WO2023038433A1 - Circular rna and use thereof - Google Patents

Circular rna and use thereof Download PDF

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Publication number
WO2023038433A1
WO2023038433A1 PCT/KR2022/013454 KR2022013454W WO2023038433A1 WO 2023038433 A1 WO2023038433 A1 WO 2023038433A1 KR 2022013454 W KR2022013454 W KR 2022013454W WO 2023038433 A1 WO2023038433 A1 WO 2023038433A1
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vector
circular rna
site
internal ribosome
ribosome entry
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PCT/KR2022/013454
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French (fr)
Korean (ko)
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박영근
조병문
카무투마니
조영란
허훈
김신영
송민아
김보영
정지윤
이지원
조현준
정미선
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진원생명과학 주식회사
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Publication of WO2023038433A1 publication Critical patent/WO2023038433A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to circular RNA and its use, and more particularly to circular RNA that is more stable than linear RNA in vivo.
  • circRNA circular RNA
  • ecircRNA exonic RNA
  • EIcircRNA exon-intron RNA
  • ciRNA intronic RNA
  • ecircRNA is the major circRNA and is mainly produced in vivo by a process called backsplicing.
  • EcircRNAs are mainly located in the cytoplasm and have a variety of functions.
  • the best-known function of circRNAs is as a microRNA (miRNA) sponge, such as the circRNA CDR1as and Sry.
  • circRNAs have also been demonstrated to serve as protein sponges and scaffolds for protein complexes.
  • circRNAs also play a role in regulating the translation and stability of mRNA levels and the activity of proteins.
  • circRNAs as biomarkers have been demonstrated to be associated with age-dependent neuronal accumulation in Drosophila, and are known to be involved in cancer and other diseases.
  • the present inventors have completed the present invention by developing a circular RNA platform with improved stability compared to the existing platform.
  • an object of the present invention is a promoter; internal ribosomal entry site (IRES); protein coding region; 3' untranslated region (UTR); poly A; And to provide a vector for production of a circular RNA comprising a 5' group I intron fragment including a 5' splicing site.
  • Another object of the present invention is an internal ribosome entry site; protein coding region; 3' UTR; poly A; and a 5' group I intronic fragment including a 5' splicing site.
  • Another object of the present invention is a vector for producing the circular RNA; Or to provide a pharmaceutical composition for the prevention or treatment of a target disease comprising; or the circular RNA.
  • Another object of the present invention is a vector for producing the circular RNA; Or to provide a vaccine composition for preventing a target disease comprising; or the circular RNA.
  • Another object of the present invention is to provide a method for expressing a protein of interest in a cell, including the step of transducing the vector or the circular RNA for production of the circular RNA into the cell.
  • Another object of the present invention is to provide a method for treating a target disease comprising administering the vector for producing the circular RNA or the circular RNA to a subject in need thereof.
  • the present invention provides a vector for the production of circular RNA comprising the following elements operably linked:
  • the present invention also provides a circular RNA comprising the following elements operably linked:
  • the present invention is a vector for producing the circular RNA; Or the circular RNA; It provides a pharmaceutical composition for the prevention or treatment of a target disease, including.
  • the present invention is a vector for producing the circular RNA; Or the circular RNA; It provides a vaccine composition for preventing a target disease comprising.
  • the present invention provides a method for expressing a target protein in a cell, comprising transducing a vector for production of the circular RNA or the circular RNA into the cell.
  • the present invention is a vector for producing the circular RNA; Or, administering the circular RNA to a subject in need thereof; provides a method for treating a disease of interest, including the.
  • the circular RNA according to the present invention has higher stability than linear RNA even after treatment with RNase and higher stability than linear RNA even after long-term storage. This means that since the circular RNA of the present invention has high stability in vitro or in vivo, it can maintain the expression of the target protein for a long time in vitro or in vivo. Therefore, the circular RNA of the present invention is a platform for preventing or treating a target disease; Or a vaccine platform for preventing infection of a target disease; it can be used in various ways.
  • FIG. 1 is a diagram showing the results of confirming mRNA-S, which is a linear RNA, and circRNA-S, which is a circular RNA, through electrophoresis.
  • Figure 2 is a diagram showing the results of confirming the stability of mRNA-S, which is a linear RNA, and circRNA-S, which is a circular RNA, according to RNase treatment.
  • FIG. 3 is a diagram showing the results of confirming the stability of mRNA-S, which is a linear RNA, and circRNA-S, which is a circular RNA, over time.
  • Figure 4 is a diagram showing the results of confirming the splicing site of pCircRNA without a 3' splicing site through splicing junction site sequencing.
  • the invention provides a vector for the production of circular RNA comprising the following elements operably linked:
  • operatively linked means a functional linkage between a nucleotide expression control sequence (eg, a promoter sequence) and another nucleotide sequence. Accordingly, the regulatory sequence may thereby regulate the transcription and/or translation of the other nucleotide sequence.
  • a nucleotide expression control sequence eg, a promoter sequence
  • a vector means a means for expressing a target gene in a host cell.
  • viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors.
  • the usable vectors include plasmids often used in the art (e.g., pGLS, pSC101, pGV1106, pACYC177, ColE1, pKT230, ME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), phages (eg, ⁇ gt4 ⁇ B, ⁇ Charon, ⁇ z1, and M13, etc.) or viruses (eg, CMV, SV40, etc.).
  • plasmids often used in the art (e.g., pGLS, pSC101, pGV1106, pACYC177, ColE1, pKT230, ME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14,
  • the vector of the present invention can typically be constructed as a vector for cloning or a vector for expression.
  • the vector those conventionally used in the art to express foreign proteins in plants, animals, or microorganisms may be used.
  • the vector may be constructed through various methods known in the art.
  • the vector may be constructed using a prokaryotic or eukaryotic cell as a host.
  • the origins of replication operating in the eukaryotic cell included in the vector are the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, the CMV origin of replication, and the BBV Including the origin of replication, etc., but is not limited thereto.
  • promoters derived from the genome of mammalian cells eg, metallotionine promoter
  • promoters derived from mammalian viruses eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter,
  • the cytomegalovirus (CMV) promoter and the TK promoter of HSV can be used, and usually have a polyadenylation sequence as a transcription termination sequence.
  • a protein coding region means a gene encoding a target protein.
  • the protein coding region may be a region encoding a protein having therapeutic efficacy, an antigen protein, an antibody, and the like, and any gene encoding a target protein may be applied.
  • the vector preferably does not contain a 3' splicing site upstream of the internal ribosome entry site.
  • the structure of a preferred vector of the present invention is shown as pCircRNA-ver2.0 in FIG. 4 .
  • the vector may be one in which the elements 1) to 6) are sequentially operably linked.
  • the vector comprises the following elements operably linked:
  • 3'UTR represented by the nucleotide sequence of SEQ ID NO: 3;
  • variants of the above-described nucleotide sequences are included within the scope of the present invention.
  • the variant of the base sequence has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the base sequence described in the sequence listing.
  • It means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence described in the sequence listing.
  • the "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to (not including).
  • the 5' group I intronic fragment containing the 5' splicing site is preferably derived from Anabaena pre-tRNA.
  • the internal ribosome entry site may be derived from Encephalomyocarditis virus (EMCV), and any internal ribosome entry site exhibiting the same activity can be applied without limitation.
  • EMCV Encephalomyocarditis virus
  • the poly A is preferably 50 to 300 bp, more preferably 100 to 200 bp, and most preferably 151 bp.
  • the vector preferably includes a modified nucleic acid, natural nucleic acid or non-natural nucleic acid, but is not limited thereto.
  • the vector can produce circular RNA by self-splicing, and it is preferable that the 5' end of the internal ribosome entry site is cleaved by self-splicing, and further Preferably, 1 to 50 nt of the 5' end of the internal ribosome entry site may be cut by self-splicing, and more preferably, 28 nt may be cut.
  • the internal ribosome entry site of the vector preferably overlaps the 5' end of the internal ribosome entry site with the 5' splicing site, more preferably the 5' end of the internal ribosome entry site. It may overlap with the 5' splicing site by 1 to 10 nt, most preferably by 7 nt.
  • the present invention provides a circular RNA comprising the following elements operably linked:
  • the circular RNA is preferably a circular RNA comprising the following elements operably linked:
  • 3'UTR represented by the nucleotide sequence of SEQ ID NO: 3;
  • the circular RNA is preferably cleaved from 1 to 50 nt of the 5' end of the internal ribosome entry site, more preferably 28 nt from the 5' end.
  • the circular RNA preferably has the 5' end of the internal ribosome entry site overlapping the 5' splicing site, more preferably the circular RNA has the 5' end of the internal ribosome entry site It may overlap 1 to 10 nt with the 5' splicing site, and most preferably may overlap 7 nt.
  • the circular RNA according to the present invention has much higher stability than linear RNA under RNase treatment conditions and long-term storage conditions. This means that the circular RNA of the present invention can stably and continuously express the target protein under various conditions. Therefore, the circular RNA of the present invention may be used in medicine for preventing or treating a target disease; Vaccine use for preventing infection of target disease; And expression method of the target protein; can be used as a platform technology in various fields including.
  • the present invention provides a vector for the production of circular RNA; Or circular RNA; It provides a pharmaceutical composition for the prevention or treatment of a target disease comprising.
  • Vectors and circular RNAs for the production of circular RNAs of the present invention include protein coding regions.
  • the protein coding region refers to a gene encoding a target protein, and when the target protein exhibits a preventive or therapeutic effect of a specific disease (ie, target disease), it can be usefully used for medicinal purposes for the target disease.
  • the present invention provides a vector for producing the circular RNA; Or the circular RNA; It provides a vaccine composition for preventing a target disease comprising.
  • a vector for producing the circular RNA; and circular RNA; the protein coding region included in may be a gene encoding an antigen protein.
  • the gene encoding the antigen protein may be a spike gene represented by the nucleotide sequence of SEQ ID NO: 5 used in Examples of the present invention, and the scope of the present invention is not limited thereto.
  • the vaccine is a veterinary vaccine containing an antigenic material, and is administered for the purpose of inducing active or passive immunity specific to a target disease.
  • the "immunologically effective amount" of PCV2 means an amount sufficient to exhibit a preventive effect of related diseases and an amount sufficient to not cause side effects or severe or excessive immune reactions, and the exact dosage concentration varies depending on the specific immunogen to be administered. It can be easily determined by a person skilled in the art according to factors well-known in the medical field, such as age, weight, health, sex, sensitivity to drugs of the subject, administration route, and administration method of the vaccination subject, and can be administered once or several times. can
  • the vaccine composition according to the present invention includes a vector for producing circular RNA as an active ingredient; and circular RNA; In addition, one or more immune enhancers or excipients or carriers suitable for constituting the vaccine composition may be included.
  • An adjuvant that may be included in the vaccine composition of the present invention refers to a substance that enhances the immune response of an injected animal, and many different adjuvants are known to those skilled in the art.
  • the immune enhancers include Freund's complete and incomplete immune enhancers, vitamin E, nonionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and non-mineral oil and Carbopol, water-in-oil emulsion immune enhancers, etc., but are limited thereto it is not going to be
  • Carriers that may be included in the vaccine composition of the present invention are known to those skilled in the art, and include, but are not limited to, proteins, sugars, and the like.
  • the above carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Carriers for intravenous injection include electrolyte replenishers, liquid and nutritional supplements, and the like, such as those based on Ringer's dextrose.
  • the vaccine composition of the present invention may further contain preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.
  • the preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B, and the like.
  • the vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween.
  • the vaccine composition of the present invention may include a protecting agent, and a protecting agent known in the art may be used without limitation, which may include lactose (LPGG) or trehalose (TPGG), It is not limited thereto.
  • the present invention provides a method for expressing a target protein in a cell, including transducing a vector for production of the circular RNA or the circular RNA into a cell.
  • the vector and circular RNA for production of the circular RNA of the present invention include a protein coding region, and genes encoding various target proteins can be applied to the protein coding region.
  • Circular RNA according to the present invention has much higher stability than linear RNA under RNase treatment conditions and long-term storage conditions, which means that the circular RNA of the present invention can stably and continuously express a target protein in cells.
  • a vector for producing the circular RNA or a method for treating a target disease comprising administering the circular RNA to a subject in need thereof is provided.
  • the subject is a subject expected to develop a disease in which the target protein exhibits therapeutic efficacy; diseased individuals; Alternatively, it may be an object that has been cured, but is not limited thereto.
  • the target protein refers to a protein encoded by a 'protein coding region' included in a vector or circular RNA for circular RNA production.
  • Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
  • pCircDNA-S and pCircDNA-eGFP were used as vectors for circular RNA production, and circular RNAs (CircDNA-S and CircDNA-eGFP) were produced using these vectors.
  • a vector for producing circular RNA includes 1) a promoter represented by the nucleotide sequence of SEQ ID NO: 1; 2) an internal ribosome entry site represented by the nucleotide sequence of SEQ ID NO: 2; 3) protein coding regions; 4) 3'UTR represented by the nucleotide sequence of SEQ ID NO: 3; 5) Poly A; and 6) a 5' Group I intron fragment including a 5' splicing site represented by the nucleotide sequence of SEQ ID NO: 4; characterized in that they are sequentially operably linked.
  • the pCircDNA-S of this experiment applied the spike gene represented by the nucleotide sequence of SEQ ID NO: 5 to the protein coding region, and the eGFP gene represented by the nucleotide sequence of SEQ ID NO: 7 was applied to pCircDNA-eGFP.
  • the 5' group I intron fragment including the 5' splicing site is derived from Anabaena pre-tRNA, and the internal ribosome entry site is derived from Encephalomyocarditis virus (EMCV).
  • poly A included in the vector is 151 bp.
  • linear RNA (mRNA-S, mRNA-eGFP) produced using vectors pDNA-S and pDNA-eGFP for linear RNA production was used.
  • the vectors pDNA-S and pDNA-eGFP for the production of the linear RNA are represented by the nucleotide sequences of SEQ ID NOs: 15 and 17, respectively, and the linear RNAs mRNA-S and mRNA-eGFP produced using them are SEQ ID NOs: 15 and 17, respectively. is represented by the nucleotide sequence of
  • IVT is the process of synthesizing RNA using T7 RNA polymerase in vitro using linearized template DNA. IVT was performed using linearized DNA according to the manufacturer's instructions of the EZTM MEGA T7 Transcription Kit (Enzynomics). Since pDNA-S and pDNA-eGFP are linear RNA forms, ARCA (8 mM) (New England Biolabs) was mixed and reacted in IVT process for mRNA stability. Each sample was reacted at 37 ° C for 1 hour and 40 minutes, and DNase I (2 ⁇ l / 1 rxn) was added and reacted at 37 ° C for 20 minutes.
  • RNA was purified using the Lithium Chloride (LiCl) Precipitation Solution included in the EZTM MEGA T7 Transcription Kit (Enzynomics). LiCl Precipitation Solution was added as much as half the volume of the reaction solution, thoroughly mixed, and maintained at -20 ° C for more than 30 minutes. It was then centrifuged at 4° C. for 15 minutes at the highest possible speed, and the supernatant was carefully removed. For washing, 700 ⁇ l of 70% EtOH was added and centrifuged again under the same conditions as above. After completely removing EtOH, the RNA pellet was resuspended in RNase-free water.
  • LiCl Precipitation Solution was added as much as half the volume of the reaction solution, thoroughly mixed, and maintained at -20 ° C for more than 30 minutes. It was then centrifuged at 4° C. for 15 minutes at the highest possible speed, and the supernatant was carefully removed. For washing, 700 ⁇ l of 70% EtOH was added and centrifuged again
  • the circular RNAs circRNA-S and circRNA-eGFP are characterized in that 28 nt of the 5' end of the internal ribosome entry site is cleaved and the 5' end of the internal ribosome entry site overlaps the 5' splicing site by 7 nt. to be
  • RNA electrophoresis was performed in the same manner as in Example 1-3. As a control group in this experiment, mRNA-S and CircRNA R -S were used. The circRNA R -S is RNA in circular form synthesized in a compact form from Houston Cincinnati Leading Medicine. RNA electrophoresis results are shown in FIG. 2 .
  • HEK293T cells were cultured in 10% FBS (Gibco), 1% penicillin/streptomycin, and Dulbecco's Modified Eagle Medium (DMEM) high glucose medium at 37°C and 5% CO 2 . 1 ⁇ g each of mRNA-eGFP or circRNA-eGFP was added to HEK293T cells according to the manufacturer's instructions. After that, Lipofectamine2000 (Invitrogen) and Opti-MEM (Gibco) were used to transfect 500,000 cells/1mL per well in a 6-well plate. After 4 hours of transfection, the medium was changed to the first culture medium, DMEM high glucose medium.
  • FBS Gibco
  • DMEM Dulbecco's Modified Eagle Medium
  • the RNAs extracted from the lysed cells are mRNA-eGFP (SEQ ID NO: 17) in linear RNA form and circRNA-eGFP (SEQ ID NO: 13) in circular RNA form.
  • RNA 1 ⁇ g was used to synthesize cDNA according to the manufacturer's instructions of the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific).
  • Realtime qPCR was performed using the synthesized cDNA to confirm the gene expression level according to the target gene.
  • Faststart SYBR green master (Roche)
  • 10X concentrated solution of the PCR primer was prepared.
  • Forward and reverse primers were used at a concentration of 0.4 ⁇ M
  • eGFP was used as the target gene
  • b-actin was used as the housekeeping gene.
  • qPCR was performed using a Lightcycler96 device (Roche). The Ct value for each sample was compared and calculated, and the gene expression level was normalized to b-actin, a housekeeping gene. The qPCR results are shown in FIG. 3 .
  • splicing junction sequencing 10 ⁇ L of splicing reaction column-purified circRNA with RNase R, 1 ⁇ L of oligodT (invitrogen), 1 ⁇ L of dNTP (Enzynomics), and 1 ⁇ L of nuclease free water (13 ⁇ L final volume) were added to 65 The secondary structure was normalized by heating at °C for 5 min and cooling on ice for 3 min.
  • the reverse transcription reaction was performed according to the manufacturer's instructions using 1 ⁇ L of SuperiorScript III reverse transcriptase (Enzynomics) for the internal region of the putative circRNA, and the specific conditions are shown in Table 3 below.
  • PCR products for sequencing were synthesized using 2X topsimple Dyemix - Tenuto (Enzynomics) and a pair of primers spanning splice junctions. Specific PCR conditions are shown in Table 4.
  • Step Temperature(°C) Time One 98 3:00 2 98 0:10 3 64 0:30 touch down(-0.5°C) 4 72 0:30 5 GOTO STEP2 9X 6 98 0:10 7 59 0:30 8 72 0:30 9 GOTO STEP 14X 10 72 2:00 11 4 ⁇
  • the synthesized DNA was purified according to the manufacturer's instructions of a DNA cleanup kit (New England Biolabs). Sanger-sequencing was requested to the company to confirm the splicing junction site, and the results are shown in FIG. 4 .

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Abstract

The present invention relates to a circular RNA and a use thereof, and more specifically to a circular RNA which is more stable in vivo than a linear RNA. The circular RNA, according to the present invention, has been confirmed through experiments to have higher stability than a linear RNA, even when treated with RNase, and to have higher stability than a linear RNA, even when stored for a long time. This means that the circular RNA of the present invention has high in vitro or in vivo stability and thus enables the in vitro or in vivo expression of a target protein to be sustained for a long time. Thus, the circular RNA of the present invention may be used in various ways as a platform for the prevention or treatment of a target disease, or as a vaccine platform for the prevention of infection of a target disease.

Description

원형 RNA 및 그의 이용Circular RNA and its uses
본 발명은 원형 RNA 및 이의 이용에 관한 것으로, 보다 상세하게는 생체 내에서 선형 RNA보다 안정한 원형 RNA에 관한 것이다.The present invention relates to circular RNA and its use, and more particularly to circular RNA that is more stable than linear RNA in vivo.
Circular RNA(circRNA)라고 불리는 새롭게 기술된 RNA는 최근 몇 년 동안 높은 처리량의 RNA 시퀀싱 기술의 발달로 인해 많은 주목을 받고 있다. 기존의 선형 RNA와 비교하여 circRNA는 3'-5' 공유적으로 닫힌 고리이며, 안정적으로 유지하기 위해 5'-캡 또는 3'-폴리 A 꼬리가 필요없다. 상기 circRNA는 광범위한 세포에서 발견되었으며 대부분은 비암호화 RNA(noncoding RNA, ncRNA)이다. circRNA의 구성 요소에 따라 circRNA는 exonic RNA(ecircRNA), exon-intron RNA(EIcircRNA) 또는 intronic RNA(ciRNA)으로 분류된다. 그 중 ecircRNA는 주요 circRNA로, 주로 in vivo에서 backsplicing이라는 과정에 의해 생성된다. EcircRNA는 주로 세포질에 위치하며 다양한 기능을 한다. circRNA의 가장 잘 알려진 기능은 circRNA CDR1as 및 Sry와 같은 microRNA(miRNA) 스폰지이다. circRNA는 또한 단백질 복합체에 대한 단백질 스폰지 및 스캐폴드 역할을 하는 것으로 입증되었다. 또한 circRNA는 mRNA 수준의 번역 및 안정성과 단백질의 활성을 조절하는 역할도 한다. 최근 바이오 마커로서 circRNA는 초파리의 연령의존적 신경 축적과 관련이 있는 것으로 입증되었으며, 암이나 다른 질병에도 관여할 수 있다고 알려져 있다.A newly described RNA called circular RNA (circRNA) has received a lot of attention in recent years due to the development of high-throughput RNA sequencing technology. Compared to conventional linear RNAs, circRNAs are 3'-5' covalently closed loops and do not require a 5'-cap or 3'-poly A tail to remain stable. The circRNAs have been found in a wide range of cells, and most of them are noncoding RNAs (ncRNAs). Depending on the components of circRNA, circRNAs are classified as exonic RNA (ecircRNA), exon-intron RNA (EIcircRNA), or intronic RNA (ciRNA). Among them, ecircRNA is the major circRNA and is mainly produced in vivo by a process called backsplicing. EcircRNAs are mainly located in the cytoplasm and have a variety of functions. The best-known function of circRNAs is as a microRNA (miRNA) sponge, such as the circRNA CDR1as and Sry. circRNAs have also been demonstrated to serve as protein sponges and scaffolds for protein complexes. circRNAs also play a role in regulating the translation and stability of mRNA levels and the activity of proteins. Recently, circRNAs as biomarkers have been demonstrated to be associated with age-dependent neuronal accumulation in Drosophila, and are known to be involved in cancer and other diseases.
이에 본 발명자들은 기존 플랫폼에 비해 안정성이 향상된 원형 RNA 플랫폼을 개발함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have completed the present invention by developing a circular RNA platform with improved stability compared to the existing platform.
따라서 본 발명의 목적은, 프로모터; 내부 리보솜 진입 부위(internal ribosomal entry site, IRES); 단백질 코딩 영역; 3' UTR(Untranslated Region); 폴리 A(poly A); 및 5' 스플라이싱 부위(5' splicing site)를 포함하는 5' 그룹 I 인트론 단편(5' group I intron fragment)을 포함하는 원형 RNA의 생산을 위한 벡터를 제공하는 것이다.Therefore, an object of the present invention is a promoter; internal ribosomal entry site (IRES); protein coding region; 3' untranslated region (UTR); poly A; And to provide a vector for production of a circular RNA comprising a 5' group I intron fragment including a 5' splicing site.
본 발명의 다른 목적은, 내부 리보솜 진입 부위; 단백질 코딩 영역; 3’ UTR; 폴리 A; 및 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편;을 포함하는 원형 RNA를 제공하는 것이다.Another object of the present invention is an internal ribosome entry site; protein coding region; 3' UTR; poly A; and a 5' group I intronic fragment including a 5' splicing site.
본 발명의 또 다른 목적은, 상기 원형 RNA 생산을 위한 벡터; 또는 상기 원형 RNA;를 포함하는, 목적 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is a vector for producing the circular RNA; Or to provide a pharmaceutical composition for the prevention or treatment of a target disease comprising; or the circular RNA.
본 발명의 또 다른 목적은, 상기 원형 RNA 생산을 위한 벡터; 또는 상기 원형 RNA;를 포함하는 목적 질환 예방용 백신 조성물을 제공하는 것이다.Another object of the present invention is a vector for producing the circular RNA; Or to provide a vaccine composition for preventing a target disease comprising; or the circular RNA.
본 발명의 또 다른 목적은, 상기 원형 RNA의 생산을 위한 벡터 또는 상기 원형 RNA;를 세포에 형질도입시키는 단계를 포함하는, 세포 내에서 목적 단백질을 발현시키는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for expressing a protein of interest in a cell, including the step of transducing the vector or the circular RNA for production of the circular RNA into the cell.
본 발명의 또 다른 목적은, 상기 원형 RNA 생산을 위한 벡터 또는 상기 원형 RNA를 이를 필요로 하는 개체에 투여하는 단계를 포함하는 목적 질환의 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating a target disease comprising administering the vector for producing the circular RNA or the circular RNA to a subject in need thereof.
상기 목적을 달성하기 위하여, 본 발명은 작동 가능하게 연결된 하기 요소를 포함하는 원형 RNA의 생산을 위한 벡터를 제공한다:In order to achieve the above object, the present invention provides a vector for the production of circular RNA comprising the following elements operably linked:
1) 프로모터;1) promoter;
2) 내부 리보솜 진입 부위(internal ribosomal entry site, IRES);2) internal ribosomal entry site (IRES);
3) 단백질 코딩 영역;3) protein coding regions;
4) 3' UTR(Untranslated Region);4) 3' UTR (Untranslated Region);
5) 폴리 A(poly A); 및5) poly A; and
6) 5' 스플라이싱 부위(5' splicing site)를 포함하는 5' 그룹 I 인트론 단편(5' group I intron fragment).6) A 5' group I intron fragment containing a 5' splicing site.
또한 본 발명은 작동 가능하게 연결된 하기 요소를 포함하는 원형 RNA를 제공한다:The present invention also provides a circular RNA comprising the following elements operably linked:
1) 내부 리보솜 진입 부위;1) an internal ribosome entry site;
2) 단백질 코딩 영역;2) protein coding regions;
3) 3' UTR;3) 3' UTR;
4) 폴리 A; 및4) Poly A; and
5) 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편.5) a 5' group I intronic fragment containing a 5' splice site.
또한 본 발명은 상기 원형 RNA 생산을 위한 벡터; 또는 상기 원형 RNA;를 포함하는, 목적 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention is a vector for producing the circular RNA; Or the circular RNA; It provides a pharmaceutical composition for the prevention or treatment of a target disease, including.
또한 본 발명은 상기 원형 RNA 생산을 위한 벡터; 또는 상기 원형 RNA;를 포함하는 목적 질환 예방용 백신 조성물을 제공한다.In addition, the present invention is a vector for producing the circular RNA; Or the circular RNA; It provides a vaccine composition for preventing a target disease comprising.
또한 본 발명은 상기 원형 RNA의 생산을 위한 벡터 또는 상기 원형 RNA를 세포에 형질도입시키는 단계를 포함하는, 세포 내에서 목적 단백질을 발현시키는 방법을 제공한다.In addition, the present invention provides a method for expressing a target protein in a cell, comprising transducing a vector for production of the circular RNA or the circular RNA into the cell.
또한 본 발명은 상기 원형 RNA 생산을 위한 벡터; 또는 상기 원형 RNA;를 이를 필요로 하는 개체에 투여하는 단계;를 포함하는 목적 질환의 치료 방법을 제공한다.In addition, the present invention is a vector for producing the circular RNA; Or, administering the circular RNA to a subject in need thereof; provides a method for treating a disease of interest, including the.
본 발명에 따른 원형 RNA는 RNase를 처리하였음에도 선형 RNA보다 안정성이 높고, 오랜시간 보관하여도 선형 RNA보다 안정성이 높은 것을 실험적으로 확인하였다. 이는 본 발명의 원형 RNA가 시험관 내 또는 생체 내에서 안정성이 높은바, 시험관 내 또는 생체 내에서 목적 단백질의 발현을 오랫동안 유지할 수 있음을 의미한다. 따라서 본 발명의 원형 RNA는 목적 질환의 예방 또는 치료를 위한 플랫폼; 또는 목적 질환의 감염 예방을 위한 백신 플랫폼;으로 다양하게 활용될 수 있다.It was experimentally confirmed that the circular RNA according to the present invention has higher stability than linear RNA even after treatment with RNase and higher stability than linear RNA even after long-term storage. This means that since the circular RNA of the present invention has high stability in vitro or in vivo, it can maintain the expression of the target protein for a long time in vitro or in vivo. Therefore, the circular RNA of the present invention is a platform for preventing or treating a target disease; Or a vaccine platform for preventing infection of a target disease; it can be used in various ways.
도 1은 전기영동을 통해 선형 RNA인 mRNA-S 및 원형 RNA인 circRNA-S를 확인한 결과를 나타낸 도이다.1 is a diagram showing the results of confirming mRNA-S, which is a linear RNA, and circRNA-S, which is a circular RNA, through electrophoresis.
도 2는 RNase 처리에 따른 선형 RNA인 mRNA-S 및 원형 RNA인 circRNA-S의 안정성을 확인한 결과를 나타낸 도이다.Figure 2 is a diagram showing the results of confirming the stability of mRNA-S, which is a linear RNA, and circRNA-S, which is a circular RNA, according to RNase treatment.
도 3은 시간에 따른 선형 RNA인 mRNA-S 및 원형 RNA인 circRNA-S의 안정성을 확인한 결과를 나타낸 도이다.3 is a diagram showing the results of confirming the stability of mRNA-S, which is a linear RNA, and circRNA-S, which is a circular RNA, over time.
도 4는 Splicing junction site sequencing을 통해 3’ splicing site가 없는 pCircRNA의 splicing site를 확인한 결과를 나타낸 도이다.Figure 4 is a diagram showing the results of confirming the splicing site of pCircRNA without a 3' splicing site through splicing junction site sequencing.
이하, 본 발명을 상세히 설명하였다.Hereinafter, the present invention has been described in detail.
본 발명의 양태에 따르면, 본 발명은 작동 가능하게 연결된 하기 요소를 포함하는 원형 RNA의 생산을 위한 벡터를 제공한다:According to an aspect of the invention, the invention provides a vector for the production of circular RNA comprising the following elements operably linked:
1) 프로모터;1) promoter;
2) 내부 리보솜 진입 부위(internal ribosomal entry site, IRES);2) internal ribosomal entry site (IRES);
3) 단백질 코딩 영역;3) protein coding regions;
4) 3' UTR(Untranslated Region);4) 3' UTR (Untranslated Region);
5) 폴리 A(poly A); 및5) poly A; and
6) 5' 스플라이싱 부위(5' splicing site)를 포함하는 5' 그룹 I 인트론 단편(5' group I intron fragment).6) A 5' group I intron fragment containing a 5' splicing site.
본 발명에 있어서, "작동적으로 연결된(operatively linked)"은 뉴클레오티드 발현 조절 서열(예를 들면, 프로모터 서열)과 다른 뉴클레오티드 서열 사이의 기능적인 결합을 의미한다. 따라서, 이에 의해 상기 조절 서열은 상기 다른 뉴클레오티드 서열의 전사 및/또는 해독을 조절할 수 있다.In the present invention, "operatively linked" means a functional linkage between a nucleotide expression control sequence (eg, a promoter sequence) and another nucleotide sequence. Accordingly, the regulatory sequence may thereby regulate the transcription and/or translation of the other nucleotide sequence.
본 발명에 있어서, 벡터(vector)는 숙주 세포에서 목적 유전자를 발현시키기 위한 수단을 의미한다. 예를 들어, 플라스미드 벡터, 코즈미드 벡터 및 박테리오파아지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스 벡터와 같은 바이러스 벡터를 포함한다. 상기 사용 가능한 벡터는 당업계에서 종종 사용되는 플라스미드(예를 들면, pGLS, pSC101, pGV1106, pACYC177, ColE1, pKT230, ME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX 시리즈, pET 시리즈 및 pUC19 등), 파지(예를 들면, λgt4λB, λCharon, λΔz1 및 M13 등) 또는 바이러스(예를 들면, CMV, SV40 등)를 조작하여 제작될 수 있다.In the present invention, a vector means a means for expressing a target gene in a host cell. Examples include viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors. The usable vectors include plasmids often used in the art (e.g., pGLS, pSC101, pGV1106, pACYC177, ColE1, pKT230, ME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), phages (eg, λgt4λB, λCharon, λΔz1, and M13, etc.) or viruses (eg, CMV, SV40, etc.).
또한 본 발명의 벡터는, 전형적으로 클로닝을 위한 벡터 또는 발현을 위한 벡터로서 구축될 수 있다. 상기 벡터는 당업계에서 식물, 동물 또는 미생물에서 외래의 단백질을 발현하는데 사용되는 통상의 것을 사용할 수 있다. 상기 벡터는 당업계에 공지된 다양한 방법을 통해 구축될 수 있다.In addition, the vector of the present invention can typically be constructed as a vector for cloning or a vector for expression. As the vector, those conventionally used in the art to express foreign proteins in plants, animals, or microorganisms may be used. The vector may be constructed through various methods known in the art.
상기 벡터는 원핵 세포 또는 진핵 세포를 숙주로 하여 구축될 수 있다. 예를 들어, 진핵 세포를 숙주로 하는 경우에는, 벡터에 포함되는 진핵 세포에서 작동하는 복제원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점, CMV 복제원점 및 BBV 복제원점 등을 포함하나, 이에 한정되는 것은 아니다. 또한, 포유동물 세포의 유전체로부터 유래된 프로모터(예를 들어, 메탈로티오닌 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터(예를 들어, 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, 사이토메갈로바이러스(CMV) 프로모터 및 HSV의 TK 프로모터가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 갖는다.The vector may be constructed using a prokaryotic or eukaryotic cell as a host. For example, when a eukaryotic cell is used as a host, the origins of replication operating in the eukaryotic cell included in the vector are the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, the CMV origin of replication, and the BBV Including the origin of replication, etc., but is not limited thereto. In addition, promoters derived from the genome of mammalian cells (eg, metallotionine promoter) or promoters derived from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus (CMV) promoter and the TK promoter of HSV can be used, and usually have a polyadenylation sequence as a transcription termination sequence.
본 발명에 있어서, 단백질 코딩 영역은 목적 단백질을 암호화하는 유전자를 의미한다. 단백질 코딩 영역은 치료 효능을 갖는 단백질, 항원 단백질, 항체 등을 코딩하는 영역일 수 있으며, 어떠한 목적 단백질을 암호화하는 유전자라도 적용 가능하다.In the present invention, a protein coding region means a gene encoding a target protein. The protein coding region may be a region encoding a protein having therapeutic efficacy, an antigen protein, an antibody, and the like, and any gene encoding a target protein may be applied.
본 발명의 구체예에서, 상기 벡터는 내부 리보솜 진입 부위의 상류(upstream)에 3' 스플라이싱 부위(3’ splicing site)를 포함하지 않는 것이 바람직하다. In an embodiment of the present invention, the vector preferably does not contain a 3' splicing site upstream of the internal ribosome entry site.
본 발명의 바람직한 벡터의 구조는 도 4의 pCircRNA-ver2.0 로 나타내었다. The structure of a preferred vector of the present invention is shown as pCircRNA-ver2.0 in FIG. 4 .
본 발명의 구체예에서, 상기 벡터는 상기 1) 내지 6)의 요소가 순차적으로 작동가능하게 연결된 것일 수 있다.In an embodiment of the present invention, the vector may be one in which the elements 1) to 6) are sequentially operably linked.
본 발명의 구체예에서, 상기 벡터는 작동 가능하게 연결된 하기 요소를 포함하는 것이 바람직하다:In an embodiment of the invention, it is preferred that the vector comprises the following elements operably linked:
1) 서열번호 1의 염기서열로 표시되는 프로모터;1) a promoter represented by the nucleotide sequence of SEQ ID NO: 1;
2) 서열번호 2의 염기서열로 표시되는 내부 리보솜 진입 부위;2) an internal ribosome entry site represented by the nucleotide sequence of SEQ ID NO: 2;
3) 단백질 코딩 영역;3) protein coding regions;
4) 서열번호 3의 염기서열로 표시되는 3' UTR;4) 3'UTR represented by the nucleotide sequence of SEQ ID NO: 3;
5) 폴리 A; 및5) Poly A; and
6) 서열번호 4의 염기서열로 표시되는 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편.6) A 5' Group I intron fragment including a 5' splicing site represented by the nucleotide sequence of SEQ ID NO: 4.
또한, 전술한 염기서열의 변이체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 염기서열의 변이체는 서열목록에 기재된 염기서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 것으로, 서열목록에 기재된 염기서열과 실질적으로 동질의 생리활성을 나타내는 서열을 의미한다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.In addition, variants of the above-described nucleotide sequences are included within the scope of the present invention. Specifically, the variant of the base sequence has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the base sequence described in the sequence listing. , It means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence described in the sequence listing. The "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to (not including).
본 발명의 구체예에서, 상기 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편은 Anabaena pre-tRNA 유래인 것이 바람직하다.In an embodiment of the present invention, the 5' group I intronic fragment containing the 5' splicing site is preferably derived from Anabaena pre-tRNA.
본 발명의 구체예에서, 상기 내부 리보솜 진입 부위는 뇌심근염 바이러스(Encephalomyocarditis virus, EMCV) 유래일 수 있고, 동질의 활성을 나타내는 내부 리보솜 진입 부위라면 제한없이 적용 가능하다.In an embodiment of the present invention, the internal ribosome entry site may be derived from Encephalomyocarditis virus (EMCV), and any internal ribosome entry site exhibiting the same activity can be applied without limitation.
본 발명의 구체예에서, 상기 폴리 A는 50 내지 300bp인 것이 바람직하고, 더 바람직하게는 100 내지 200bp일 수 있으며, 가장 바람직하게는 151bp인 것이 바람직하다.In an embodiment of the present invention, the poly A is preferably 50 to 300 bp, more preferably 100 to 200 bp, and most preferably 151 bp.
본 발명의 구체예에서, 상기 벡터는 변형된 핵산, 천연 핵산 또는 비천연 핵산을 포함하는 것이 바람직하나, 이에 제한되지 않는다.In an embodiment of the present invention, the vector preferably includes a modified nucleic acid, natural nucleic acid or non-natural nucleic acid, but is not limited thereto.
본 발명의 구체예에서, 상기 벡터는 자가 스플라이싱(self-splicing)에 의해 원형 RNA를 생산할 수 있고, 내부 리보솜 진입 부위의 5' 말단이 자가 스플라이싱에 의해 절단되는 것이 바람직하고, 더 바람직하게는 자가 스플라이싱에 의해 내부 리보솜 진입 부위의 5' 말단의 1 내지 50 nt가 절단되는 것일 수 있고, 더욱 바람직하게는 28 nt가 절단되는 것일 수 있다.In an embodiment of the present invention, the vector can produce circular RNA by self-splicing, and it is preferable that the 5' end of the internal ribosome entry site is cleaved by self-splicing, and further Preferably, 1 to 50 nt of the 5' end of the internal ribosome entry site may be cut by self-splicing, and more preferably, 28 nt may be cut.
본 발명의 바람직한 구체예에서, 상기 벡터의 내부 리보솜 진입 부위는 내부 리보솜 진입 부위의 5' 말단이 5' 스플라이싱 부위와 중첩되는 것이 바람직하고, 더 바람직하게는 내부 리보솜 진입 부위의 5' 말단이 5' 스플라이싱 부위와 1 내지 10 nt 중첩되는 것일 수 있고, 가장 바람직하게는 7 nt 중첩되는 것일 수 있다.In a preferred embodiment of the present invention, the internal ribosome entry site of the vector preferably overlaps the 5' end of the internal ribosome entry site with the 5' splicing site, more preferably the 5' end of the internal ribosome entry site. It may overlap with the 5' splicing site by 1 to 10 nt, most preferably by 7 nt.
본 발명의 다른 양태에 따르면, 본 발명은 작동 가능하게 연결된 하기 요소를 포함하는 원형 RNA를 제공한다:According to another aspect of the present invention, the present invention provides a circular RNA comprising the following elements operably linked:
1) 내부 리보솜 진입 부위;1) an internal ribosome entry site;
2) 단백질 코딩 영역;2) protein coding regions;
3) 3' UTR;3) 3' UTR;
4) 폴리 A; 및4) Poly A; and
5) 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편.5) a 5' group I intronic fragment containing a 5' splice site.
본 발명의 구체예에서, 상기 원형 RNA는 작동 가능하게 연결된 하기 요소를 포함하는 원형 RNA인 것이 바람직하다:In an embodiment of the present invention, the circular RNA is preferably a circular RNA comprising the following elements operably linked:
1) 서열번호 1의 염기서열로 표시되는 프로모터;1) a promoter represented by the nucleotide sequence of SEQ ID NO: 1;
2) 서열번호 6의 염기서열로 표시되는 내부 리보솜 진입 부위;2) an internal ribosome entry site represented by the nucleotide sequence of SEQ ID NO: 6;
3) 단백질 코딩 영역;3) protein coding regions;
4) 서열번호 3의 염기서열로 표시되는 3' UTR;4) 3'UTR represented by the nucleotide sequence of SEQ ID NO: 3;
5) 폴리 A; 및5) Poly A; and
6) 서열번호 4의 염기서열로 표시되는 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편.6) A 5' Group I intron fragment including a 5' splicing site represented by the nucleotide sequence of SEQ ID NO: 4.
본 발명의 구체예에서, 상기 원형 RNA는 내부 리보솜 진입 부위의 5' 말단의 1 내지 50 nt가 절단된 것이 바람직하고, 더욱 바람직하게는 28 nt가 절단된 것일 수 있다.In an embodiment of the present invention, the circular RNA is preferably cleaved from 1 to 50 nt of the 5' end of the internal ribosome entry site, more preferably 28 nt from the 5' end.
본 발명의 구체예에서, 상기 원형 RNA는 내부 리보솜 진입 부위의 5' 말단이 5' 스플라이싱 부위와 중첩된 것이 바람직하고, 더 바람직하게는 상기 원형 RNA는 내부 리보솜 진입 부위의 5' 말단이 5' 스플라이싱 부위와 1 내지 10 nt 중첩된 것일 수 있고, 가장 바람직하게는 7 nt 중첩된 것일 수 있다.In an embodiment of the present invention, the circular RNA preferably has the 5' end of the internal ribosome entry site overlapping the 5' splicing site, more preferably the circular RNA has the 5' end of the internal ribosome entry site It may overlap 1 to 10 nt with the 5' splicing site, and most preferably may overlap 7 nt.
본 발명에 따른 원형 RNA는 RNase 처리 조건 및 장기 보관 조건에서 선형 RNA보다 안정성이 매우 높은 것을 실험적으로 확인하였다. 이는 본 발명의 원형 RNA가 다양한 조건에서도 목적 단백질을 안정적이고 지속적으로 발현시킬 수 있음을 의미한다. 따라서 본 발명의 원형 RNA는 목적 질환의 예방 또는 치료를 위한 의약 용도; 목적 질환의 감염 예방을 위한 백신 용도; 및 목적 단백질의 발현 방법;을 포함한 다양한 분야에서 플랫폼 기술로 활용될 수 있다.It was experimentally confirmed that the circular RNA according to the present invention has much higher stability than linear RNA under RNase treatment conditions and long-term storage conditions. This means that the circular RNA of the present invention can stably and continuously express the target protein under various conditions. Therefore, the circular RNA of the present invention may be used in medicine for preventing or treating a target disease; Vaccine use for preventing infection of target disease; And expression method of the target protein; can be used as a platform technology in various fields including.
본 발명의 또 다른 양태에 따르면, 본 발명은 원형 RNA의 생산을 위한 벡터; 또는 원형 RNA;를 포함하는 목적 질환의 예방 또는 치료용 약학적 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a vector for the production of circular RNA; Or circular RNA; It provides a pharmaceutical composition for the prevention or treatment of a target disease comprising.
본 발명의 원형 RNA의 생산을 위한 벡터 및 원형 RNA는 단백질 코딩 영역을 포함한다. 상기 단백질 코딩 영역은 목적 단백질을 암호화하는 유전자를 의미하는데, 상기 목적 단백질이 특정 질환(즉, 목적 질환)의 예방 또는 치료 효과를 나타내는 경우 목적 질환에 대한 의약 용도로 유용하게 활용될 수 있다.Vectors and circular RNAs for the production of circular RNAs of the present invention include protein coding regions. The protein coding region refers to a gene encoding a target protein, and when the target protein exhibits a preventive or therapeutic effect of a specific disease (ie, target disease), it can be usefully used for medicinal purposes for the target disease.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 원형 RNA 생산을 위한 벡터; 또는 상기 원형 RNA;를 포함하는 목적 질환 예방용 백신 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a vector for producing the circular RNA; Or the circular RNA; It provides a vaccine composition for preventing a target disease comprising.
본 발명의 구체예에서, 상기 원형 RNA 생산을 위한 벡터; 및 원형 RNA;에 포함된 단백질 코딩 영역은 항원 단백질을 코딩하는 유전자일 수 있다. 상기 항원 단백질을 코딩하는 유전자는 본 발명의 실시예에서 사용한 서열번호 5의 염기서열로 표시되는 스파이크 유전자일 수 있으며, 이에 본 발명의 범위가 제한되지 않는다.In an embodiment of the present invention, a vector for producing the circular RNA; and circular RNA; the protein coding region included in may be a gene encoding an antigen protein. The gene encoding the antigen protein may be a spike gene represented by the nucleotide sequence of SEQ ID NO: 5 used in Examples of the present invention, and the scope of the present invention is not limited thereto.
본 발명에 있어서, 백신은 항원 물질을 포함하는 수의학용 백신으로, 목적 질환에 대하여 특이적이고 능동 또는 수동의 면역성을 유도하기 위한 목적으로 투여된다.In the present invention, the vaccine is a veterinary vaccine containing an antigenic material, and is administered for the purpose of inducing active or passive immunity specific to a target disease.
본 발명에 따른 백신 조성물의 투여는 면역학적 유효량으로 투여할 수 있다. 상기 "면역학적 유효량"이란 PCV2는 관련된 질병의 예방 효과를 나타낼 수 있을 정도의 충분한 양과 부작용이나 심각한 또는 과도한 면역반응을 일으키지 않을 정도의 양을 의미하며, 정확한 투여 농도는 투여될 특정 면역원에 따라 달라지며, 예방 접종 대상의 연령, 체중, 건강, 성별, 개체의 약물에 대한 민감도, 투여 경로, 투여 방법 등 의학 분야에 잘 알려진 요소에 따라 당업자에 의해 용이하게 결정될 수 있으며, 1회 내지 수회 투여할 수 있다.Administration of the vaccine composition according to the present invention can be administered in an immunologically effective amount. The "immunologically effective amount" of PCV2 means an amount sufficient to exhibit a preventive effect of related diseases and an amount sufficient to not cause side effects or severe or excessive immune reactions, and the exact dosage concentration varies depending on the specific immunogen to be administered. It can be easily determined by a person skilled in the art according to factors well-known in the medical field, such as age, weight, health, sex, sensitivity to drugs of the subject, administration route, and administration method of the vaccination subject, and can be administered once or several times. can
본 발명에 따른 백신 조성물은 유효 성분인 원형 RNA 생산을 위한 벡터; 및 원형 RNA; 이외에도 백신 조성물을 구성하는 데 적절한 하나 이상의 면역 증강제 또는 부형제 또는 담체를 포함할 수 있다.The vaccine composition according to the present invention includes a vector for producing circular RNA as an active ingredient; and circular RNA; In addition, one or more immune enhancers or excipients or carriers suitable for constituting the vaccine composition may be included.
본 발명의 백신 조성물에 포함될 수 있는 면역증강제는 주사한 동물의 면역 반응을 증대시키는 물질을 의미하는 것으로, 다수의 상이한 면역증강제가 기술 분야의 당업자에게 공지되어 있다. 상기 면역증강제는 프로인트 완전 및 불완전 면역 증강제, 비타민 E, 비이온성 차단 폴리머, 뮤라밀디펩티드, Quil A, 광유 및 무광물유 및 카보폴(Carbopol), 유중수형 유제 면역증강제 등을 포함하며, 이에 제한되는 것은 아니다.An adjuvant that may be included in the vaccine composition of the present invention refers to a substance that enhances the immune response of an injected animal, and many different adjuvants are known to those skilled in the art. The immune enhancers include Freund's complete and incomplete immune enhancers, vitamin E, nonionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and non-mineral oil and Carbopol, water-in-oil emulsion immune enhancers, etc., but are limited thereto it is not going to be
본 발명의 백신 조성물에 포함될 수 있는 담체는 기술 분야의 당업자에게 공지되어 있으며, 단백질, 설탕 등을 포함하지만, 이로 제한되는 것은 아니다. 상기의 담체는 수용액 또는 비-수용액, 현탁액, 및 에멀전 일 수 있다. 비-수용액 담체의 예는 프로필렌 글리콜, 폴리에틸렌 글리콜, 식용유 예컨대 올리브 오일, 및 주사 가능한 유기 에스테르 예컨대 에틸 올리에이트를 들 수 있다. 수용액 담체는 식염수 및 완충배지를 포함하는, 물, 알콜/수용액, 에멀전 또는 현탁액을 포함한다. 비경구 담체는 염화나트륨 용액, 링거 덱스트로스, 덱스트로스 및 염화나트륨, 유산처리 링거 또는 고정 오일을 포함한다. 정맥주사용 담체는 예컨대 링거 덱스트로스를 기본으로 하는 것과 같은 전해질 보충제, 액체 및 영양 보충제 등을 포함한다.Carriers that may be included in the vaccine composition of the present invention are known to those skilled in the art, and include, but are not limited to, proteins, sugars, and the like. The above carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Carriers for intravenous injection include electrolyte replenishers, liquid and nutritional supplements, and the like, such as those based on Ringer's dextrose.
본 발명의 백신 조성물은 방부제 및 기타 첨가제 예컨대 예를 들면 항미생물제제, 항산화제, 킬레이트제, 불활성 가스 등과 같은 것을 추가로 포함할 수 있다. 상기 방부제는 포르말린, 티메로살, 네오마이신, 폴리믹신 B 및 암포테리신 B 등을 포함한다. 본 발명의 백신 조성물은 하나 이상의 적절한 유화제, 예로서 스판(Span) 또는 트윈(Tween)을 포함할 수 있다. 또한 본 발명의 백신 조성물은 보호제를 포함할 수 있으며, 당업계에 공지된 보호제를 제한 없이 사용할 수 있고, 이는 락토오스(Lactose; LPGG) 또는 트레할로오스(Trehalose; TPGG)를 포함할 수 있으나, 이에 제한되는 것은 아니다.The vaccine composition of the present invention may further contain preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like. The preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B, and the like. The vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween. In addition, the vaccine composition of the present invention may include a protecting agent, and a protecting agent known in the art may be used without limitation, which may include lactose (LPGG) or trehalose (TPGG), It is not limited thereto.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 원형 RNA의 생산을 위한 벡터 또는 상기 원형 RNA를 세포에 형질도입시키는 단계;를 포함하는, 세포 내에서 목적 단백질을 발현시키는 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for expressing a target protein in a cell, including transducing a vector for production of the circular RNA or the circular RNA into a cell.
본 발명의 원형 RNA의 생산을 위한 벡터 및 원형 RNA는 단백질 코딩 영역을 포함하는데, 상기 단백질 코딩 영역에는 다양한 목적 단백질을 암호화하는 유전자를 적용할 수 있다. The vector and circular RNA for production of the circular RNA of the present invention include a protein coding region, and genes encoding various target proteins can be applied to the protein coding region.
본 발명에 따른 원형 RNA는 RNase 처리 조건 및 장기 보관 조건에서 선형 RNA보다 안정성이 매우 높은바, 이는 본 발명의 원형 RNA가 세포 내에서 목적 단백질을 안정적이고 지속적으로 발현시킬 수 있다.Circular RNA according to the present invention has much higher stability than linear RNA under RNase treatment conditions and long-term storage conditions, which means that the circular RNA of the present invention can stably and continuously express a target protein in cells.
본 발명의 또 다른 양태에 따르면, 상기 원형 RNA의 생산을 위한 벡터 또는 상기 원형 RNA를 이를 필요로 하는 개체에 투여하는 단계를 포함하는 목적 질환의 치료 방법을 제공한다.According to another aspect of the present invention, a vector for producing the circular RNA or a method for treating a target disease comprising administering the circular RNA to a subject in need thereof is provided.
본 발명의 구체예에서, 상기 개체는 목적 단백질이 치료 효능을 나타내는 질환이 발병할 것으로 예상되는 개체; 발병한 개체; 또는 완치판정을 받은 개체일 수 있으나, 이에 제한되지 않는다. 구체적으로, 상기 목적 단백질은 원형 RNA 생산을 위한 벡터 또는 원형 RNA에 포함된 ‘단백질 코딩 영역’으로 암호화되는 단백질을 의미한다.In an embodiment of the present invention, the subject is a subject expected to develop a disease in which the target protein exhibits therapeutic efficacy; diseased individuals; Alternatively, it may be an object that has been cured, but is not limited thereto. Specifically, the target protein refers to a protein encoded by a 'protein coding region' included in a vector or circular RNA for circular RNA production.
중복되는 내용은 본 명세서의 복잡성을 고려하여 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 하였다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention was described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. In vitro transcription(IVT)을 통한 linear 및 circular RNA 준비 Example 1. Preparation of linear and circular RNA through in vitro transcription (IVT)
본 실험에서는 원형 RNA 생산을 위한 벡터로 pCircDNA-S 및 pCircDNA-eGFP를 이용하였으며, 이를 이용하여 원형 RNA(CircDNA-S, CircDNA-eGFP)를 생산하였다. 원형 RNA 생산을 위한 벡터는 1) 서열번호 1의 염기서열로 표시되는 프로모터; 2) 서열번호 2의 염기서열로 표시되는 내부 리보솜 진입 부위; 3) 단백질 코딩 영역; 4) 서열번호 3의 염기서열로 표시되는 3' UTR; 5) 폴리 A; 및 6) 서열번호 4의 염기서열로 표시되는 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편;이 순차적으로 작동 가능하게 연결된 것을 특징으로 한다. 본 실험의 pCircDNA-S는 상기 단백질 코딩 영역 부분에 서열번호 5의 염기서열로 표시되는 스파이크 유전자를 적용하였고, pCircDNA-eGFP는 서열번호 7의 염기서열로 표시되는 eGFP 유전자를 적용하였다. 상기 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편은 Anabaena pre-tRNA 유래이고, 상기 내부 리보솜 진입 부위는 뇌심근염 바이러스(Encephalomyocarditis virus, EMCV) 유래이다. 또한 상기 벡터에 포함된 폴리 A는 151 bp이다. In this experiment, pCircDNA-S and pCircDNA-eGFP were used as vectors for circular RNA production, and circular RNAs (CircDNA-S and CircDNA-eGFP) were produced using these vectors. A vector for producing circular RNA includes 1) a promoter represented by the nucleotide sequence of SEQ ID NO: 1; 2) an internal ribosome entry site represented by the nucleotide sequence of SEQ ID NO: 2; 3) protein coding regions; 4) 3'UTR represented by the nucleotide sequence of SEQ ID NO: 3; 5) Poly A; and 6) a 5' Group I intron fragment including a 5' splicing site represented by the nucleotide sequence of SEQ ID NO: 4; characterized in that they are sequentially operably linked. The pCircDNA-S of this experiment applied the spike gene represented by the nucleotide sequence of SEQ ID NO: 5 to the protein coding region, and the eGFP gene represented by the nucleotide sequence of SEQ ID NO: 7 was applied to pCircDNA-eGFP. The 5' group I intron fragment including the 5' splicing site is derived from Anabaena pre-tRNA, and the internal ribosome entry site is derived from Encephalomyocarditis virus (EMCV). Also, poly A included in the vector is 151 bp.
본 실험의 대조군은 선형 RNA 생산을 위한 벡터 pDNA-S 및 pDNA-eGFP를 이용하여 생산된 선형 RNA(mRNA-S, mRNA-eGFP)를 사용하였다. 상기 선형 RNA 생산을 위한 벡터 pDNA-S 및 pDNA-eGFP는 각각 서열번호 15 및 17의 염기서열로 표시되고, 이를 이용하여 생산된 선형 RNA인 mRNA-S 및 mRNA-eGFP는 각각 서열번호 15 및 17의 염기서열로 표시된다.As a control group in this experiment, linear RNA (mRNA-S, mRNA-eGFP) produced using vectors pDNA-S and pDNA-eGFP for linear RNA production was used. The vectors pDNA-S and pDNA-eGFP for the production of the linear RNA are represented by the nucleotide sequences of SEQ ID NOs: 15 and 17, respectively, and the linear RNAs mRNA-S and mRNA-eGFP produced using them are SEQ ID NOs: 15 and 17, respectively. is represented by the nucleotide sequence of
1-1. pDNA-S와 pDNA-eGFP, pCircDNA-S, pCircDNA-eGFP의 linearization 1-1. Linearization of pDNA-S and pDNA-eGFP, pCircDNA-S and pCircDNA-eGFP
IVT를 수행하기 앞서 plasmid DNA를 사용하여 각 시료의 linearization을 진행하였다. 구체적으로, 각각의 시료 30 μg을 사용하여 restriction enzyme(2U/μg), 5x enzyme reaction buffer, nuclease free water를 시료의 총 부피가 300 μL가 되도록 혼합하였다. 각각의 시료를 37°C, overnight incubation을 실시하였다. 시료의 정제를 위해 UltraPure™ Phenol:Chloroform:Isoamyl Alcohol(25:24:1, v/v)(Invitrogen)을 시료와 동일한 부피로 넣어 inverting한 후, 13,000 rpm에서 10분간 원심분리하였다. 수층을 따서 새로운 E.P tube에 옮겨 담은 후, 수층 volume의 2.5배 100% EtOH와 1/10 3M NaOAc(pH5.2)를 넣어 inverting 한 후, 가능한 최대 속도로 4℃에서 15분간 원심분리하였다. 상층액을 조심스럽게 제거하고, washing을 위해 70% EtOH 500 μl를 넣고 13,000 rpm에서 5분간 원심분리하였다. EtOH를 완벽하게 제거한 후, RNase-free water로 DNA pellet을 resuspension하였다. Nanodrop(Thermo-fisher scientific)을 이용하여 농도와 순도를 측정하였다.Prior to performing IVT, linearization of each sample was performed using plasmid DNA. Specifically, 30 μg of each sample was used to mix restriction enzyme (2U/μg), 5x enzyme reaction buffer, and nuclease free water so that the total volume of the sample was 300 μL. Each sample was subjected to overnight incubation at 37 °C. To purify the sample, UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (Invitrogen) was put in the same volume as the sample, inverted, and centrifuged at 13,000 rpm for 10 minutes. After picking up the water layer and transferring it to a new E.P tube, 2.5 times the volume of the water layer, 100% EtOH and 1/10 3M NaOAc (pH 5.2) were added, inverted, and then centrifuged at the maximum possible speed at 4°C for 15 minutes. The supernatant was carefully removed, and 500 μl of 70% EtOH was added for washing, followed by centrifugation at 13,000 rpm for 5 minutes. After completely removing EtOH, the DNA pellet was resuspended in RNase-free water. Concentration and purity were measured using Nanodrop (Thermo-fisher scientific).
1-2. In vitro transcription(IVT)1-2. In vitro transcription (IVT)
IVT는 Linearization 된 주형 DNA를 이용하여 시험관 내에서 T7 RNA polymerase를 사용하여 RNA를 합성하는 과정이다. EZ™ MEGA T7 Transcription Kit(Enzynomics)의 제조업체의 지침에 따라 linearization DNA를 이용하여 IVT를 실시하였다. pDNA-S와 pDNA-eGFP는 linear RNA form이기 때문에 mRNA 안정성을 위해 IVT 과정에 ARCA(8mM)(New England Biolabs)를 혼합하여 반응시켰다. 각 시료는 37°C에서 1시간 40분 반응시키고, DNase I(2 μl/1 rxn)을 넣어 37°C에서 20분 동안 반응시켰다. EZ™ MEGA T7 Transcription Kit(Enzynomics)에 포함되어 있는 Lithium Chloride(LiCl) Precipitation Solution을 이용하여 RNA를 정제하였다. LiCl Precipitation Solution을 반응액의 절반 부피만큼 넣고 완전히 혼합하였고, -20℃에서 30분 이상 유지하였다. 그 후 가능한 최대 속도로 4℃에서 15분간 원심분리하였고, 상층액을 조심스럽게 제거하였다. 세척을 위해 70% EtOH 700 μl를 넣고 위와 같은 조건으로 재 원심분리하였다. EtOH를 완벽하게 제거한 후 RNase-free water로 RNA pellet을 resuspension하였다. Nanodrop(Thermo-fisher scientific)을 이용하여 수득된 circRNA-S(서열번호 11), circRNA-eGFP(서열번호 13), mRNA-S(서열번호 15) 및 mRNA-eGFP(서열번호 17)의 농도와 순도를 측정하였다. IVT is the process of synthesizing RNA using T7 RNA polymerase in vitro using linearized template DNA. IVT was performed using linearized DNA according to the manufacturer's instructions of the EZ™ MEGA T7 Transcription Kit (Enzynomics). Since pDNA-S and pDNA-eGFP are linear RNA forms, ARCA (8 mM) (New England Biolabs) was mixed and reacted in IVT process for mRNA stability. Each sample was reacted at 37 ° C for 1 hour and 40 minutes, and DNase I (2 μl / 1 rxn) was added and reacted at 37 ° C for 20 minutes. RNA was purified using the Lithium Chloride (LiCl) Precipitation Solution included in the EZ™ MEGA T7 Transcription Kit (Enzynomics). LiCl Precipitation Solution was added as much as half the volume of the reaction solution, thoroughly mixed, and maintained at -20 ° C for more than 30 minutes. It was then centrifuged at 4° C. for 15 minutes at the highest possible speed, and the supernatant was carefully removed. For washing, 700 μl of 70% EtOH was added and centrifuged again under the same conditions as above. After completely removing EtOH, the RNA pellet was resuspended in RNase-free water. Concentrations of circRNA-S (SEQ ID NO: 11), circRNA-eGFP (SEQ ID NO: 13), mRNA-S (SEQ ID NO: 15) and mRNA-eGFP (SEQ ID NO: 17) obtained using Nanodrop (Thermo-fisher scientific) Purity was measured.
상기 원형 RNA인 circRNA-S 및 circRNA-eGFP는 내부 리보솜 진입 부위의 5' 말단의 28 nt가 절단되고, 내부 리보솜 진입 부위의 5’ 말단이 5’ 스플라이싱 부위와 7 nt가 중첩된 것을 특징으로 한다.The circular RNAs circRNA-S and circRNA-eGFP are characterized in that 28 nt of the 5' end of the internal ribosome entry site is cleaved and the 5' end of the internal ribosome entry site overlaps the 5' splicing site by 7 nt. to be
1-3. RNA denatured gel electrophoresis 1-3. RNA denatured gel electrophoresis
(1) Running Buffer 및 Gel components (1) Running Buffer and Gel components
IVT가 완료된 mRNA의 integrity를 확인하기 위해 Mupid-One(mupid)의 Gel 상에서 분리하였다. 구체적으로, Running buffer를 총 1L의 부피로 D.W 948 mL, 1M sodium phosphate 10 mL, 37% HCHO(formaldehyde) 42 mL를 혼합하여 제조하였다. Agarose gel은 아래의 표 1과 같은 조성으로 제조하였다.In order to confirm the integrity of the IVT-completed mRNA, it was separated on Mupid-One (mupid) Gel. Specifically, running buffer was prepared by mixing 948 mL of D.W, 10 mL of 1M sodium phosphate, and 42 mL of 37% HCHO (formaldehyde) in a total volume of 1 L. Agarose gel was prepared with the composition shown in Table 1 below.
1 ea1ea
AgaroseAgarose 0.5 g0.5 g
1M sodium phosphate1M sodium phosphate 1 mL1mL
37% HCHO37% HCHOs 4 mL4 mL
D.WD.W. 45 mL45 mL
(2) mRNA-S 와 circRNA-S의 준비 (2) Preparation of mRNA-S and circRNA-S
RiboRuler High Range RNA Ladder(Thermo-fisher scientific) 2 μL와 2x RNA loading dye(Thermo-fisher scientific) 4 μL의 부피로 혼합하여 준비하였다. 시료는 0.5 - 2μg을 준비하여 2X RNA loading dye, nuclease free water를 총 8 μL의 부피가 되도록 혼합하여 70°C에서 5분간 반응시켰다. 각각의 시료를 gel에 loading한 후 100 V로 1시간 전기영동 실시하였다. 전기영동 밴드를 확인하기 위해 Agarose gel을 Gel-doc system(Azure)으로 확인하였다. 전기영동 결과는 도 1에 나타내었다.It was prepared by mixing 2 μL of RiboRuler High Range RNA Ladder (Thermo-fisher scientific) and 4 μL of 2x RNA loading dye (Thermo-fisher scientific). 0.5 - 2 μg of sample was prepared, 2X RNA loading dye and nuclease free water were mixed to a total volume of 8 μL, and reacted at 70 ° C for 5 minutes. After loading each sample on a gel, electrophoresis was performed at 100 V for 1 hour. To confirm the electrophoretic band, the agarose gel was confirmed using the Gel-doc system (Azure). The electrophoresis results are shown in FIG. 1 .
도 1에 나타낸 바와 같이, linear RNA form인 mRNA-S 및 circular RNA form인 circRNA-S를 확인하였다.As shown in Figure 1, mRNA-S in linear RNA form and circRNA-S in circular RNA form were identified.
실시예 2. RNase R 처리에 따른 circRNA와 linearRNA의 비교 Example 2. Comparison of circRNA and linearRNA according to RNase R treatment
각각의 시료 20 μg을 물에 희석(43 μl 최종 부피)한 다음 65 ℃에서 3분 동안 가열하고, 3분 동안 냉각하였다. 20U RNase R 및 10μL의 10x RNase R buffer(Epicenter)를 첨가하고, 반응물을 37 ℃에서 15 분 동안 반응시켰다. 그 후 추가 10U RNase R을 반응 중간에 첨가하였다. 반응이 끝난 시료는 RNA cleanup kit(NEB)로 purification을 수행하였다. 정제한 3종의 RNA sample을 nanodrop(Thermo Fisher Scientific) 으로 농도를 측정하였다. 또한 실시예 1-3과 동일한 방법으로 RNA 전기영동을 수행하였다. 본 실험의 대조군은 mRNA-S 및 CircRNAR-S를 사용하였다. 상기 CircRNAR-S는 휴스턴 메소디스트 리딩 메드신 (Houston Methodist Leading Medicine)으로부터 compact하게 붙여진 형태로 합성된 circular form의 RNA이다. RNA 전기영동 결과는 도 2에 나타내었다.20 μg of each sample was diluted in water (43 μl final volume) then heated at 65° C. for 3 minutes and cooled for 3 minutes. 20 U RNase R and 10 μL of 10x RNase R buffer (Epicenter) were added, and the reaction was reacted at 37° C. for 15 minutes. An additional 10 U RNase R was then added in the middle of the reaction. After the reaction, the sample was purified with RNA cleanup kit (NEB). The concentration of three types of purified RNA samples was measured by nanodrop (Thermo Fisher Scientific). In addition, RNA electrophoresis was performed in the same manner as in Example 1-3. As a control group in this experiment, mRNA-S and CircRNA R -S were used. The circRNA R -S is RNA in circular form synthesized in a compact form from Houston Methodist Leading Medicine. RNA electrophoresis results are shown in FIG. 2 .
도 2에 나타낸 바와 같이, linear RNA form인 mRNA-S는 RNase R 처리에 따라 분해된 것을 확인하였다. 그러나 circular RNA form인 CircRNA-S 및 pCircRNAR-S은 RNase R 처리에도 불구하고 밴드가 확인되었다. 상기 결과는 in vitro에서 circular RNA가 mRNA보다 더 오래 유지된다는 것을 의미한다.As shown in Figure 2, it was confirmed that mRNA-S, which is a linear RNA form, was degraded by RNase R treatment. However, circRNA-S and pCircRNA R -S, which are circular RNA forms, showed bands despite RNase R treatment. The above result means that circular RNA is maintained longer than mRNA in vitro.
실시예 3. 시간에 따른 circular RNA의 stability 확인Example 3. Checking the stability of circular RNA over time
3-1. Cell culture 및 transfection 3-1. Cell culture and transfection
HEK293T 세포를 10% FBS(Gibco), 1% penicillin/streptomycin, Dulbecco's Modified Eagle Medium(DMEM) high glucose 배지에서 37 ℃ 및 5% CO2에서 배양하였다. 제조업체의 지침에 따라 HEK293T 세포에 mRNA-eGFP 또는 circRNA-eGFP를 각각 1μg씩 첨가하였다. 그 후 Lipofectamine2000(Invitrogen)과 Opti-MEM(Gibco)을 사용하여 6 well plate에 well당 500,000 Cell/1mL로 transfection하였다. Transfection 4시간 이후에 처음 배양 배지인 DMEM high glucose 배지로 변경하였다. Day1, Day3 일 차에 Trizol(Invitrogen)을 사용하여 RNA 추출을 위한 cell lysis를 하였다. Trizol(Invitrogen)의 제조업체 지침에 따라 Lysis된 세포로부터 RNA를 추출하였다. Lysis된 세포로부터 추출된 RNA는 linear RNA form인 mRNA-eGFP(서열번호 17) 및 circular RNA form인 circRNA-eGFP(서열번호 13)이다.HEK293T cells were cultured in 10% FBS (Gibco), 1% penicillin/streptomycin, and Dulbecco's Modified Eagle Medium (DMEM) high glucose medium at 37°C and 5% CO 2 . 1 μg each of mRNA-eGFP or circRNA-eGFP was added to HEK293T cells according to the manufacturer's instructions. After that, Lipofectamine2000 (Invitrogen) and Opti-MEM (Gibco) were used to transfect 500,000 cells/1mL per well in a 6-well plate. After 4 hours of transfection, the medium was changed to the first culture medium, DMEM high glucose medium. On Day 1 and Day 3, cell lysis was performed for RNA extraction using Trizol (Invitrogen). RNA was extracted from lysed cells according to the manufacturer's instructions of Trizol (Invitrogen). The RNAs extracted from the lysed cells are mRNA-eGFP (SEQ ID NO: 17) in linear RNA form and circRNA-eGFP (SEQ ID NO: 13) in circular RNA form.
3-2. RNA Reverse transcription3-2. RNA reverse transcription
추출된 RNA 1μg을 사용하여 RevertAid First Strand cDNA Synthesis Kit(Thermo Scientific)의 제조업체 지침에 따라 cDNA를 합성하였다. 1 μg of extracted RNA was used to synthesize cDNA according to the manufacturer's instructions of the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific).
3-3. Realtime qPCR 3-3. Realtime qPCR
Target gene에 따른 유전자 발현양을 확인하기 위해 합성된 cDNA를 이용하여 realtime qPCR을 진행하였다. Faststart SYBR green master(Roche), 10X concentrated solution of the PCR primer를 준비하였다. Forward 및 reverse primer는 0.4μM의 농도로 사용하고, target gene은 eGFP, housekeeping gene은 b-actin을 사용하였다. 1.5mL E.P tube를 ice에 두고, well에 표 2와 같이 총 20μl PCR Mix를 준비하였다.Realtime qPCR was performed using the synthesized cDNA to confirm the gene expression level according to the target gene. Faststart SYBR green master (Roche), 10X concentrated solution of the PCR primer was prepared. Forward and reverse primers were used at a concentration of 0.4 μM, eGFP was used as the target gene, and b-actin was used as the housekeeping gene. Place the 1.5mL E.P tube on ice, and prepare a total of 20μl PCR Mix in the well as shown in Table 2.
1 rxn1 rxn
FastStart SYBR Green MasterFastStart SYBR Green Master 10 μL10 µL
cDNA(10ng)cDNA (10 ng) 2 μL2 μL
Primer-F(0.4μM)Primer-F (0.4μM) 0.8 μL0.8 µL
Primer-R(0.4μM)Primer-R (0.4 μM) 0.8 μL0.8 µL
D.WD.W. 6.4 μL6.4 µL
TotalTotal 20 μL20 µL
PCR MIX를 잘 섞어 준비된 plate에 loading 한 후, Lightcycler96 기기(Roche)를 사용하여 qPCR을 수행하였다. 각 샘플에 대한 Ct값을 비교하여 계산하고, 유전자 발현 수준을 housekeeping gene인 b-actin으로 normalization을 하였다. qPCR 결과는 도 3에 나타내었다.After mixing the PCR MIX well and loading it on the prepared plate, qPCR was performed using a Lightcycler96 device (Roche). The Ct value for each sample was compared and calculated, and the gene expression level was normalized to b-actin, a housekeeping gene. The qPCR results are shown in FIG. 3 .
도 3에 나타낸 바와 같이, linear RNA form인 mRNA-eGFP는 시간이 지남에 따라 농도가 감소(즉, 분해)된 것을 확인하였다. 그러나 circular RNA form인 CircRNA-eGFP는 시간이 지났음에도 농도가 유지되는 것을 확인하였다. 상기 결과는 circular RNA가 mRNA보다 더욱 안정하다는 것을 의미한다.As shown in Figure 3, it was confirmed that the concentration of mRNA-eGFP, which is a linear RNA form, decreased (ie, degraded) over time. However, it was confirmed that the circular RNA form, CircRNA-eGFP, maintained its concentration over time. This result means that circular RNA is more stable than mRNA.
실시예 4. Splicing junction site sequencing Example 4. Splicing junction site sequencing
본 실험에서는 5’ 스플라이싱 사이트만 포함하는 pCircRNA-ver2.0로 생산된 원형 RNA의 스플라이싱 사이트를 분석하였다. 본 실험의 대조군으로는 3’ 및 5’ 스플라이싱 사이트를 모두 포함하는 벡터(pCircRNA-ver1.0)를 사용하였다.In this experiment, the splicing site of circular RNA produced with pCircRNA-ver2.0 containing only the 5' splicing site was analyzed. As a control for this experiment, a vector (pCircRNA-ver1.0) containing both 3' and 5' splicing sites was used.
4-1. 역전사 및 cDNA 합성과 PCR 4-1. Reverse transcription and cDNA synthesis and PCR
스플라이싱 접합 시퀀싱을 위해, RNase R로 circRNA를 컬럼 정제한 스플라이싱 반응물 10 μL과 oligodT(invitrogen) 1 μL, dNTP(Enzynomics) 1 μL, nuclease free water 1 μL(13 μL 최종 부피)을 65 ℃에서 5 분 동안 가열하고 얼음에서 3 분 동안 냉각하여 2차 구조를 표준화하였다. 역전사 반응을 추정 circRNA 내부 영역에 SuperiorScript III reverse transcriptase(Enzynomics) 1 μL을 사용하여 제조업체의 지침에 따라 수행하였으며, 구체적인 조건은 하기 표 3에 나타내었다.For splicing junction sequencing, 10 μL of splicing reaction column-purified circRNA with RNase R, 1 μL of oligodT (invitrogen), 1 μL of dNTP (Enzynomics), and 1 μL of nuclease free water (13 μL final volume) were added to 65 The secondary structure was normalized by heating at °C for 5 min and cooling on ice for 3 min. The reverse transcription reaction was performed according to the manufacturer's instructions using 1 μL of SuperiorScript III reverse transcriptase (Enzynomics) for the internal region of the putative circRNA, and the specific conditions are shown in Table 3 below.
. .
Temperature(℃)Temperature(℃) Time(min)Time(min)
2525 2020
5050 6060
7070 1515
44 ∞∞
2X topsimple Dyemix - Tenuto(Enzynomics)와 스플라이스 접합부에 걸쳐있는 한 쌍의 프라이머를 사용하여 시퀀싱을 위한 PCR 산물을 합성하였다. 구체적인 PCR 조건은 표 4에 나타내었다.PCR products for sequencing were synthesized using 2X topsimple Dyemix - Tenuto (Enzynomics) and a pair of primers spanning splice junctions. Specific PCR conditions are shown in Table 4.
StepStep Temperature(℃)Temperature(℃) TimeTime
1One 9898 3:003:00
22 9898 0:100:10
33 6464 0:30 touch down(-0.5℃)0:30 touch down(-0.5℃)
44 7272 0:300:30
55 GOTO STEP2GOTO STEP2 9X9X
66 9898 0:100:10
77 5959 0:300:30
88 7272 0:300:30
99 GOTO STEPGOTO STEP 14X14X
1010 7272 2:002:00
1111 44 ∞∞
DNA cleanup kit(New England Biolabs)의 제조업체의 지침을 따라 합성된 DNA를 정제하였다. Sanger-sequencing을 업체에 의뢰하여 splicing junction site를 확인하였으며, 그 결과는 도 4에 나타내었다.The synthesized DNA was purified according to the manufacturer's instructions of a DNA cleanup kit (New England Biolabs). Sanger-sequencing was requested to the company to confirm the splicing junction site, and the results are shown in FIG. 4 .
도 4에 나타낸 바와 같이, 3’ splicing site가 없는 pCircRNA(CircRNA-ver2.0)는 스플라이싱이 정상적으로 일어난 것을 확인하였다. 보다 상세하게는, 스플라이싱 결과로 생성된 원형 RNA는 IRES 부위의 절단이 관찰되었고, IRES 및 5’ splicing site가 7 nt 중첩된 것을 확인하였다.As shown in Figure 4, it was confirmed that pCircRNA (CircRNA-ver2.0) without a 3' splicing site was spliced normally. More specifically, in the circular RNA produced as a result of splicing, cleavage of the IRES site was observed, and it was confirmed that the IRES and the 5' splicing site overlapped by 7 nt.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다. In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (21)

  1. 작동 가능하게 연결된 하기 요소를 포함하는 원형 RNA의 생산을 위한 벡터:A vector for the production of circular RNA comprising the following operably linked elements:
    1) 프로모터;1) promoter;
    2) 내부 리보솜 진입 부위(internal ribosomal entry site, IRES);2) internal ribosomal entry site (IRES);
    3) 단백질 코딩 영역;3) protein coding regions;
    4) 3' UTR(Untranslated Region);4) 3' UTR (Untranslated Region);
    5) 폴리 A(poly A); 및5) poly A; and
    6) 5' 스플라이싱 부위(5' splicing site)를 포함하는 5' 그룹 I 인트론 단편(5' group I intron fragment).6) A 5' group I intron fragment containing a 5' splicing site.
  2. 제1항에 있어서, 상기 벡터는 내부 리보솜 진입 부위의 상류(upstream)에 3' 스플라이싱 부위(3’ splicing site)를 포함하지 않는 것인, 벡터.The vector according to claim 1, wherein the vector does not include a 3' splicing site upstream of the internal ribosome entry site.
  3. 제1항에 있어서, 상기 벡터는 상기 1) 내지 6)의 요소가 순차적으로 작동가능하게 연결된 것인, 벡터.The vector according to claim 1, wherein the elements of 1) to 6) are sequentially and operably linked.
  4. 제1항에 있어서,According to claim 1,
    상기 벡터는 작동 가능하게 연결된 하기 요소를 포함하는, 벡터:A vector, wherein the vector comprises the following elements operably linked:
    1) 서열번호 1의 염기서열로 표시되는 프로모터;1) a promoter represented by the nucleotide sequence of SEQ ID NO: 1;
    2) 서열번호 2의 염기서열로 표시되는 내부 리보솜 진입 부위;2) an internal ribosome entry site represented by the nucleotide sequence of SEQ ID NO: 2;
    3) 단백질 코딩 영역;3) protein coding regions;
    4) 서열번호 3의 염기서열로 표시되는 3' UTR;4) 3'UTR represented by the nucleotide sequence of SEQ ID NO: 3;
    5) 폴리 A; 및5) Poly A; and
    6) 서열번호 4의 염기서열로 표시되는 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편.6) A 5' Group I intron fragment including a 5' splicing site represented by the nucleotide sequence of SEQ ID NO: 4.
  5. 제1항에 있어서, 상기 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편은 Anabaena pre-tRNA 유래인, 벡터.The vector according to claim 1, wherein the 5' group I intron fragment including the 5' splicing site is derived from Anabaena pre-tRNA.
  6. 제1항에 있어서, 상기 내부 리보솜 진입 부위는 뇌심근염 바이러스(Encephalomyocarditis virus, EMCV) 유래인, 벡터.The vector according to claim 1, wherein the internal ribosome entry site is derived from Encephalomyocarditis virus (EMCV).
  7. 제1항에 있어서, 상기 폴리 A는 50 내지 300bp인, 벡터.The vector according to claim 1, wherein the poly A is 50 to 300 bp.
  8. 제1항에 있어서, 상기 벡터는 변형된 핵산, 천연 핵산 또는 비천연 핵산을 포함하는 것인, 벡터.The vector according to claim 1, wherein the vector comprises a modified nucleic acid, a natural nucleic acid or a non-natural nucleic acid.
  9. 제1항에 있어서, 상기 벡터는 자가 스플라이싱(self-splicing)에 의해 원형 RNA를 생산할 수 있고, 내부 리보솜 진입 부위의 5' 말단이 자가 스플라이싱에 의해 절단되는 것인, 벡터.The vector according to claim 1, wherein the vector can produce circular RNA by self-splicing, and the 5' end of the internal ribosome entry site is cut by self-splicing.
  10. 제9항에 있어서, 상기 벡터의 내부 리보솜 진입 부위는 자가 스플라이싱에 의해 내부 리보솜 진입 부위의 5' 말단의 1 내지 50 nt가 절단되는 것인, 벡터.The vector according to claim 9, wherein 1 to 50 nt of the 5' end of the internal ribosome entry site of the vector is cut by self-splicing.
  11. 제10항에 있어서, 상기 벡터의 내부 리보솜 진입 부위는 내부 리보솜 진입 부위의 5' 말단이 5' 스플라이싱 부위와 중첩되는 것인, 벡터.The vector according to claim 10, wherein the 5' end of the internal ribosome entry site of the vector overlaps the 5' splicing site.
  12. 제11항에 있어서, 상기 벡터의 내부 리보솜 진입 부위는 내부 리보솜 진입 부위의 5' 말단이 5' 스플라이싱 부위와 1 내지 10 nt 중첩되는 것인, 벡터.The vector according to claim 11, wherein the 5' end of the internal ribosome entry site of the vector overlaps the 5' splicing site by 1 to 10 nt.
  13. 작동 가능하게 연결된 하기 요소를 포함하는 원형 RNA:Circular RNA comprising the following elements operably linked:
    1) 내부 리보솜 진입 부위;1) an internal ribosome entry site;
    2) 단백질 코딩 영역;2) protein coding regions;
    3) 3' UTR;3) 3' UTR;
    4) 폴리 A; 및4) Poly A; and
    5) 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편.5) a 5' group I intronic fragment containing a 5' splice site.
  14. 제13항에 있어서,According to claim 13,
    상기 원형 RNA는 작동 가능하게 연결된 하기 요소를 포함하는, 원형 RNA:Circular RNA comprising the following elements operably linked:
    1) 서열번호 1의 염기서열로 표시되는 프로모터;1) a promoter represented by the nucleotide sequence of SEQ ID NO: 1;
    2) 서열번호 6의 염기서열로 표시되는 내부 리보솜 진입 부위;2) an internal ribosome entry site represented by the nucleotide sequence of SEQ ID NO: 6;
    3) 단백질 코딩 영역;3) protein coding regions;
    4) 서열번호 3의 염기서열로 표시되는 3' UTR;4) 3'UTR represented by the nucleotide sequence of SEQ ID NO: 3;
    5) 폴리 A; 및5) Poly A; and
    6) 서열번호 4의 염기서열로 표시되는 5' 스플라이싱 부위를 포함하는 5' 그룹 I 인트론 단편.6) A 5' Group I intron fragment including a 5' splicing site represented by the nucleotide sequence of SEQ ID NO: 4.
  15. 제13항에 있어서, 상기 원형 RNA는 내부 리보솜 진입 부위의 5' 말단의 1 내지 50 nt가 절단된 것인, 원형 RNA.The circular RNA according to claim 13, wherein 1 to 50 nt of the 5' end of the internal ribosome entry site is cut off.
  16. 제13항에 있어서, 상기 원형 RNA는 내부 리보솜 진입 부위의 5' 말단이 5' 스플라이싱 부위와 중첩된 것인, 원형 RNA.The circular RNA according to claim 13, wherein the 5' end of the internal ribosome entry site overlaps with the 5' splicing site.
  17. 제16항에 있어서, 상기 원형 RNA는 내부 리보솜 진입 부위의 5' 말단이 5' 스플라이싱 부위와 1 내지 10 nt 중첩된 것인, 원형 RNA.The circular RNA according to claim 16, wherein the 5' end of the internal ribosome entry site overlaps the 5' splicing site by 1 to 10 nt.
  18. 제1항의 원형 RNA의 생산을 위한 벡터; 또는 제13항의 원형 RNA;를 포함하는 목적 질환의 예방 또는 치료용 약학적 조성물.A vector for production of the circular RNA of claim 1; Or the circular RNA of claim 13; a pharmaceutical composition for the prevention or treatment of a target disease comprising a.
  19. 제1항의 원형 RNA의 생산을 위한 벡터; 또는 제13항의 원형 RNA;를 포함하는 목적 질환 예방용 백신 조성물.A vector for production of the circular RNA of claim 1; Or the circular RNA of claim 13; vaccine composition for preventing the target disease comprising.
  20. 제1항의 원형 RNA의 생산을 위한 벡터 또는 제13항의 원형 RNA를 세포에 형질도입시키는 단계를 포함하는, 세포 내에서 목적 단백질을 발현시키는 방법.A method for expressing a target protein in a cell, comprising transducing the vector for production of the circular RNA of claim 1 or the circular RNA of claim 13 into a cell.
  21. 제1항의 원형 RNA의 생산을 위한 벡터 또는 제13항의 원형 RNA를 이를 필요로 하는 개체에 투여하는 단계를 포함하는 목적 질환의 치료 방법.A method of treating a target disease comprising administering the vector for production of the circular RNA of claim 1 or the circular RNA of claim 13 to a subject in need thereof.
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WO2017222911A1 (en) * 2016-06-20 2017-12-28 The Board Of Trustees Of The Leland Stanford Junior University Circular rnas and their use in immunomodulation
WO2019094486A1 (en) * 2017-11-07 2019-05-16 The University Of North Carolina At Chapel Hill Methods and compositions for circular rna molecules
KR20210018323A (en) * 2018-06-06 2021-02-17 매사추세츠 인스티튜트 오브 테크놀로지 Circular RNA for translation in eukaryotic cells
KR20210057019A (en) * 2018-07-24 2021-05-20 메이오 파운데이션 포 메디칼 에쥬케이션 앤드 리써치 Circularized engineered RNA and methods

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