Polycistron self-amplifying RNA and preparation method therefor

Abstract

The present invention provides a polycistron self-amplifying RNA and a preparation method therefor. According to the present invention, based on the non-structural protein sequence and subgenomic promoter of the alphavirus-derived saRNA, a first target gene is connected behind a first subgenomic promoter which is partially overlapped with nsP1234, and then subgenomic promoters and other target gene frames are repeatedly connected, achieving the expression of a plurality of target genes, and achieving self-amplification of the plurality of target genes.

Description

Polycistronic self-amplifying RNA and methods of making TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a polycistronic self-amplifying RNA and methods of making. BACKGROUND

[0002] mRNA vaccines are broadly divided into non-replicating mRNA vaccines and self-amplifying RNA vaccines. Compared with non-replicating mRNA vaccines, self-amplifying RNA (saRNA) can replicate itself using its own RNA sequence as a template. A lower dose of saRNA can achieve similar levels of expression of immunogenic proteins and equivalent protection against viruses. In addition, the use of a lower dose of saRNA will minimize the use of delivery materials (such as cationic liposomes), thereby helping to control costs and potential side effects.

[0003] saRNA is longer than traditional mRNA, up to 9-12 kb. This is because in addition to the most basic mRNA elements, such as the cap, 5'UTR, 3'UTR and PolyA tail, saRNA also contains a non-structural protein sequence derived from Alphavirus and a 26S subgenomic promoter upstream of the gene of interest (GOI). The non-structural protein sequence encodes four non-structural proteins (nsP1, nsP2, nsP3, nsP4) from the virus, as shown in Figure 4A. The absence of viral structural proteins makes saRNA unable to produce infectious viruses. After being delivered to the cytosol of cells, the released saRNA has the ability to translate and bind to host ribosomes to produce four functional components of the RNA-dependent RNA polymerase (RDRP) or viral genome replication device: nsP1, nsP2, nsP3 and nsP4.

[0004] As shown in Figure 1, the translation of the non-structural proteins of saRNA in host cells is a multi-stage process. Early saRNA enters the host cell, using the cell's translation machinery to express two precursor proteins P123 and P1234, most of the translation products are P123, only a few are P1234. P1234 is cleaved into P123 and nsP4 by the activity of nsP2 protease. As an early replication complex, P123+nsP4 replicates negative strand RNA as a positive strand RNA template. Once the P123+nsP4 complex accumulates, P123 is trans-cleaved to form nsP1, P23 and nsP4 complex, which can synthesize negative and positive strand RNA. With the cis-acting cleavage of P23 (generating nsP2 and nsP3), a stable late replication complex consisting of all non-structural proteins is formed. Then the four single-component non-structural proteins are converted into RDRP, which is responsible for the synthesis of positive strand genomic and subgenomic mRNA. The synthesis of negative strand genomic RNA transcribes positive strand subgenomic mRNA through subgenomic promoter, and then translates the target gene. Compared with traditional mRNA technology, saRNA indeed has great potential and can show better RNA methods. At present, more and more researches on saRNA are carried out, but so far there has been no attempt to use saRNA for independent multi-cistronic simultaneous self-amplification and expression.

[0005] Multiple subunits often exist in prokaryotes, one transcript can be translated into multiple proteins. Eukaryotes, however, usually use single subunit strategy. In molecular cloning, we can simulate the characteristics of prokaryotes, use multiple subunit elements in eukaryotes, and express multiple genes in series. At present, IRES (Internal Ribosome Entry Site, IRES) elements or self-cleaving 2A peptide (2A) elements are widely used in eukaryotic cells. IRES elements have the function of independent recruitment of ribosomes without cap structure, and can initiate the translation of downstream genes. However, IRES has a major drawback, the expression level of the downstream subunit is relatively low compared with the upstream subunit (usually 10%-20% of the upstream). In order to overcome the shortcomings of IRES elements, scientists have modified self-cleaving 2A peptides into multiple subunit vectors. These short peptides are believed to work by causing ribosomes to skip the synthesis of the C-terminal peptide bond of the 2A element. Due to its unique "cutting" mechanism, an upstream protein fused with a 2A peptide tail and a downstream protein with an N-terminal proline are finally obtained. The advantage of 2A element is that the subunits belong to the same coding frame, and the expression strength tends to be consistent. However, the disadvantage is that it will increase an extra 2A peptide residual sequence at the N- or C-terminus of the target protein, which may have some impact on the function of the target protein. However, due to the advantages and disadvantages of IRES and 2A peptide, sometimes people still need to find new methods to express multiple subunits in vitro experiments.

[0006] SUMMARY

[0007] The present application provides a kind of multiple subunit self-amplification RNA, its structure is as shown in formula (I):

[0008] Wherein, SGP-n unit and GOI-n unit constitute repeat framework, n is the integer greater than 1, and the sequence of UTR unit, nsP1234 / SGP-1 unit, each SGP-n unit in repeat framework all come from alphavirus.

[0009] The repeat framework refers to the combination of the SGP unit and the GOI unit. The self-amplifying RNA provided by the present application is a self-amplifying RNA in which the 5' end part of the first SGP-1 unit is partially overlapped with the 3' end part of the nsP1234 unit, that is, the first SGP-1 unit and the nsP1234 unit have a partially overlapped sequence. In addition to the SGP-1 unit and the GOI-1 unit connected thereto which are overlapped with the nsP1234 unit, the SGP-n unit and the GOI-n unit are independent of each other and exist in a tandem form, and therefore the combination of the SGP-n unit and the GOI-n unit is referred to as a repeat framework, but the specific sequence within each repeat framework can be different. In some embodiments, the self-amplifying RNA comprises one repeat framework, that is, repeat framework 1 (containing SGP+GOI-2), in which GOI-1 and GOI-2 express different proteins, respectively. In some embodiments, the self-amplifying RNA comprises two repeat frameworks, that is, repeat framework 1 (containing SGP+GOI-2) and repeat framework 2 (containing SGP+GOI-3), in which GOI-1, GOI-2 and GOI-3 express different proteins, respectively. In some embodiments, the self-amplifying RNA comprises three repeat frameworks, that is, repeat framework 1 (containing SGP+GOI-2), repeat framework 2 (containing SGP+GOI-3) and repeat framework 3 (containing SGP+GOI-4), in which GOI-1, GOI-2, GOI-3 and GOI-4 express different proteins, respectively. The "-1" and "-n" in SGP-1 and SGP-n are used to mark the serial number of the SGP unit in formula (I), and the same applies to the GOI unit.

[0010] The polycistronic / multicistronic self-amplifying RNA (psaRNA / msaRNA) provided by the present application is a self-amplifying RNA in which the sequences of the units are independent of each other except that the 3' end of the nsP1234 unit and the 5' end part of the first SGP unit are partially overlapped.

[0011] Alphaviruses are a class of enveloped, single-stranded, positive-sense RNA viruses transmitted by arthropods such as mosquitoes, and belong to the Togaviridae family. They can widely infect humans, birds, mice, horses and other animals and cause related diseases, such as Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV) and Sindbis virus (SINV).

[0012] The polycistronic / multicistronic self-amplifying RNA (psaRNA / msaRNA) designed in the present application can independently self-amplify and express proteins of multiple genes on one RNA strand while retaining the self-amplifying effect of traditional single-cistronic saRNA. The method is simple and easy to implement, and can be customized to express multiple target genes in one cell without distinction.

[0013] Further, n is any one integer from 2 to 5. Specifically, n can be 2, 3, 4 or 5.

[0014] Further, the alphavirus in the self-amplifying RNA is selected from one or more of VEEV, SFV or SINV.

[0015] In some embodiments, the sequences of the UTR unit, the nsP1234 unit and each SGP unit of the self-amplifying RNA are derived from the same alphavirus. In some embodiments, the sequences of the UTR unit, the nsP1234 / SGP-1 unit and each SGP-n unit of the self-amplifying RNA are derived from VEEV. In some embodiments, the sequences of the UTR unit, the nsP1234 / SGP-1 unit and each SGP-n unit of the self-amplifying RNA are derived from SFV. In some embodiments, the sequences of the UTR unit, the nsP1234 / SGP-1 unit and each SGP-n unit of the self-amplifying RNA are derived from SINV.

[0016] In some embodiments, the alphavirus source of at least one of the UTR unit, the nsP1234 / SGP-1 unit and each SGP-n unit of the self-amplifying RNA is different from that of the other units. In some embodiments, the alphavirus source of the UTR unit of the self-amplifying RNA is different from that of the nsP1234 / SGP-1 unit and each SGP-n unit. In some embodiments, the alphavirus source of the nsP1234 / SGP-1 unit of the self-amplifying RNA is different from that of the UTR unit and each SGP-n unit. In some embodiments, the self-amplifying RNA comprises two repeat frames, and SGP-2 and SGP-3 in the two repeat frames are respectively derived from different alphaviruses.

[0017] In some embodiments, the sequence of the 5' UTR unit is set forth in SEQ ID NO: 1, 8, or 15; the sequence of the 3' UTR unit is set forth in SEQ ID NO: 7, 14, or 21; the sequence of the SGP unit is set forth in SEQ ID NO: 6, 13, or 20; and the sequence of the nsP1234 / SGP-1 unit is a sequence of an nsP1234 sequence and the sequence of the SGP unit connected in tandem, except that a portion of the sequence of the SGP unit that overlaps with a 3' end of the sequence of the nsP1234 sequence is removed, wherein the sequence of the nsP1234 is a sequence of an RNA encoding an nsP1 protein, an nsP2 protein, an nsP3 protein, and an nsP4 protein connected in tandem, wherein the sequence of the RNA encoding the nsP1 protein is set forth in SEQ ID NO: 2, 9, or 16, the sequence of the RNA encoding the nsP2 protein is set forth in SEQ ID NO: 3, 10, or 17, the sequence of the RNA encoding the nsP3 protein is set forth in SEQ ID NO: 4, 11, or 18, and the sequence of the RNA encoding the nsP4 protein is set forth in SEQ ID NO: 5, 12, or 19.

[0018] The present application also provides a plasmid, which is a plasmid capable of transcribing any one of the self-amplifying RNAs described above.

[0019] Further, the plasmid further comprises a T7 promoter. In some embodiments, the T7 promoter is located in front of (i.e., in the 5' direction of) the sequence of the 5' UTR unit.

[0020] In some embodiments, the plasmid further comprises a truncated human RNA polymerase I promoter. In some embodiments, the truncated human RNA polymerase I promoter is located in front of the T7 promoter.

[0021] In some embodiments, when designing and constructing the plasmid, a truncated human RNA polymerase I promoter is designed before the T7 promoter, which can directly transcribe the self-amplifying RNA to play a role after directly transfecting the plasmid into eukaryotic cells. The T7 promoter can be used to transcribe the artificially synthesized self-amplifying RNA in vitro, followed by the 5'UTR and nsP1234 sequences derived from the alphavirus, wherein the 5' end of the first subgenomic promoter and the 3' end of the nsP4 sequence overlap, the target gene 1 (such as the green fluorescent protein reporter gene: NeonGreen) is designed after the first subgenomic promoter, the sequence framework of the subgenomic promoter and other target genes (such as the red fluorescent protein reporter gene: mTagRFP) is connected again (repeatable) after the last codon of NeonGreen, and then the 3'UTR and polyA tail derived from the alphavirus are connected, followed by a single enzyme digestion site, and then the RNA polymerase I terminator (SEQ ID NO: 28) and the T7 terminator (SEQ ID NO: 26) are designed.

[0022] In some embodiments, when designing and constructing the plasmid, a truncated human RNA polymerase I promoter is designed before the T7 promoter, which can directly transcribe the self-amplifying RNA to play a role after directly transfecting the plasmid into eukaryotic cells. The T7 promoter can be used to transcribe the artificially synthesized self-amplifying RNA in vitro, followed by the 5'UTR and nsP1234 sequences derived from the alphavirus, wherein the 5' end of the first subgenomic promoter and the 3' end of the nsP4 sequence overlap, the target gene 1 (such as the green fluorescent protein reporter gene: NeonGreen) is designed after the first subgenomic promoter, the sequence framework of the subgenomic promoter and other target genes (such as the red fluorescent protein reporter gene: mTagRFP) is connected again (repeatable) after the last codon of NeonGreen, and then the 3'UTR and polyA tail derived from the alphavirus are connected, followed by a single enzyme digestion site, and then the RNA polymerase I terminator (SEQ ID NO: 28) and the T7 terminator (SEQ ID NO: 26) are designed.

[0023] The present application also provides a preparation method of the above-mentioned self-amplifying RNA, comprising the following steps:

[0024] S1: constructing any one of the plasmids described above;

[0025] In some embodiments, when constructing the plasmid of the multi-cistronic self-amplifying RNA, the non-structural protein sequence on the plasmid is derived from VEEV and SFV.

[0026] In some embodiments, the sequence of the gene of interest GOI-1 is designed after the sequence of the non-structural protein of the virus, and then the viral subgenomic promoter and the sequence of the gene of interest GOI-2 are connected.

[0027] In some embodiments, the sequence of the gene of interest GOI-1 is designed after the first SGP-1 sequence; then the second SGP-2 sequence and the sequence of the gene of interest GOI-2 are connected; and then the third SGP-3 sequence and the sequence of the gene of interest GOI-3 are connected.

[0028] S2: The plasmid is replicated and extracted;

[0029] S3: The extracted plasmid is linearized and cut to obtain a linearized plasmid;

[0030] In some embodiments, the plasmid is linearized by using a restriction endonuclease to perform a cutting reaction on the plasmid expressing the multi-cistronic self-amplifying RNA after the polyA tail.

[0031] S4: The linearized plasmid is transcribed to obtain self-amplifying RNA.

[0032] In some embodiments, the preparation of the multi-cistronic self-amplifying RNA (psaRNA / msaRNA) is performed by using an in vitro co-transcription capping method.

[0033] In another aspect, the present application also provides a treatment method, comprising: delivering the self-amplifying RNA or the plasmid according to any one of the above to or into contact with a cell in need of treatment under the condition that the effect molecule is effectively expressed. The cell is treated by the translated effect molecule.

[0034] The multi-cistronic self-amplifying RNA (psaRNA / msaRNA) designed by the present application has a structural formula as shown in formula (I), and comprises multiple GOIs. By setting the SGP in front of each GOI, the independent self-amplification and protein expression of the multi-cistronic RNA on one RNA chain can be performed while the self-amplification effect of the traditional single-cistronic saRNA is retained. This method is simple and easy to implement, and can be customized to express multiple genes of interest in one cell without distinction. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to better understand the present application and more clearly show how to implement the present application, the features of the embodiments according to the present application are described by way of examples and with reference to the accompanying drawings, in which:

[0036] Figure 1: Schematic diagram of replication and expression of conventional saRNA in cells;

[0037] Figure 2: Schematic diagram of replication and expression of psaRNA in cells;

[0038] Figure 3: Replication and expression of split VEEV1234-psaRNA and VEEVsgp-repotersRNA in cells;

[0039] Figure 4: A, schematic diagram of the conventional saRNA structural elements; B, schematic diagram of VEEV-psaRNA polycistronic self-amplification element containing 2 reporters and only VEEV sequences; C, schematic diagram of SFV-VEEV-psaRNA polycistronic self-amplification element containing 2 reporters and hybrid SFV and VEEV sequences; D, schematic diagram of split VEEV1234-psaRNA + VEEVsgp-repotersRNA containing 2 reporters; E, schematic diagram of VEEV-psaRNA containing 3 reporters;

[0040] Figure 5: Gel electrophoresis detection of VEEV-psaRNA, SFV-VEEV-psaRNA, VEEV1234-psaRNA and VEEVsgp-repotersRNA;

[0041] Figure 6: A, fluorescence detection after plasmid and RNA molecule transfection of VEEV-psaRNA, SFV-VEEV-psaRNA and VEEV1234-psaRNA + VEEVsgp-repotersRNA combination (containing 2 reporters: green fluorescent protein and red fluorescent protein); B, fluorescence detection after plasmid-5 transfection (containing 3 reporters: blue fluorescent protein, green fluorescent protein and red fluorescent protein). DETAILED DESCRIPTION

[0042] Definitions: To provide a clear and consistent understanding of the terminology used in the specification and claims of the present application, a number of terms are first defined below. Additionally, unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs.

[0043] The use of the word "a" or "an" can mean "one," but it is also known that with "one or more" or "at least one." Similarly, the word "another" can mean at least a second or more.

[0044] The word "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, "have" and "has"), "including" (and any form of including, "includes" and "include") "containing" (and any form of containing, "contains" and "contain") as used herein, are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0045] As used herein, "self-amplifying RNA", "self-replicating RNA", "self- amplifying RNA" and "saRNA" are used interchangeably and refer to an important distinction from ordinary mRNA in that it is capable of self-replicating using its own RNA sequence as a template. While ordinary mRNA encodes a protein of interest for expression, using ribosomes in the cell to complete translation and protein production, saRNA carries with it a sequence that is capable of expressing an RNA polymerase (referred to as RNA-dependent RNA polymerase, RdRP) in addition to expressing the protein of interest. This RNA polymerase, once produced, is capable of using the saRNA as a template to produce more copies of the saRNA.

[0046] As used herein, "polycistronic self-amplifying RNA", "multicistronic self- amplifying RNA", "psaRNA" and "msaRNA" are used interchangeably and refer to a saRNA that has multiple genes GOI for expression.

[0047] As used herein, "SG Promoter", "SG-Promoter", "SG- Promoter", "SGP" and "SG-P" are used interchangeably and refer to a subgenomic promoter.

[0048] As used herein, nsp1234 is a combined sequence encoding four non-structural proteins nsPl, nsP2, nsP3 and nsP4 from a virus.

[0049] EXAMPLE: The present application will be more readily understood by reference to the following examples, which are offered by way of illustration and are not intended to limit the scope of the present application.

[0050] Unless otherwise defined or specified by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application.

[0051] While the application has been described in detail with respect to the embodiments thereof, it will be apparent to those skilled in the art that various alterations, modifications, and improvements can be made therein without departing from the spirit and scope of the application. Accordingly, it is intended that all such alterations, modifications, and improvements be included within the scope of the application as defined by the following claims.

[0052] The experimental methods not specifically described in the present application are performed according to the specific methods in the book of Molecular Cloning: A Laboratory Manual (Fourth Edition) by J. Sambrook or according to the relevant product instructions. When used in the present application, all terms should be understood according to their ordinary meanings known in the art, unless otherwise specified. The biological reagents used in the present application, unless otherwise specified, can be obtained from commercial channels.

[0053] Example 1: Design and construction of plasmid for multi-cistronic self-amplifying RNA

[0054] The 5'UTR sequence, nsP1234 sequence, SG-Promoter sequence, and 3'UTR sequence of alphaviruses such as VEEV, SFV, and SINV were searched in the NCBI database, and then the UTR sequence, nsP1234 sequence, SG-Promoter sequence of VEEV virus, and other elements such as NeonGreen sequence and mTagRFP sequence were inserted into the pUC plasmid backbone. Two promoters were designed before the 5'UTR sequence, one was a T7 promoter for in vitro transcription to prepare saRNA, and the other was a truncated human RNA polymerase I promoter for verification of plasmid expression in eukaryotic cells.

[0055] Plasmid-1 is a homozygous plasmid derived from VEEV virus (as shown in Figure 4B). The 5'UTR sequence is shown in SEQ ID NO: 1, the RNA coding sequence of nsP1 protein is shown in SEQ ID NO: 2, the RNA coding sequence of nsP2 protein is shown in SEQ ID NO: 3, the RNA coding sequence of nsP3 protein is shown in SEQ ID NO: 4, the RNA coding sequence of nsP4 protein is shown in SEQ ID NO: 5, and the RNA coding sequence of nsP4 protein is partially overlapped with the SG-Promoter sequence of VEEV virus at the end, while the independent SGP sequence is shown in SEQ ID NO: 6, the 3'UTR sequence is shown in SEQ ID NO: 7, the RNA coding sequence of NeoGreen protein is shown in SEQ ID NO: 22, and the RNA coding sequence of mTagRFP protein is shown in SEQ ID NO: 23.

[0056] Plasmid-2 is a hybrid plasmid derived from SFV virus and VEEV virus (as shown in Fig. 4, C), which is also composed of pUC plasmid backbone, and contains T7 promoter (SEQ ID NO: 25) and truncated human RNA polymerase I promoter (SEQ ID NO: 27) before 5'UTR, the 5'UTR sequence (SEQ ID NO: 8), and the RNA coding sequences of nsP1234 (SEQ ID NO: 9-12) are derived from SFV virus, and the RNA coding sequence of nsP4 is partially overlapped with the SG-Promoter sequence of SFV virus at the end, followed by insertion of NeonGreen sequence (SEQ ID NO: 22) downstream, followed by insertion of the SG-Promoter sequence of VEEV virus (SEQ ID NO: 6), mTagRFP sequence (SEQ ID NO: 23), and 3'UTR sequence of VEEV virus (SEQ ID NO: 7).

[0057] Plasmid-3 and plasmid-4 are split from plasmid 1 (as shown in Fig. 4, D). Plasmid-3 contains the expression elements of non-structural proteins of VEEV virus (VEEV-P1234), which is obtained by self-ligation after removing the NeonGreen-SGP-mTagRFP sequence from plasmid-1 by enzyme digestion, only retaining the VEEV virus nsP1234 expression framework and the UTR and polyA tail sequences before and after, i.e. plasmid-3 is a VEEV1234-psaRNA plasmid; plasmid-4 is obtained by self-ligation after removing the nsP1234 protein sequence from plasmid-1 by enzyme digestion, only retaining the T7 promoter-5'UTR-SGP-NeonGreen-SGP-mTagRFP-3'UTR-PolyA sequence, i.e. plasmid-4 is a VEEVsgp-repoters plasmid, which is also constructed by the method of seamless cloning.

[0058] Plasmid-5 (as shown in Fig. 4, E) is a repeat unit of VEEV SG promoter (SEQ ID NO: 6) + mTagBFP sequence (SEQ ID NO: 24) added on the basis of plasmid-1, which is used to verify whether the three target proteins can be expressed.

[0059] After the above plasmid design is completed, the plasmid can be obtained by gene synthesis.

[0060] Example 2: Preparation of capped psaRNA by co-transcription method

[0061] Plasmid-1, plasmid-2, plasmid-3, plasmid-4 and plasmid-5 were respectively transformed into DH5alpha strain, and single colonies were picked and cultured overnight at 37°C in a shaking incubator. The bacteria were collected, and a large amount of plasmid was obtained by an endotoxin-free plasmid maxi kit (Beijing Tiangen Biochemical Technology Co., Ltd.). The enzyme cutting system was prepared according to the instructions, that is, the plasmid was mixed uniformly with specific restriction enzymes and their buffers, and reacted for 2 hours at working temperature to completely linearize the plasmid. After complete linearization, the ordinary DNA product purification recovery kit (Beijing Tiangen Biochemical Technology Co., Ltd.) was used for recovery of the linearized plasmid.

[0062] After linearizing the transcription template plasmid using restriction enzymes, in vitro transcription of RNA was initiated by T7 RNA polymerase, and co-transcriptional capping of psaRNA was performed using a co-transcriptional capping T7 in vitro transcription kit (Wuhan Hanhai New Enzyme Biological Technology Co., Ltd.). The reaction system is shown in Table 1.

[0063] Table 1 Co-transcriptional capping reaction system

[0064] 37°C for 2h, after the reaction was completed, 2μL DNase I was added, and 37°C digestion was performed for 30min to remove the DNA plasmid template. The purification after transcription was performed according to the following steps:

[0065] (1) Mix the IVT reaction solution and 5M LiCl solution at a volume ratio of 1:1, mix well, and stand at -20°C for 30min, centrifuge at 12000rpm for 10min, observe the bottom precipitate, and remove the supernatant;

[0066] (2) Place the sample on ice, add 1mL of pre-cooled 70% ethanol, shake or blow the precipitate to suspend, centrifuge at 4°C, 12000rpm for 10min, discard the supernatant, and repeat the operation twice;

[0067] (3) Without disturbing the precipitate, try to absorb the residual ethanol as much as possible, dry in a biological safety cabinet, and then add an appropriate amount of nuclease-free water to dissolve the precipitate.

[0068] The RNA obtained by in vitro transcription of the linearized plasmid above passes through agarose electrophoresis to obtain a single band, reaching the purity standard. As shown in Figure 5, M represents the molecular weight Marker, and bands 1, 2, 3 and 4 are respectively: VEEV-psaRNA, SFV-VEEV-psaRNA, VEEV1234-psaRNA and VEEVsgp-repotersRNA samples.

[0069] Example 3: Cell transfection verifies the function of multi-cistronic self-amplification RNA

[0070] In order to verify whether the multi-cistronic self-amplification RNA obtained by the present application can express two target genes in the same cell, since a truncated human RNA polymerase I promoter is designed when the plasmid is constructed, the promoter can start the transcription function of the plasmid in eukaryotic cells, the successfully constructed plasmid and prepared saRNA are subjected to cell transfection, and whether two target genes or three target genes are expressed is observed under a fluorescence microscope.

[0071] The specific implementation process is as follows: 2x106cells / well of HEK-293T cells (ATCC number: CRL-3216) are inoculated in a 24-well plate, and cultured at 37°C, 5% CO2, and the reagents involved are all from commercial products. When the cell confluence reaches 70% to 80%, the prepared plasmid and saRNA are introduced into the cells by transfection, and the transfection method can be selected, for example, liposome transfection and PEI transfection, etc., and the uBrigene PEI kit (Yiming Biology) transfection is selected in this embodiment. After 48 hours of transfection, fluorescence microscopy is performed. 6 The cells / well of HEK-293T cells (ATCC number: CRL-3216) are inoculated in a 24-well plate, and cultured at 37°C, 5% CO2, and the reagents involved are all from commercial products. When the cell confluence reaches 70% to 80%, the prepared plasmid and saRNA are introduced into the cells by transfection, and the transfection method can be selected, for example, liposome transfection and PEI transfection, etc., and the uBrigene PEI kit (Yiming Biology) transfection is selected in this embodiment. After 48 hours of transfection, fluorescence microscopy is performed.

[0072] The results are shown in Figure 6A. Whether the plasmid-1, plasmid-2 or VEEV-psaRNA, SFV-VEEV-psaRNA is used for transfection, the green fluorescent protein and the red fluorescent protein can be expressed in the same cell. Figure 6A shows that the plasmid or RNA containing the complete VEEV-psaRNA or SFV-VEEV-psaRNA self-amplification element can produce a complete negative strand after entering the cell, so that the positive strand RNA containing only the red fluorescent protein can be independently amplified (see Figure 2), thereby expressing the corresponding red color. The above results prove that the psaRNA provided by the present application can express two different proteins, and the expression of the red fluorescent protein itself also proves that the psaRNA provided by the present application can be multi-cistronic self-amplified.

[0073] For the homozygous VEEV-psaRNA derived from VEEV virus, the expression intensity of the green fluorescent protein and the red fluorescent protein is relatively uniform, indicating that the two cistrons are successfully expressed after being independently self-amplified. Since the two SGP before and after are derived from VEEV virus, they are matched with the four non-structural proteins derived from VEEV, and the expression intensity of the two proteins is similar.

[0074] For the hybrid SFV-VEEV-psaRNA from SFV virus and VEEV virus, the green fluorescent protein expression is strong, and the red fluorescent protein expression is slightly weak, which may be due to the first SG promoter before the green fluorescent protein is from SFV virus, which is naturally matched with the four SFV-derived non-structural proteins contained in the plasmid, has a stronger driving ability of SFV-derived SG promoter self-amplification, and obtains higher expression. The second SG promoter in the plasmid is from VEEV, although it can still interact with the four non-structural proteins of SFV virus to play a self-amplification function, but it does not have a high matching degree, and the self-amplification strength is relatively low, so the red fluorescent protein expression is also weak.

[0075] Therefore, the present application finds that when selecting matched various known alphavirus non-structural proteins, subgenomic promoters and UTRs (such as SFV, VEEV, SINV, etc., the relevant sequences of SINV are shown in SEQ ID NO: 15-21), the phenomenon of uneven multi-cistronic expression caused by IRES can be overcome, and no additional sequences need to be added at both ends of the target gene like 2A peptides, that is, the synchronous and uniform expression of multi-cistronic genes can be realized, which has a good application prospect in the synchronous and uniform expression of multi-cistronic genes without additional sequences.

[0076] As a comparison, the combination of split plasmid-3 and plasmid-4 or the combination of VEEV1234-psaRNA and VEEVsgp-repotersRNA can only observe weak green fluorescent protein and no red fluorescent protein, which shows that the split plasmid-3 has independent expression elements for expressing the four non-structural proteins of VEEV virus in the cell, but the combination of VEEV1234-psaRNA and VEEVsgp-repotersRNA cannot effectively produce RNA negative strand containing the reporter gene, so as to further amplify the generation of positive strand RNA containing only red fluorescent protein (see FIG. 3), so the red fluorescence is not expressed, and the green fluorescence is expressed from the RNA directly transcribed by the human RNA polymerase I promoter on the VEEVsgp-repoters plasmid. The RNA is a positive strand RNA containing the green fluorescent protein expression frame and the red fluorescent protein expression frame in sequence. The ribosome can start the translation of the first coding region (i.e., the green fluorescent protein) near the 5' end of the positive strand RNA (but cannot be amplified, so the green fluorescent protein can be expressed but the intensity is weak). Since the NeonGreen terminal contains a stop codon, the translation is terminated after the ribosome encounters the stop codon, and the red fluorescent protein behind it is no longer expressed.

[0077] For the transfected three-cistronic plasmid-5, the B result in FIG. 6 shows that the blue fluorescent protein, green fluorescent protein and red fluorescent protein can be expressed in the same cell.

[0078] In summary, the present application, based on the traditional saRNA, ingeniously uses the SG promoter of VEEV virus and SFV virus, not only retains the expression and self-amplification function of the GOI adjacent to the nsP1234 unit in the traditional saRNA, but also enables the expression and self-amplification of other GOIs not adjacent to the nsP1234 unit, successfully performs independent self-amplification and protein expression of multiple GOIs / cistrons in cells, and does not introduce additional sequences on the amino acid sequence of the target gene. The present application further finds that when the matching various known alphavirus non-structural proteins, subgenomic promoters and UTRs are selected, the synchronous and uniform expression of multiple cistrons can be realized.

[0079] Although the present application is described in detail with reference to the embodiments thereof, the embodiments are provided for illustration purposes and not to limit the present application. Other embodiments obtained according to the principles of the present application all fall within the scope defined by the claims of the present application.

[0080] Some nucleotide sequences (5'-3') involved herein are:

[0081] SEQ ID NO: 1: VEEV virus 5' UTR sequence

[0082] SEQ ID NO: 2: RNA coding sequence of nsP1 of VEEV virus

[0083] SEQ ID NO: 3: RNA coding sequence of nsP2 of VEEV virus

[0084] SEQ ID NO: 4: RNA coding sequence of nsP3 of VEEV virus

[0085] SEQ ID NO: 5: RNA coding sequence of nsP4 of VEEV virus (the bold sequence at the 3' end is the part overlapping with the VEEV virus SG promoter sequence)

[0086] SEQ ID NO: 6: VEEV virus SG promoter sequence

[0087] SEQ ID NO: 7: VEEV virus 3' UTR sequence

[0088] SEQ ID NO: 8: SFV virus 5' UTR sequence

[0089] SEQ ID NO: 9: RNA coding sequence for SFV viral nsPl

[0090] SEQ ID NO: 10: RNA coding sequence for SFV viral nsP2

[0091] SEQ ID NO: 11: RNA coding sequence for SFV viral nsP3

[0092] SEQ ID NO: 12: RNA coding sequence for SFV viral nsP4 (3' end bold sequence is overlapping with VEEV viral SG promoter sequence)

[0093] SEQ ID NO: 13: SFV viral SG promoter sequence

[0094] SEQ ID NO: 14: SFV viral 3'-UTR sequence

[0095] SEQ ID NO: 15: SINV viral 5'-UTR sequence

[0096] SEQ ID NO: 16: RNA coding sequence for SINV viral nsPl

[0097] SEQ ID NO: 17: RNA coding sequence for SINV viral nsP2

[0098] SEQ ID NO: 18: RNA coding sequence for SINV viral nsP3

[0099] SEQ ID NO: 19: RNA coding sequence for SINV viral nsP4 (3' end bold sequence is overlapping with VEEV viral SG promoter sequence)

[0100] SEQ ID NO: 20: SINV viral SG promoter sequence

[0101] SEQ ID NO: 21: SINV viral 3'UTR sequence

[0102] SEQ ID NO: 22: RNA coding sequence for NeonGreen

[0103] SEQ ID NO: 23: RNA coding sequence for mTagRFP

[0104] SEQ ID NO: 24: RNA coding sequence for mTagBFP

[0105] SEQ ID NO: 25: T7 promoter sequence

[0106] SEQ ID NO: 26: T7 terminator sequence

[0107] SEQ ID NO: 27: Human RNA polymerase I promoter sequence

[0108] SEQ ID NO: 28: RNA polymerase I terminator sequence

Claims

1. A multi-cistronic self-amplifying RNA having the structure according to formula (I): wherein The SGP-n units and the GOI-n units constitute a repeating framework, n is an integer greater than 1, and the sequences of the 5' UTR unit, the 3' UTR unit, the nsP1234 / SGP-1 unit, and each SGP-n unit in the repeating framework are derived from an alphavirus.

2. The self-amplifying RNA of claim 1, wherein, The n is not greater than 5.

3. The self-amplifying RNA of claim 1, wherein, The alphavirus is selected from the group consisting of VEEV, SFV, and SINV.

4. The self-amplifying RNA of claim 1, wherein, The sequences of the UTR unit, the nsP1234 / SGP-1 unit, and each SGP unit are derived from the same alphavirus.

5. The self-amplifying RNA of claim 1, wherein, The sequences of the UTR unit, the nsP1234 / SGP-1 unit, and each SGP unit are derived from different alphaviruses.

6. The self-amplifying RNA of claim 1, wherein, The GOI-1 unit and each GOI-n unit in the repeating framework are different genes of interest.

7. The self-amplifying RNA of claim 1, wherein, The sequence of the 5' UTR unit is shown in SEQ ID NO: 1, 8, or 15.

8. The self-amplifying RNA of claim 1, wherein, The sequence of the 3' UTR unit is shown in SEQ ID NO: 7, 14, or 21.

9. The self-amplifying RNA of claim 1, wherein, The sequence of the SGP unit is shown in SEQ ID NO: 6, 13, or 20.

10. The self-amplifying RNA of claim 1, wherein, The sequence of the nsP1234 / SGP-1 unit is a sequence of the SGP unit and a sequence of nsP1234 connected in series, and a portion of the sequence of the SGP unit at the 5' end overlapping with a portion of the sequence of nsP1234 at the 3' end is removed, wherein the sequence of nsP1234 is a sequence of RNA coding for an nsP1 protein, an nsP2 protein, an nsP3 protein, and an nsP4 protein connected in series; the sequence of RNA coding for the nsP1 protein is shown in SEQ ID NO: 2, 9, or 16; the sequence of RNA coding for the nsP2 protein is shown in SEQ ID NO: 3, 10, or 17; the sequence of RNA coding for the nsP3 protein is shown in SEQ ID NO: 4, 11, or 18; and the sequence of RNA coding for the nsP4 protein is shown in SEQ ID NO: 5, 12, or 19.

11. A plasmid comprising a sequence coding for the self-amplifying RNA according to any one of claims 1 to 10.

12. The plasmid of claim 11, wherein, The plasmid further comprises a T7 promoter upstream of the sequence coding for the self-amplifying RNA.

13. The plasmid of claim 12, wherein, The plasmid further comprises a truncated human RNA polymerase I promoter upstream of the sequence coding for the self-amplifying RNA.

14. A method for preparing the self-amplifying RNA according to any one of claims 1 to 10, comprising the following steps: S1: constructing the plasmid according to any one of claims 11 to 13; S2: replicating and extracting the plasmid; S3: linearizing the extracted plasmid to obtain a linearized plasmid; S4: transcribing the linearized plasmid to obtain the self-amplifying RNA.

15. A method of treatment comprising: The self-amplifying RNA according to any one of claims 1 to 10 or the plasmid according to any one of claims 11 to 13 is delivered to or contacted with a cell in need of treatment in the case of effective expression of an effector molecule.

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