRNATM-GFP
生产细胞的比例大幅提高(图5)，并且产能也很可观(3.15μg/ml)。When only reRNA TM -GFP was fed as a seed, after 48 hours, only a few cells had fluorescent expression of GFP (Figure 5), and there was almost no production capacity. When LCMV-GP plasmid is used as an auxiliary material, reRNA TM -GFP can be effectively delivered into the cells, and the GFP in the cells is clearly visible. With the same feeding material, reRNA TM -GFP can be delivered as reRNA TM -GFP. The proportion of producing cells was greatly increased (Figure 5), and the production capacity was also considerable (3.15 μg/ml).
实施例3Example 3
参考图6，以外壳蛋白稳转细胞株制备外壳蛋白包裹的reRNATM药物，具体步骤如下：Referring to Figure 6, coat protein-coated reRNA TM drugs are prepared using coat protein stably transduced cell lines. The specific steps are as follows:
1、将含有VSV-G基因(对应的氨基酸序列如SEQ ID NO：7所示)的慢病毒进行HEK293细胞的侵染，加入嘌呤霉素进行阳性细胞株的筛选，而后进行单克隆阳性细胞株的筛选，筛选出VSV-G基因表达高的细胞株进行稳定传代建库。1. Infect HEK293 cells with lentivirus containing the VSV-G gene (the corresponding amino acid sequence is shown in SEQ ID NO: 7), add puromycin to screen positive cell lines, and then select monoclonal positive cell lines. Through screening, cell lines with high VSV-G gene expression were selected for stable passage and library construction.
2、在稳定传代建库后，将细胞复苏，经培养后投料reRNATM-GFP种子(制备方法参考实施例1)，投料之后继续培养48小时，收取上清作为收获液，进行纯化。2. After stable passage and bank establishment, resuscitate the cells, add reRNA TM -GFP seeds after culture (refer to Example 1 for the preparation method), continue culturing for 48 hours after feeding, and collect the supernatant as the harvest liquid for purification.
非稳转细胞株为不含有VSV-G蛋白的HEK 293细胞中，仅向该细胞中投料reRNATM-GFP种子，48小时之后，只有很少的细胞会有GFP的荧光表达(图7)，产能极低，而稳转细胞株中由于含有VSV-G蛋白，可以有效地递送reRNATM-GFP进入细胞，可作为生产细胞的细胞比例大幅增加，几乎为100％，产能也很可观(5.94μg/ml)。The non-stably transduced cell line is HEK 293 cells that do not contain VSV-G protein. Only reRNA TM -GFP seeds are fed into the cells. After 48 hours, only a few cells have fluorescent expression of GFP (Figure 7). The production capacity is extremely low. However, because the stably transduced cell line contains VSV-G protein, it can effectively deliver reRNA TM -GFP into the cells. The proportion of cells that can be used as production cells is greatly increased, almost 100%, and the production capacity is also considerable (5.94 μg /ml).
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," "some embodiments," "an example," "specific examples," 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 (16)
- 一种重组细胞，其特征在于，包括：A recombinant cell, characterized by including:第一核酸分子，所述第一核酸分子包括自复制RNA分子；a first nucleic acid molecule, the first nucleic acid molecule comprising a self-replicating RNA molecule;第二核酸分子，所述第二核酸分子编码递送载体蛋白。A second nucleic acid molecule encoding a delivery vector protein.
- 根据权利要求1所述的重组细胞，其特征在于，所述自复制RNA分子包括：The recombinant cell according to 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.
- 根据权利要求2所述的重组细胞，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 recombinant cell according to claim 2, wherein at least one of the N protein, the P protein, and the L protein is independently derived from a virus of the family Rhabdoviridae.
- 根据权利要求2所述的重组细胞，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 recombinant cell 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.
- 根据权利要求1所述的重组细胞，其特征在于，所述递送载体蛋白包括非人源蛋白和人源蛋白；The recombinant cell according to claim 1, wherein the delivery carrier 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.
- 根据权利要求5所述的重组细胞，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、淋巴细胞脉络丛脑膜炎病毒的至少之一。The recombinant cell according to claim 5, wherein the virus includes poxvirus, rabies virus, flavivirus, measles virus, coronavirus, vesicular stomatitis virus, Newcastle disease virus, lymphocytic choriomeningitis At least one of the viruses.
- 根据权利要求1所述的重组细胞，其特征在于，所述递送载体蛋白选自水疱性口炎病毒受体和/或淋巴细胞性脉络丛脑膜炎病毒外壳蛋白。The recombinant cell according to claim 1, wherein the delivery carrier protein is selected from the group consisting of vesicular stomatitis virus receptor and/or lymphocytic choriomeningitis virus coat protein.
- 根据权利要求1所述的重组细胞，其特征在于，所述第一核酸分子和所述第二核酸分子分别独立地以游离形式或者整合至基因组的形式存在于所述重组细胞中。The recombinant cell according to claim 1, wherein the first nucleic acid molecule and the second nucleic acid molecule exist independently in the recombinant cell in a free form or in a form integrated into the genome.
- 根据权利要求1所述的重组细胞，其特征在于，所述第一核酸分子和所述第二核酸分子分别独立地以质粒的形式存在于所述重组细胞中。The recombinant cell according to claim 1, wherein the first nucleic acid molecule and the second nucleic acid molecule independently exist in the recombinant cell in the form of plasmids.
- 根据权利要求9所述的重组细胞，其特征在于，所述第一核酸分子和所述第二核酸分子存在于同一质粒上。 The recombinant cell according to claim 9, wherein the first nucleic acid molecule and the second nucleic acid molecule exist on the same plasmid.
- 一种制备权利要求1～10任一项所述重组细胞的方法，其特征在于，包括：A method for preparing the recombinant cells according to any one of claims 1 to 10, characterized by comprising:使细胞携带第一核酸分子和第二核酸分子，causing the cell to carry the first nucleic acid molecule and the second nucleic acid molecule,所述第一核酸分子和第二核酸分子是如权利要求1～10任一项所述重组细胞中所限定的。The first nucleic acid molecule and the second nucleic acid molecule are as defined in the recombinant cell of any one of claims 1 to 10.
- 根据权利要求11所述的方法，其特征在于，包括：The method according to claim 11, characterized in that it includes:将所述第一核酸分子和所述第二核酸分子的至少之一以质粒形式引入至所述细胞中。At least one of the first nucleic acid molecule and the second nucleic acid molecule is introduced into the cell in plasmid form.
- 根据权利要求12所述的方法，其特征在于，所述引入是通过将所述质粒采用转染方式进行的。The method according to claim 12, characterized in that the introduction is carried out by transfection of the plasmid.
- 根据权利要求11所述的方法，其特征在于，所述第二核酸分子是以慢病毒或CRISPR/Cas9的形式提供。The method of claim 11, wherein the second nucleic acid molecule is provided in the form of lentivirus or CRISPR/Cas9.
- 根据权利要求11所述的方法，其特征在于，所述细胞选自HEK 293细胞系、Vero细胞系、CHO-K1细胞或NIH-3T3细胞。The method according to claim 11, characterized in that the cells are selected from HEK 293 cell lines, Vero cell lines, CHO-K1 cells or NIH-3T3 cells.
- 一种制备自复制RNA分子组合物的方法，其特征在于，所述自复制RNA分子包括自复制RNA和递送载体蛋白，所述方法包括：A method for preparing a self-replicating RNA molecule composition, characterized in that the self-replicating RNA molecule includes self-replicating RNA and a delivery carrier protein, and the method includes:培养权利要求1～10任一项所述的重组细胞；和Cultivate the recombinant cells described in any one of claims 1 to 10; and获取所述自复制RNA分子组合物。 Obtain the self-replicating RNA molecule composition.
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