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WO2025046039A1 - Procédés de fabrication d'arn circulaires - Google Patents

Procédés de fabrication d'arn circulaires Download PDF

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Publication number
WO2025046039A1
WO2025046039A1 PCT/EP2024/074233 EP2024074233W WO2025046039A1 WO 2025046039 A1 WO2025046039 A1 WO 2025046039A1 EP 2024074233 W EP2024074233 W EP 2024074233W WO 2025046039 A1 WO2025046039 A1 WO 2025046039A1
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ribozyme
sequence
interest
gene
modified
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Yifei DU
Venkatraman Ramakrishnan
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United Kingdom Research and Innovation
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United Kingdom Research and Innovation
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    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular

Definitions

  • the invention relates to methods for making circular RNAs.
  • the invention also relates to modified ribozymes for making circular RNAs, nucleic acid molecules for making circular RNAs, and kits for making circular RNAs.
  • Ribosomes are molecular machines that translate the genetic code carried by messenger RNAs (mRNA) into functional proteins. When exogenous mRNA is introduced into cells, the gene of interest (GOI) encoded by that mRNA can be expressed, forming the basis of mRNA therapeutics.
  • mRNA therapeutics did not receive significant investment due to challenges such as mRNA instability, high innate immunogenicity, and inefficient in vivo delivery (Pardi, N. et al. (2016) Nat Rev Drug Discov 17 , 261-279).
  • Recent advances in mRNA delivery and nucleotide modification techniques have led to the successful application of mRNA therapeutics, particularly in the field of vaccines.
  • other types of mRNA therapeutics such as protein replacement therapy, still face challenges due to mRNA instability.
  • Circular RNAs have emerged as a promising solution to address the instability problem of mRNAs in therapeutic applications. Due to their covalently closed structure, circRNAs are inherently resistant to exonucleases, making them more stable compared to linear mRNAs (Jeck, W. R. & Sharpless, N. E. (2014) Nat Biotechnol 32, 453-461). CircRNAs are widely present in human cells and can serve as microRNA and protein sponges, as well as templates for peptide or protein production (Liu, C. X. & Chen, L. L. (2022) Cell 185, 2016-2034).
  • circRNAs are primarily generated through back splicing within cells, they can also be synthesized in vitro using chemical or enzymatic ligation methods or through group I intron-based circularization methods (Petkovic, S. & Muller, S. (2015) Nucleic Acids Res 43, 2454-2465).
  • PIE permuted intron-exon method
  • TAC trans ribozyme-based circularization
  • Figure 1A-C Puttaraju, M. & Been, M. D. (1992) Nucleic Acids Res 20, 5357-5364; Wesselhoeft, R. A., Kowalski, P. S.
  • the invention provides a method for producing a circular gene of interest, the method comprising: a) providing a nucleic acid molecule, wherein the nucleic acid molecule comprises, in the 5’ to 3’ direction, a first bridge sequence, and a gene of interest, wherein the first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme, b) providing a modified ribozyme comprising a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme, and c) combining the nucleic acid molecule and the modified ribozyme under conditions suitable for circularization to occur.
  • the invention provides a circular RNA obtained by the methods described herein.
  • the invention provides a modified ribozyme for use in a method of circularizing an RNA molecule, the modified ribozyme comprising, in the 5’ to 3’ direction: a) a first extended guide sequence (EGS), b) a first loop sequence, and c) a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme.
  • EGS extended guide sequence
  • the invention provides a nucleic acid molecule containing a gene of interest for use in a method of circularizing the gene of interest, wherein the nucleic acid molecule comprises, in the 5’ to 3’ direction: a) a bridge sequence comprising a sequence corresponding to a 3’ portion of a ribozyme sequence, b) the gene of interest, c) a loop sequence, and d) an extended guide sequence.
  • the invention provides a kit for circularizing a gene of interest, the kit comprising a modified ribozyme and a nucleic acid molecule containing the gene of interest, wherein the modified ribozyme comprises a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme, and wherein the nucleic acid molecule containing the gene of interest comprises, in the 5’ to 3’ direction: a) a bridge sequence comprising a 3’ portion of a ribozyme, and b) the gene of interest.
  • the modified ribozyme may be a modified ribozyme of the third aspect.
  • the nucleic acid molecule may be a nucleic acid molecule of the fourth aspect.
  • the invention provides a DNA molecule encoding a modified ribozyme as defined herein.
  • the invention provides a DNA molecule encoding a nucleic acid molecule containing a gene of interest as defined herein.
  • the invention provides a kit for circularizing a gene of interest, the kit comprising a DNA molecule of the sixth aspect and a DNA molecule of the seventh aspect.
  • Figure 1 Comparison of prior art approaches and trans excision ribozyme-based circularisation (TERIC).
  • A Schematic depicting splicing by the group I intron from cyanobacterium Anabaena (Ana), resulting in a linear molecule comprising joined exons.
  • B Schematic depicting circularization of a gene of interest by the permuted intron-exon (PIE) method.
  • C Schematic depicting circularization of a gene of interest using the TRIC approach.
  • D Schematic depicting splicing by a trans excision ribozyme (TER).
  • E Schematic depicting circularization of a gene of interest using the TERIC approach.
  • Figure 2 (A) Secondary structure of the Ana group I intron. Lower case characters are exon sequences. TER V1 and TER V2 labels indicate points from which a 3’ portion of the intron is removed. (B) Details for designing of trans excision ribozymes (TERs) and corresponding precursor of GOIs (pGs) using the cyanobacterium Anabaena tRNALeu group I intron as an example.
  • TERs trans excision ribozymes
  • pGs corresponding precursor of GOIs
  • FIG. 3 (A) Splicing reactions of TERIC V1 and V2 were loaded to a 0.8% native agarose gel. The TRIC V2-CVB3-EGFP was loaded as a positive control. (B) pG, TER and circularized sample of TERIC V2 were loaded to a 6M urea-1 .5% agarose gel. Circular CVB3-EGFP was present in the circularized TERIC sample. (C) RT-PCR of the pG and circularized sample of TERIC V2. Primers used here are indicated in Figure 2B. (D-E) Sequence of TERs and pGs of TERIC V1 (SEQ ID NOs: 5 and 6) and V2 (SEQ ID NOs: 7 and 8).
  • FIG. 4 Optimization of (A) TER to pG ratio, (B) concentration of pGs, and (C) duration of circularization. 0.8% native agarose gel was used. In the case of (B) and (C), TERs have run out of the gel. TERIC V2 shows generally better circularization efficiency than the TERIC V1 , although both achieve circularization. The most efficient circularization protocol for TERIC V2 was identified as 200- 400 nM pG and 20-40 min of circularization in the presence of 2mM GTP and 4 times amount of TERs to pGs.
  • FIG. 5 (A) Circularization efficiencies between pGs with the eACA elements or the native tRNA sequences are compared. The results show the eACA is important for TERIC to work. (B) Sequence of the TERIC-pG-tRNA (SEQ ID NO: 9).
  • FIG. 6 (A) Modified and unmodified precursors of PIE, TRIC, and TERIC were subject to circularization and loaded onto a 0.8% native agarose. No modified circular RNA can be seen for any of these three constructs. (B) IGSs of TRIC and TERIC are mutated from UUGAG to CCGCC and the circZnf609 was selected as the target sequence. Mutation of IGS to CCGCC for the TERIC restored ribozyme activity as indicated by the presence of circular modified RNAs. (C) Sequences of the TER (1 ,226)-IGS CCGCC (SEQ ID NO: 10) and pG_circZnf609 (SEQ ID NO: 11).
  • Figure 7 Schematic overview of the TERIC approach.
  • Figure 8 The extended anticodon arm (eACA). As long as a stem loop structure can be found where the loop contains a uracil in 3 rd position and the stem is >5bp, a circularization site can be assembled. Thus, circularization sites can be either placed in UTR or CDS.
  • eACA extended anticodon arm
  • Figure 9 Circularization of pGs using either 3’-truncated TER or 5’ and-3’-truncated TER.
  • the 3’ bridge sequence in pGs is either identical to the truncated portion of the ribozyme or contains mutations. Circularization was performed at a 4:1 TER/pG ratio at 55 °C for 20 minutes and then analyzed on a 0.8% native agarose gel.
  • the invention provides a new method for circularizing genes of interest to produce circular RNAs.
  • the invention is based at least in part on the discovery that portions of a ribozyme can be relocated to a gene of interest to allow the ribozyme to interact with the gene of interest, and facilitate splicing in trans. This results in the circularization of the gene of interest, while the ribozyme itself can be reused.
  • the invention enables the circularisation of genes of interest which contain modified nucleotides.
  • a truncated ribozyme may be designed to lack a 3’ portion of its sequence.
  • a bridge sequence can then be designed which corresponds to this missing portion of the ribozyme.
  • the bridge sequence allows the truncated ribozyme to interact with the gene of interest and restore its tertiary structures, permitting splicing and circularisation of the gene of interest.
  • the methods comprise providing a nucleic acid molecule comprising a bridge sequence and the gene of interest, and a modified ribozyme.
  • the bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme.
  • the modified ribozyme comprises a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme.
  • the methods described herein further comprise combining the nucleic acid molecule and modified ribozyme under conditions suitable for circularization to occur, in order to produce a circular gene of interest.
  • Ribozymes such as group I introns, are capable of folding to form tertiary structures consisting of paired segments, termed P1-P10 (as shown in Figure 2A).
  • the methods of the invention exploit these self-interactions by relocating parts of the ribozyme sequence to a gene of interest.
  • the truncated ribozyme which might lack a 3’ portion of its sequence, or a 3’ portion and a 5’ portion, is then able to interact with the relocated parts of the ribozyme sequence on the gene of interest (the bridge sequence(s)) to restore its tertiary structure.
  • modified ribozymes which generally comprise a truncated ribozyme.
  • truncated ribozyme it is generally meant a ribozyme in which a 3’ portion of the corresponding wild-type ribozyme sequence has been removed.
  • a truncated ribozyme may also refer to a ribozyme in which a 3’ portion and a 5’ portion of the corresponding wild-type ribozyme sequence have been removed.
  • the ribozyme can be any ribozyme capable of acting as a trans excision ribozyme.
  • the ribozyme is derived from or is a group I intron.
  • suitable ribozymes are the Tetrahymena ribosomal intron, T4 phage thymidylate synthase intron, Anabaena (Ana) pre-tRNA intron, Azoarcus sp. BH72 lie tRNA intron, and Staphylococcus phage Twort ribonucleotide reductase intron. Sequences of these ribozymes are shown below, with the internal guide sequence (IGS) shown with underlined, shaded letters.
  • IGS internal guide sequence
  • Staphylococcus phage Twort ribonucleotide reductase intron Staphylococcus phage Twort ribonucleotide reductase intron:
  • Truncated ribozymes generally do not comprise a 3’ portion of a corresponding wild-type ribozyme.
  • a 3’ portion corresponding to nucleotides 227-249 may be removed.
  • the 3’ portion can also be shorter than this, for example corresponding to nucleotides 242-249.
  • a 3’ portion it is generally meant a 3’ end portion, such that the remaining ribozyme is essentially truncated at the 3’ end.
  • the 3’ portion which is removed is generally between 1 and 1500 nucleotides in length.
  • the truncated ribozyme may not comprise a 3’ portion of a corresponding wild-type ribozyme which is between 1 and 1250, 1 and 1000, 1 and 750, 1 and 500, 1 and 400, 1 and 300, 1 and 250, 1 and 200, 1 and 150, 1 and 100, 1 and 50, 5 and 1500, 5 and 1250, 5 and 1000, 5 and 750, 5 and 500, 5 and 400, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 50, 10 and 1500, 10 and 1250, 10 and 1000, 10 and 750, 10 and 500, 10 and 400, 10 and 300, 10 and 250, 10 and 200, 10 and 150, 10 and 100, 10 and 50, or 10 and 30 nucleotides
  • the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme which is between 1 and 30 nucleotides in length.
  • the 3’ portion which is missing or removed may be 23 nucleotides in length.
  • the 3’ portion which is removed may be 8 nucleotides in length.
  • the truncated ribozyme may not comprise a 3’ portion which is between 1 and 249 nucleotides in length.
  • the 3’ portion may variously be referred to as a portion which is “removed” from the ribozyme.
  • the method does not necessarily include a step of removing this portion (or a 5’ portion) from the ribozyme, since the ribozyme can be reused and therefore provided in an already truncated form.
  • the 3’ portion which is missing from the truncated ribozyme may generally be one that is involved in the formation of a paired segment in a wild-type ribozyme.
  • the 3’ portion may be one that forms, or is involved in the formation of, a P9 region in a wild-type ribozyme.
  • the ribozyme is an Ana group I intron ribozyme
  • the ribozyme may comprise nucleotides 1-226 of SEQ ID NO: 1 or SEQ ID NO: 49 or a variant thereof.
  • the ribozyme may comprise the nucleotide sequence of SEQ ID NO: 12. or a variant thereof.
  • the ribozyme may comprise nucleotides 1-241 of SEQ ID NO: 1 or SEQ ID NO: 49 or a variant thereof.
  • the ribozyme may comprise the nucleotide sequence of SEQ ID NO: 13 or a variant thereof.
  • the truncated ribozymes described herein may also not comprise a 5’ portion of a corresponding wildtype ribozyme.
  • a 5’ portion it is generally meant a 5’ end portion, such that the remaining ribozyme is essentially truncated at the 5’ end.
  • the 5’ portion which is removed is generally between 1 and 1500 nucleotides in length.
  • the truncated ribozyme may not comprise a 5’ portion of a corresponding wild-type ribozyme which is between 1 and 1250, 1 and 1000, 1 and 750, 1 and 500, 1 and 400, 1 and 300, 1 and 250, 1 and 200, 1 and 150, 1 and 100, 1 and 50, 5 and 1500, 5 and 1250, 5 and 1000, 5 and 750, 5 and 500, 5 and 400, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 50, 10 and 1500, 10 and 1250, 10 and 1000, 10 and 750, 10 and 500, 10 and 400, 10 and 300, 10 and 250, 10 and 200, 10 and 150, 10 and 100, 10 and 50, or 10 and 30 nucleotides in length.
  • the truncated ribozyme does not comprise a 5’ portion of a corresponding wild-type ribozyme which is between 1 and 30 nucleotides in length.
  • the 5’ portion may variously be referred to as a portion which is “removed” from the ribozyme.
  • the method does not necessarily include a step of removing this portion from the ribozyme, since the ribozyme can be reused and therefore provided in an already truncated form.
  • the 5’ portion that is missing from the truncated ribozyme may generally be one that forms an internal guide sequence (IGS).
  • IGS internal guide sequence
  • the IGS forms P1 and P10 regions by complementary base pairing. This is shown in Figure 2A.
  • the ribozyme used in the approaches described herein should generally match the ribozyme from which the bridge sequence on the gene of interest is derived.
  • the bridge sequence added to the gene of interest should also be derived from the Ana group I intron, although this is not absolutely necessary as long as the bridge sequence enables interaction with the truncated ribozyme.
  • the sequence corresponding to a 3’ portion of a ribozyme of the first bridge sequence may be different to the sequence of the 3’ portion of a corresponding wild-type ribozyme that is missing or absent from the truncated ribozyme.
  • the sequence of the first bridge sequence may differ from the missing 3’ portion of the corresponding wild-type ribozyme by insertion, addition, substitution or deletion of 1 nucleotide, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 nucleotides.
  • the sequence of the first bridge sequence may comprise a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the missing 3’ portion.
  • Sequence comparison may be made over the full-length of the relevant sequence described herein using a standard algorithm, such as GAP, BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147'. 195- 197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters.
  • GAP GAP
  • BLAST which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410
  • FASTA which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448
  • Smith-Waterman algorithm Smith and Waterman (1981) J. Mol Biol. 147'. 195- 197
  • TBLASTN program of Altschul e
  • the length of the bridge sequence corresponds to the length of the 3’ portion (or 5’ portion) which is removed from the ribozyme, although this is not a requirement. Differences in length between the missing 3’ portion (and 5’ portion) and the bridge sequence are tolerated well.
  • the missing 3’ portion (and 5’ portion) may be 1 , 2, 3, 4, 5, or 10 or more nucleotides longer or shorter than the bridge sequence.
  • a bridge sequence may comprise a sequence corresponding to a 3’ portion of a ribozyme, such as a 3’ end portion of a ribozyme. This may be referred to as a 3’ bridge sequence, or a first bridge sequence.
  • a bridge sequence may also comprise a sequence corresponding to a 5’ portion of a ribozyme, such as a 5’ end portion of a ribozyme. This may be referred to as a 5’ bridge sequence, or a second bridge sequence.
  • the first bridge sequence may be between 5 and 1500, 5 and 1000, 5 and 750, 5 and 500, 5 and 450, 5 and 400, 5 and 350, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 50 nucleotides in length. In many cases, the first bridge sequence may be between 5 and 30 nucleotides in length. For example, when an Ana group I intron is used, the first bridge sequence may be between 2 and 248 nucleotides in length. The first bridge sequence may be between 5 and 30 nucleotides in length, for example when an Ana group I intron is used. The first bridge sequence may be 8 nucleotides in length. The first bridge sequence may be 23 nucleotides in length.
  • the function of the bridge sequence is to facilitate interaction with a modified ribozyme as described herein.
  • the first bridge sequence generally comprises a portion which is capable of complementary base pairing with a 3’ portion of a ribozyme, such as a portion between 1 and 10 nucleotides in length.
  • the first bridge sequence may be capable of forming a paired segment with a modified ribozyme as described herein, or with a 3’ portion of a ribozyme.
  • the first bridge sequence may be capable of forming a P9 region with a 3’ portion of a ribozyme.
  • the first bridge sequence may comprise a portion which is capable of complementary base pairing with nucleotides 207-212 of SEQ ID NO: 1 or SEQ ID NO: 49 or a variant thereof.
  • the precise nucleotide sequence of the first bridge sequence is not critical to the invention, as long as the nucleotide sequence of the first bridge sequence enables interaction with the modified ribozyme.
  • sequence corresponding to the 3’ portion of a ribozyme of the first bridge sequence may be identical or different to the sequence of the 3’ portion of a corresponding wild-type ribozyme that is missing or absent from the truncated ribozyme.
  • the first bridge sequence may comprise a sequence corresponding to nucleotide residues 242-249 of SEQ ID NO: 1 or SEQ ID NO: 49 or a variant thereof.
  • the first bridge sequence may comprise the nucleotide sequence set forth in SEQ ID NO: 14 or a variant thereof.
  • the first bridge sequence may comprise a sequence corresponding to nucleotide residues 227-249 of SEQ ID NO: 1 or SEQ ID NO: 49 or a variant thereof.
  • the first bridge sequence may comprise the nucleotide sequence set forth in SEQ ID NO: 15 or a variant thereof.
  • the bridge sequence is joined (either directly or indirectly) to the 3’ end of a gene of interest.
  • the length of the second bridge sequence may vary depending on the ribozyme from which it is derived.
  • the second bridge sequence is generally at least two nucleotides in length.
  • the second bridge sequence is generally less than 1500 nucleotides in length.
  • the second bridge sequence may be between 2 and 1000, 2 and 750, 2 and 500, 2 and 450, 2 and 400, 2 and 350, 2 and 300, 2 and 250, 2 and 200, 2 and 150, 2 and 100, 2 and 50, or 2 and 30 nucleotides in length.
  • the second bridge sequence may be between 5 and 1500, 5 and 1000, 5 and 750, 5 and 500, 5 and 450, 5 and 400, 5 and 350, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 50 nucleotides in length. In many cases, the second bridge sequence may be between 5 and 30 nucleotides in length.
  • the second bridge sequence may comprise a portion that is capable of complementary base pairing with a 5’ portion of a modified ribozyme.
  • bridge sequences are described as “corresponding to” equivalent portions of ribozyme sequences, they do not need to be identical to those sequences. Any bridge sequence may be used that allows interaction with the modified ribozyme such that circularisation can occur.
  • a bridge sequence that “corresponds to” a portion of a ribozyme sequence may have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with a portion of a ribozyme sequence as defined herein.
  • the wild-type Ana ribozyme is modified to remove a 3’ portion that is 23 nucleotides in length, and corresponds to residues 227-249 of SEQ ID NO: 1 or SEQ ID NO: 49 (wild-type Ana) or a variant thereof.
  • This modified ribozyme then comprises residues 1-226 of SEQ ID NO: 1 or SEQ ID NO: 49 or a variant thereof.
  • a corresponding first bridge sequence which comprises a sequence corresponding to residues 227-249 of SEQ ID NO: 1 , is then added to a 5’ end of a gene of interest.
  • the first bridge sequence can interact with (i.e. complementary base pair with) the remaining portion of the ribozyme, for example forming the P9 region shown in Figure 2A.
  • the IGS of the modified ribozyme can form P1 and P10 regions with the gene of interest.
  • the gene of interest refers to the sequence which is to be circularized.
  • the GOI can comprise a coding sequence, coding for a peptide or protein, or can be a noncoding sequence.
  • the GOI can also comprise a combination of coding and noncoding sequence.
  • gene of interest encompasses sequences which include additional sequence elements, such as internal ribosome entry site (IRES) sequences, multiple siRNA target sites (msiTS), spacer sequences such as polyAC sequences, start codons, stop codons, and any other sequence elements known to be useful in the art for producing circular RNA.
  • IRS internal ribosome entry site
  • miTS multiple siRNA target sites
  • spacer sequences such as polyAC sequences, start codons, stop codons, and any other sequence elements known to be useful in the art for producing circular RNA.
  • Suitable IRESs for use in the invention include CVB3, CroV, CSFV, the DNA sequences of which are set out below.
  • the TERIC method is suitable for genes of interest of any length. TERIC is particularly suitable for long genes of interest.
  • “long” is generally considered to mean a sequence of at least 500 nucleotides.
  • the gene of interest may be at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, or at least 8000 nucleotides in length.
  • the gene of interest may also comprise an extended anticodon arm (eACA) sequence.
  • the eACA may be part of the gene of interest sequence (for example, it may be naturally occurring within the gene of interest), but for clarity is referred to separately herein.
  • An extended anticodon arm (eACA) sequence is one which is capable of forming a stem-loop structure (also known as a hairpin or hairpin loop). Stem-loop structures form when two regions of single-stranded RNA which are generally complementary to each other (when read in opposite directions) base-pair with each other. The base-pairing results in a double helix structure ending in an unpaired loop.
  • the natural propensity of eACA sequences to form stem-loop structures can be utilised to enable and enhance circularisation of a gene of interest.
  • a nucleic acid molecule to be circularized Prior to circularization, a nucleic acid molecule to be circularized comprises an eACA sequence in two separate portions. A first portion of the eACA sequence is positioned at or near the 5’ end of the gene of interest, and a second portion of the eACA sequence is positioned at or near the 3’ end of the gene of interest, as shown in Figure 2B and Figure 7.
  • splicing by the ribozyme causes the first and second portions of the eACA sequence to be covalently joined, in order to create a circular version of the gene of interest.
  • the first and second portions are joined to form the eACA sequence, which is generally capable of forming a stem-loop structure as shown in Figure 2B and Figure 7.
  • the first portion of the eACA sequence may comprise a first eACA stem portion and a first eACA loop portion.
  • the second portion of the eACA sequence may comprise a second eACA stem portion and a second eACA loop portion.
  • stem portion it is meant a part of the first (or second) portion of the eACA sequence that is capable of forming the stem of a stem-loop structure.
  • loop portion it is meant a part of the first (or second) portion of the eACA sequence that is capable of forming the loop of a stem-loop structure.
  • a stem-loop forming structure can be identified in a gene of interest, and that gene of interest can be rearranged to provide a first part of the stem-loop forming structure at one end, and the second part at the other end. This gene of interest can subsequently be used for circularization.
  • the stem and loop portions of the eACA sequence are capable of forming a stem-loop structure in the nucleic acid molecules described herein.
  • the eACA can be of any nucleotide sequence.
  • the last nucleotide in the second eACA loop portion is one which can form a wobble base pair with a corresponding nucleotide in the internal guide sequence described herein.
  • the last nucleotide in the second eACA loop portion is a uracil, and forms a wobble base pair with a corresponding guanine in the internal guide sequence.
  • the last nucleotide in the second eACA loop portion may also be cytosine and form a wobble base pair with adenosine.
  • the last nucleotide in the second eACA loop portion is one which can form a canonical base pair with a corresponding nucleotide in the internal guide sequence or one which does not form a wobble or canonical base pair with a corresponding nucleotide in the internal guide sequence.
  • the first and second eACA stem portions may be complementary to each other, though this is not strictly necessary, and some non-complementarity may be tolerated.
  • the first and second eACA stem portions are generally each at least 5 nucleotides in length but can be a short as 1 nucleotide in length each.
  • the stem portion lengths can be adapted depending on the gene of interest to be circularized. For example, longer stem lengths (such as lengths greater than 15 nt) may be advantageous if circularizing long (>500 nt) genes of interest.
  • first and second stem portions may each be at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25 or at least 30 nucleotides in length.
  • first and second stem portions may each be at least 15 or at least 25 nucleotides in length.
  • the first and second stem portions need not be the same length, for example one stem portion may be one or two nucleotides shorter than the other, provided a stem-loop structure can still be formed.
  • the anticodon arm loop of the Ana tRNA group I intron is naturally 7 nucleotides in length. Consequently, in the circular RNAs described herein, the loop of the stem-loop structure may be 7 nucleotides in length, particularly when the ribozyme used is, or is derived from, the Ana group I intron. If other group I introns are used, the loop of the stem-loop structure may have a different nucleotide length.
  • the loop of the stem-loop structure is generally between 3 and 10 nucleotides in length. In many cases, the loop of the stem-loop structure is at least 5 nucleotides in length, particularly if the ribozyme is or is derived from the Ana group I intron.
  • the first eACA loop portion comprises 4 nucleotides
  • the second eACA loop portion comprises 3 nucleotides.
  • the first and second eACA stem portions may each be at least 15 nucleotides in length
  • the first eACA loop portion may be 4 nucleotides in length
  • the second eACA loop portion may be 3 nucleotides in length.
  • the first portion of the eACA sequence may comprise as few as 5 nucleotides (for example, 1 stem nucleotide and 4 loop nucleotides).
  • the second portion of the eACA sequence may comprise as few as 4 nucleotides (for example, 1 stem nucleotides and 3 loop nucleotides).
  • the first portion of the eACA sequence may comprise, for example, 19 nucleotides (e.g. 15 stem nucleotides and 4 loop nucleotides), or 29 nucleotides (e.g. 25 stem nucleotides and 4 loop nucleotides).
  • the second portion of the eACA sequence may comprise, for example, 18 nucleotides (e.g. 15 stem nucleotides and 3 loop nucleotides), or 28 nucleotides (e.g. 25 stem nucleotides and 3 loop nucleotides).
  • An exemplary first portion of the eACA sequence comprising a 1 nucleotide stem and a 4 nucleotide loop may comprise the nucleotide sequence 5 -NNNNN-3’, wherein N is any nucleotide
  • an exemplary second portion of the eACA sequence comprising a 1 nucleotide stem and a 3 nucleotide loop may comprise the nucleotide sequence 5’-NNNU-3’, wherein N is any nucleotide.
  • a possible ACA sequence is GATCACCACTTTAAGGTGATC (SEQ ID NO: 19).
  • the NLuc GOI is rearranged such that the ACA sequence is provided in two portions (a 5’ first portion and a 3’ second portion).
  • the first portion of the eACA sequence comprises the sequence TTAAGGTGATC (SEQ ID NO: 20).
  • the second portion of the eACA sequence comprises the sequence GATCACCACT (SEQ ID NO: 21).
  • the first and second eACA loop portions base pair with the internal guide sequence (IGS) to form the P1 and P10 regions, which are critical for ribozyme activity.
  • the first eACA loop portion positioned towards the 5’ end of the gene of interest, base pairs with the IGS to form the P10 region. It is not necessary for all the nucleotides in the first eACA loop portion to form the P10 region, and in some cases only two nucleotides of the first eACA loop portion form the P10 region.
  • the second eACA loop portion positioned towards the 3’ end of the gene of interest, base pairs with the IGS to form the P1 region.
  • the last nucleotide of the second eACA loop portion (i.e. the nucleotide at the 3’ end of the second eACA loop portion) may form a wobble base pair with a corresponding nucleotide in the IGS.
  • the wobble base pair is a GU wobble base pair, with G in the IGS and U in the second eACA loop portion.
  • the wobble base pair provides the circularization site, such that once circularized, the nucleotide at the 3’ end of the second eACA loop portion forms the third nucleotide in the loop of the eACA stem-loop structure. This is depicted in Figure 8.
  • the last nucleotide of the second eACA loop portion may form a canonical base pair with a corresponding nucleotide in the internal guide sequence. In other embodiments, the last nucleotide of the second eACA loop portion may not form a wobble or canonical base pair with a corresponding nucleotide in the internal guide sequence.
  • the P1 region may also be formed by base pairing of the IGS with a region adjacent to the second eACA loop portion in the 3’ direction, known as the “P1 extension”. If present, the P1 extension typically comprises between 2 and 4 nucleotides, which base pair with the IGS. The P1 region may therefore be formed by the P1 extension and second eACA loop portion base pairing with the IGS, as shown in Figure 2B and Figure 7. Consequently, in some embodiments, the second portion of the eACA sequence and the P1 extension together are capable of forming a P1 region. If the P1 extension is not present, the P1 region is formed by only the second eACA loop portion base pairing with the IGS. P1 extensions have been described in Olson & Muller (2012) RNA 18:581-589. The contents of which are incorporated herein by reference. Generally, if an extended guide sequence (EGS) is used, the P1 extension region will be present.
  • EGS extended guide sequence
  • a particular advantage of the TERIC method is that it can utilise eACA sequences which are already present in the gene of interest. For example, if a stem-loop forming eACA sequence can be found in a gene of interest, this gene can be circularized efficiently without introducing any additional sequences. This in turn means that the resulting circular RNA is far less likely to be immunogenic.
  • An eACA sequence i.e. a stem-loop forming structure
  • the gene of interest is rearranged such that the eACA sequence is split into two portions, one at each end of the gene of interest.
  • This rearranged gene is then cloned into a TERIC construct for circularisation.
  • An example of this is the protein coding circular RNA T2A Nano Luciferase.
  • This circular RNA already comprises an eACA sequence in its natural sequence. This means a circularisation site can be introduced using the naturally occurring eACA sequence, without the need to perform mutations or introduce additional sequence.
  • Codon redundancy means that mutations can be made to the nucleotide sequence of the GOI without affecting the resulting peptide sequence. Consequently, an eACA sequence can be provided in the GOI without requiring the introduction of additional sequences. Instead, only selective mutation of the existing sequence is needed, following the rules of codon redundancy.
  • the circularization site can be created by introducing additional nucleotides. For example, as shown in Figure 8, 5 nucleotides (light grey nt) could be introduced to create a stem portion of the eACA sequence, using the existing sequence (black nt) of the GOI to provide the remainder of the eACA.
  • the GOI may comprise, in the 5’ to 3’ direction: a stop codon, a polyAC sequence, multiple siRNA target sites (msiTS), an IRES, a start codon, and the coding sequence including the eACA.
  • the GOI may comprise, in the 5’ to 3’ direction: multiple siRNA target sites (msiTS), an IRES, a start codon, a coding sequence, a stop codon, a polyAC sequence, and the eACA.
  • the first and/or second portions of the eACA sequence may naturally occur in the gene of interest. In other words, they may be part of the gene of interest and so are present without having to mutate the existing sequence or introduce additional sequence.
  • all or part of the eACA sequence may be derived from human ribosomal RNA (rRNA).
  • rRNA ribosomal RNA
  • the use of human rRNA has the potential to provide circular RNAs which are less immunogenic.
  • first and second portions of the eACA sequence in the gene of interest has little impact on circularization.
  • One portion of the eACA sequence may be identified in or placed in a coding sequence, whilst the other may be identified in or placed in an untranslated region. It is not necessary for both portions to be in the coding sequence, for example, or for both portions to be in the untranslated region.
  • nucleic acid molecules containing a gene of interest wherein the nucleic acid molecule comprises, in the 5’ to 3’ direction: a) a first bridge sequence, wherein the first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme, and b) the gene of interest.
  • the nucleic acid molecule comprises, in the 5’ to 3’ direction: a) a first bridge sequence, wherein the first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme, b) a first portion of an extended anticodon arm (eACA) sequence, c) the gene of interest, and d) a second portion of the eACA sequence.
  • first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme
  • eACA extended anticodon arm
  • nucleic acid molecules containing a gene of interest comprising, in the 5’ to 3’ direction: a) a first bridge sequence, wherein the first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme, b) the gene of interest, and c) a second bridge sequence, wherein the second bridge sequence comprises a sequence corresponding to a 5’ portion of a ribozyme.
  • the nucleic acid molecule comprises, in the 5’ to 3’ direction: a) a first bridge sequence, wherein the first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme, b) a first portion of an extended anticodon arm (eACA) sequence, c) the gene of interest, d) a second portion of the eACA sequence, and e) a second bridge sequence, wherein the second bridge sequence comprises a sequence corresponding to a 5’ portion of a ribozyme.
  • a first bridge sequence wherein the first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme
  • eACA extended anticodon arm
  • nucleic acid molecules Further components that may also be included in the nucleic acid molecules, such as extended guide sequences and loop sequences, are described below.
  • a nucleic acid molecule, bridge sequence, gene of interest, ribozyme, ribozyme portion or truncated ribozyme, extended guide sequence, loop sequence, homology arm, extended anticodon arm (eACA) sequence or other sequence described herein may comprise or consist of the amino acid sequence of a reference sequence set out herein or may be a variant of a reference sequence set out herein.
  • a variant sequence may have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the reference sequence.
  • a variant sequence may differ from the reference sequence by insertion, addition, substitution or deletion of 1 nucleotide, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 nucleotides.
  • the gene of interest may comprise at least one modified nucleotide.
  • the gene of interest may comprise any amount of modified nucleotides considered useful for the desired application of the circular RNA (e.g. therapeutic or research applications). Accordingly, the % of nucleotides in the gene of interest which are modified may vary from 0% to 100%. In some cases, the gene of interest may be fully modified, in that 100% of the nucleotides are modified.
  • the gene of interest may comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% modified nucleotides.
  • the modification may be a base modification.
  • the modification may be a ribose modification.
  • the modification may be selected from the group consisting of: m 5 C (5-methylcytidine); m 5 U (5-methyluridine); m 6 A (N 6 -methyladenosine); s 2 U (2-thiouridine); T (pseudouridine); N 1 T (N 1 -methylpseudouridine); Um (2'-O-methyluridine); m 1 A (1 -methyladenosine); m 2 A (2-methyladenosine); Am (2'-O-methyladenosine); ms 2 m 6 A (2-methylthio-N 6 -methyladenosine); i 6 A (N 6 -isopentenyladenosine); ms 2 i6A (2-methylthio-N 6 isopentenyladenosine); io 6 A (N 6 -(cis- hydroxyisopenteny
  • the gene of interest may comprise at least one modified nucleotide, wherein the modification is a m 6 A (N 6 -methyladenosine) modification.
  • the gene of interest may comprise at least 95% modified nucleotides, wherein the modification is a m 6 A (N 6 -methyladenosine) modification.
  • the gene of interest may comprise 100% modified nucleotides, wherein the modification is a m 6 A (N 6 - methyladenosine) modification.
  • the gene of interest may comprise at least one modified nucleotide, wherein the modification is a N 1 T (N 1 -methylpseudouridine) modification.
  • the gene of interest may comprise at least 95% modified nucleotides, wherein the modification is a N 1 1 (N 1 -methylpseudouridine) modification.
  • the gene of interest may comprise 100% modified nucleotides, wherein the modification is a N 1 1 (N 1 - methylpseudouridine) modification.
  • the gene of interest may comprise at least one modified nucleotide, wherein the modification is a m 5 C (5-methylcytidine) modification.
  • the gene of interest may comprise at least 95% modified nucleotides, wherein the modification is a m 5 C (5-methylcytidine) modification.
  • the gene of interest may comprise 100% modified nucleotides, wherein the modification is a m 5 C (5-methylcytidine) modification.
  • both the internal guide sequence of the ribozyme and the sequence of the gene of interest can be adapted to minimise the disruptive effect of modifications on interaction between ribozyme and GOL
  • the IGS can be adapted to be CG rich, for example comprising 60% or more, 80% or more, or 100% CG content.
  • CG rich for example comprising 60% or more, 80% or more, or 100% CG content.
  • corresponding adaptations should be made to the sequence of the gene of interest, in order to maintain complementary base pairing. The precise nature of the sequence alterations or adaptations will depend on the nucleotide modifications that are desired in the resulting circular RNA.
  • the nucleic acid molecules and modified ribozymes described herein may further each comprise an extended guide sequence (EGS).
  • a modified ribozyme may comprise a first EGS
  • a nucleic acid molecule containing the gene of interest may comprise a second EGS.
  • the first and the second EGS may be capable of complementary base pairing to each other.
  • the function of the EGS is generally to increase the length of the complementary base-pairing region between the ribozyme and the nucleic acid molecule containing the gene of interest.
  • the modified ribozymes described herein may comprise a first EGS positioned at a 5’ end of the truncated ribozyme.
  • the nucleic acid molecules containing the gene of interest described herein may comprise a second EGS positioned at a 3’ end of the nucleic acid molecule.
  • the first and second EGS may be partly or fully complementary to each other. Generally, mismatches are tolerated well and do not materially affect circularization. Accordingly, the first EGS may be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% complementary to the second EGS. Generally, the first EGS may be substantially complementary to the second EGS, such as at least 70% complementary. If present, the first and second EGS may each be between 1 and 500 nucleotides in length. For example, the first and second EGS may each be between 10 and 50 nucleotides in length. The first and second EGS may each be 20, 30, or 40 nucleotides in length.
  • An exemplary first EGS sequence is GGUCAAUCGGUUGGCUUCCG (SEQ ID NO: 22).
  • An exemplary second EGS sequence is CGGAAGCCAACCGAUUGACC (SEQ ID NO: 23).
  • the ribozymes and nucleic acid molecules described herein may further comprise loop sequences, such as a first loop sequence and a second loop sequence.
  • the first and second loops may be configured to act as spacers.
  • the first and second loops may act as spacers between, in the ribozyme, the internal guide sequence (IGS) and the first extended guide sequence (EGS), and in the nucleic acid molecule containing the gene of interest, the gene of interest and the second EGS.
  • the loop sequences are preferably not complementary to each other, such that there is little or no base pair interaction between the first and second loop sequences. Because of the low or non-complementarity between the two loop sequences, the base-paired P1 region remains at a fixed length.
  • the loop sequences are substantially non-complementary.
  • the loop sequences may have less than 30%, less than 20%, less than 10%, or less than 5% complementarity.
  • the first and second loop sequences may each be between 1 and 10 nucleotides in length. It is not necessary for the first and second loop sequences to have the same number of nucleotides, and in fact the TERIC method works well when the first and second loop sequences are different lengths.
  • the first loop sequence may be 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length.
  • the second loop sequence may be 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length.
  • a preferred combination is a 6 nucleotide first loop sequence and a 5 nucleotide second loop sequence.
  • Another preferred combination is a 3 nucleotide first loop sequence and a 2 nucleotide second loop sequence.
  • the first loop sequence is positioned 3’ to the first EGS and 5’ to the truncated ribozyme, in other words, in between the first EGS and the truncated ribozyme.
  • the second loop sequence is positioned 3’ to the gene of interest, and 5’ to the second EGS, in other words, between the gene of interest and the second EGS in the nucleic acid molecule containing the gene of interest. If the P1 extension region is present, the second loop sequence is 3’ to the P1 extension.
  • Circularization can be achieved using only a first loop sequence, positioned in between the first EGS and the truncated ribozyme, without a second loop sequence.
  • first loop sequence positioned between the first EGS and the truncated ribozyme
  • second loop sequence positioned between the second portion of the gene of interest and the second EGS.
  • An exemplary sequence for the first loop sequence is AAATAA.
  • An exemplary sequence for the second loop sequence is ACACC.
  • modified ribozymes comprising, in the 5’ to 3’ direction: a) an extended guide sequence (EGS) b) a loop sequence, and c) a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme.
  • EGS extended guide sequence
  • modified ribozymes comprising, in the 5’ to 3’ direction: a) an extended guide sequence (EGS) b) a loop sequence, and c) a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme and does not comprise a 5’ portion of a corresponding wildtype ribozyme.
  • EGS extended guide sequence
  • nucleic acid molecules containing a gene of interest comprising, in the 5’ to 3’ direction: a) a bridge sequence comprising a sequence corresponding to a 3’ portion of a ribozyme, b) the gene of interest, c) a loop sequence, and d) an extended guide sequence.
  • nucleic acid molecules containing a gene of interest comprising, in the 5’ to 3’ direction: a) a first bridge sequence comprising a sequence corresponding to a 3’ portion of a ribozyme, b) the gene of interest, c) a second bridge sequence comprising a sequence corresponding to a 5’ portion of a ribozyme, d) a loop sequence, and e) an extended guide sequence.
  • nucleic acid molecules containing a gene of interest comprising, in the 5’ to 3’ direction: a) a bridge sequence comprising a 3’ portion of a ribozyme, b) a first portion of an extended anticodon arm (eACA) sequence, c) the gene of interest, d) a second portion of the eACA sequence; e) a loop sequence, and f) an extended guide sequence.
  • a bridge sequence comprising a 3’ portion of a ribozyme
  • eACA extended anticodon arm
  • nucleic acid molecules containing a gene of interest comprising, in the 5’ to 3’ direction: a) a first bridge sequence comprising a sequence corresponding to a 3’ portion of a ribozyme, b) a first portion of an extended anticodon arm (eACA) sequence, c) the gene of interest, d) a second bridge sequence comprising a sequence corresponding to a 5’ portion of a ribozyme, e) a second portion of the eACA sequence; f) a loop sequence, and g) an extended guide sequence.
  • a first bridge sequence comprising a sequence corresponding to a 3’ portion of a ribozyme
  • eACA extended anticodon arm
  • the ribozyme and the nucleic acid molecule comprising the GOI may further be provided with homology arms.
  • the purpose of the homology arms is to enable the 5’ end of the GOI to interact with the 3’ end of the ribozyme, as shown in Figure 2B.
  • Figure 2B labels the homology arm on the GOI as “HR-G” and the homology arm on the ribozyme as “HR-R”.
  • the homology arms perform a similar function to the EGS, by extending the region of complementary base pairing between the GOI and the ribozyme.
  • the first and second homology arms may be partly or fully complementary to each other.
  • the first homology arm may be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% complementary to the second homology arm.
  • the homology arms are substantially complementary to each other, such as at least 70% complementary to each other.
  • the homology arms can be of any length which facilitates circularisation, and do not necessarily have to be of the same length.
  • the homology arm on the GOI can be longer or shorter than the homology arm on the ribozyme.
  • the homology arms may each be at least 1 , 2, 3, 4, 5, 10, 15, 20, 30, 50, 100, 150, 200, 250, 300 or 500 nucleotides in length.
  • the homology arms may each be between 1 and 50, 1 and 40, 1 and 30, 1 and 20, 5 and 50, 5 and 40, 5 and 30, 5 and 20, 10 and 50, 10 and 40, 10 and 30, or 10 and 20 nucleotides in length. In some cases, the homology arms are each 20 nucleotides in length.
  • HR-R ribozyme: CAGGACAACAGCATCACTAG (SEQ ID NO: 47)
  • the homology arm is generally placed at the 3’ end. In the case of the nucleic acid molecule containing the gene of interest, the homology arm is generally placed at the 5’ end.
  • a modified ribozyme as described herein may comprise, in a 5’ to 3’ direction: a) a first extended guide sequence (EGS), b) a first loop sequence, c) a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme, and d) a homology arm sequence.
  • EGS extended guide sequence
  • a nucleic acid molecule comprising a gene of interest as described herein may comprise, in a 5’ to 3’ direction: a) a homology arm sequence, b) a bridge sequence comprising a sequence corresponding to a 3’ portion of a ribozyme sequence, c) the gene of interest, d) a loop sequence, and e) an extended guide sequence.
  • the methods generally comprise providing a bridge sequence (as described herein) at a 5’ end of the gene to be circularized, and optionally also providing a second bridge sequence at a 3’ end of the gene to be circularised.
  • the bridge sequence(s) can be added to the gene of interest by methods known in the art.
  • a DNA template can be synthesised which comprises a bridge sequence 5’ to the gene of interest, and/or a bridge sequence 3’ to the gene of interest.
  • the first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme
  • the second bridge sequence comprises a sequence corresponding to a 5’ portion of a ribozyme.
  • the methods further comprise providing a modified ribozyme, as described elsewhere herein.
  • the modified ribozyme is one in which a 3’ portion of the ribozyme has been removed.
  • the modified ribozyme may comprise a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme.
  • the modified ribozyme may correspond to nucleotides 1-226, or 1-242 of SEQ ID NO: 1 , with nucleotides 227-249 or 243-249 removed, respectively.
  • the modified ribozyme may also comprise a truncated ribozyme wherein the truncated ribozyme does not comprise a 5’ portion of a corresponding wild-type ribozyme.
  • the truncated ribozyme may be truncated at both the 5’ and 3’ ends compared to a wild-type ribozyme.
  • the methods comprise combining the gene of interest and the modified ribozyme under conditions suitable for circularization to occur.
  • Circularisation protocols are generally known in the art.
  • such a step may be carried out for between 10 and 60 minutes, in some cases between 20 and 40 minutes.
  • Circularization may be achieved by heating the gene of interest and modified ribozyme together, for example to between 50°C and 60°C.
  • Circularization may be achieved by heating the mixture of the gene of interest and the modified ribozyme to about 55°C for about 20 minutes.
  • the gene of interest and the modified ribozyme may be combined and/or heated together in any suitable circularization buffer known in the art.
  • Methods for producing a circular gene of interest may comprise: a) providing a nucleic acid molecule, wherein the nucleic acid molecule comprises, in the 5’ to 3’ direction, a first bridge sequence, and a gene of interest, wherein the first bridge sequence comprises a sequence corresponding to a 3’ portion of a ribozyme, b) providing a modified ribozyme comprising a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme, and c) combining the nucleic acid molecule and the modified ribozyme under conditions suitable for circularization to occur.
  • the ratio of modified ribozyme to gene of interest is generally 1 :1 or greater.
  • the ratio of modified ribozyme to gene of interest may be 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , or greater.
  • the ratio of modified ribozyme to gene of interest may be 4:1 .
  • the concentration of gene of interest is generally between about 100 nM and about 500 nM. In some embodiments, the concentration of gene of interest may be between 200 nM and 400 nM.
  • steps of the methods described herein may be preceded by steps providing DNA templates encoding any of the modified ribozymes or genes of interest. Such methods may also include a step of in vitro transcription of the DNA templates to provide RNA precursors. The methods may also include a step of refolding the modified ribozymes and genes of interest following in vitro transcription and prior to the addition of a circularization buffer.
  • the methods described herein may further comprise a step of recovering the modified ribozyme.
  • the recovered modified ribozyme may then be used in a future reaction.
  • Suitable methods for recovering the modified ribozyme include separation and purification by gel filtration or gel extraction.
  • the modified ribozyme can be recovered and/or purified at the same time as recovering the desired circular RNA.
  • the TER can be immobilised on a solid surface, for example covalently linked to a bead or plate, or other suitable surface known in the art.
  • the gene of interest can be added, circularised and washed out for recovery of the circular RNA, while the TER remains bound to the solid surface and can be reused for a second and further rounds of circularisation with new genes of interest.
  • circular RNAs obtainable by the methods described above.
  • the invention also provides circular RNAs comprising a sequence encoding a gene of interest, wherein the circular RNA comprises at least one modified nucleotide residue as described herein, and wherein the circular RNA does not comprise exogenous exon sequences, and/or does not comprise any ribozyme-derived sequence.
  • the invention also provides circular RNAs comprising a sequence encoding a gene of interest, wherein the circular RNA comprises an eACA sequence, and wherein the circular RNA comprises at least one modified nucleotide residue. In some cases, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the nucleotide residues in the circular RNA may be modified.
  • kits for circularizing a gene of interest comprise a modified ribozyme and a nucleic acid molecule containing the gene of interest, as described herein.
  • Kits for circularizing a gene of interest may comprise a modified ribozyme and a nucleic acid molecule containing the gene of interest, wherein the modified ribozyme comprises a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme, and wherein the nucleic acid molecule containing the gene of interest comprises, in the 5’ to 3’ direction: a) a bridge sequence comprising a sequence corresponding to a 3’ portion of a ribozyme, and b) the gene of interest.
  • Kits for circularizing a gene of interest may comprise a modified ribozyme and a nucleic acid molecule containing the gene of interest, wherein the modified ribozyme comprises, in the 5’ to 3’ direction: a) a first extended guide sequence (EGS), b) a first loop sequence, c) an internal guide sequence (IGS), and d) a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme, and wherein the nucleic acid molecule containing the gene of interest comprises, in the 5’ to 3’ direction: a) a bridge sequence comprising a sequence corresponding to a 3’ portion of the ribozyme, b) a first portion of an extended anticodon arm (eACA) sequence, c) the gene of interest, d) a second portion of the eACA sequence; e) a second loop sequence, and f) a second
  • the disclosure also encompasses DNA precursor molecules encoding any of the modified ribozymes, genes of interest, and nucleic acid molecules containing genes of interest described herein.
  • any of the ribozymes or nucleic acid molecules described herein can be provided as DNA templates, which subsequently undergo in vitro transcription (IVT) in order to provide ribozymes and RNA molecules that can be circularized.
  • kits comprising a first DNA molecule encoding a modified ribozyme and a second DNA molecule encoding a nucleic acid molecule.
  • nucleic acid molecule further comprises a second bridge sequence located 3’ of the gene of interest, wherein the second bridge sequence comprises a sequence corresponding to a 5’ portion of a ribozyme, and wherein the truncated ribozyme does not comprise a 5’ portion of a corresponding wild-type ribozyme.
  • the 5’ portion of the corresponding wild-type ribozyme comprises a sequence corresponding to residues 1-10 of SEQ ID NO: 1 or 49.
  • the gene of interest comprises at least one modified nucleotide.
  • the method of clause 23 or 24, wherein the at least one modified nucleotide is selected from the group consisting of N6-methyladenosine, N1-methyl-pseudouridine, or combinations thereof.
  • the modified ribozyme further comprises a first extended guide sequence (EGS) at a 5’ end
  • the nucleic acid molecule further comprises a second EGS at a 3’ end.
  • EGS extended guide sequence
  • the method of clause 26, wherein the first and second EGS are substantially complementary to each other.
  • step (c) is performed for between 20 and 40 minutes.
  • step (c) comprises heating the mixture of the nucleic acid molecule and the modified ribozyme.
  • step (c) comprises heating the mixture of the nucleic acid molecule and the modified ribozyme to between 50°C and 60°C.
  • step (c) comprises heating the mixture of the nucleic acid molecule and the modified ribozyme to about 55°C for about 20 minutes.
  • a modified ribozyme for use in a method of circularizing an RNA molecule comprising, in the 5’ to 3’ direction: a) a first extended guide sequence (EGS), b) a first loop sequence, and c) a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme.
  • EGS extended guide sequence
  • nucleic acid molecule of clause 43 further comprising a second bridge sequence at a 3’ end, wherein the second bridge sequence comprises a sequence corresponding to a 5’ portion of a ribozyme sequence.
  • kits for circularizing a gene of interest comprising a modified ribozyme and a nucleic acid molecule containing the gene of interest, wherein the modified ribozyme comprises a truncated ribozyme, wherein the truncated ribozyme does not comprise a 3’ portion of a corresponding wild-type ribozyme, and wherein the nucleic acid molecule containing the gene of interest comprises, in the 5’ to 3’ direction: a) a bridge sequence comprising a 3’ portion of a ribozyme, and b) the gene of interest.
  • a kit comprising a first DNA molecule encoding the modified ribozyme of the kit of clause 41 or 42, and a second DNA molecule encoding the nucleic acid molecule containing the gene of interest of the kit of clause 43 or 44.
  • a circular RNA comprising a sequence encoding a gene of interest, wherein the circular RNA comprises at least one modified nucleotide residue, and wherein the circular RNA does not comprise exogenous exon sequences.
  • a circular RNA comprising a sequence encoding a gene of interest, wherein the circular RNA comprises an eACA sequence, and wherein the circular RNA comprises at least one modified nucleotide residue.
  • the plasmids TRIC V1-CVB3-EGFP (SEQ ID NO: 24), TRIC V2-CVB3-EGFP (SEQ ID NO: 25), TRIO V2-circZnf609 (SEQ ID NO: 26), PIE-CVB3-EGFP (SEQ ID NO: 27), and PIE-circZnf609 (SEQ ID NO: 28) were generated as described in GB2308675.4.
  • the inventors amplified them in TOP10 competent cells and purified them using the QIAGEN Maxi Plus plasmid purification kit. The purified plasmids were then linearized using EcoR V and cleaned through phenol:chloroform:isoamyl alcohol extraction.
  • IVT In vitro transcriptions
  • 1X IVT buffer included 80 mM HEPES-K (pH 7.5), 2 mM spermidine, 40 mM DTT, and 24 mM MgCh.
  • concentration of MgCh in 1X IVT buffer was adjusted to 14 mM.
  • the IVT reactions were incubated at 37 °C for 3-5 hours, followed by digestion with RNase-free DNase I for 20 minutes. To remove any precipitation, 100 mM EDTA was added to achieve a final concentration of 25 mM. Subsequently, an equal volume of 7.5 M lithium chloride was added to precipitate the RNAs. This precipitation step was performed for a duration of 30 minutes to overnight at -20 °C. The resulting precipitates were centrifuged at 13,000 rpm/min for at least 20 minutes, and the RNA pellets were washed with 75% alcohol, air-dried, and dissolved in DEPC-treated H2O.
  • Circular RNA synthesis
  • RNAs underwent a refolding process. They were initially denatured at 95 °C for 2 minutes and then annealed on ice for 3 minutes. The circularization step was carried out in a 10 pl reaction volume and was terminated by adding 2 pl of 100 mM EDTA.
  • RNAs at a final concentration of 200 nM were combined with 10X circularization buffer (composed of 500 mM Tris-HCI, pH 7.4, 100 mM MgCh, 10 mM DTT, and 20 mM GTP). The mixture was heated at 55 °C for 8 minutes to allow circularization.
  • 10X circularization buffer composed of 500 mM Tris-HCI, pH 7.4, 100 mM MgCh, 10 mM DTT, and 20 mM GTP.
  • protocol A TERs and pGs were mixed and refolded as described above in DEPC-treated H2O. Subsequently, they were supplied with the circularisation buffer for 20min of circularization at 55 °C.
  • protocol B the refolding of TSRs and pGs took place in the circularisation buffer, followed by mixing and 20min of circularization at 55 °C.
  • the splicing conditions such as the concentration of GTP, the ratio of TER to pG, the concentration of pGs, and the reaction duration, were optimized accordingly.
  • Reverse transcriptase and DNA polymerase used here are the SuperScrip IV Reverse Transcriptase (Thermo Fisher) and the Q5 High-Fidelity DNA Polymerase (NEB). Manufacturer’s manuals were followed for reverse transcription and PCR.
  • the pG and circularized pG of TERIC V2 were used as templates for reserve transcription using the RT-PCR_Reverse (GTGAACCGCATCGAGCTG (SEQ ID NO: 43)) as the reverse primer.
  • RT-PCR_Forward TTTGCTGTATTCAACTTAACAATGAATTGTAATG (SEQ ID NO: 44)
  • RT-PCR_Reverse RT-PCR product was gel extracted and submitted for Sanger sequencing.
  • RNA sample ( ⁇ 1 OOng) was mixed with equal volume of urea loading buffer (NEB) and denatured at 95 °C for 2 min. Gels were stained in 10ml 1X TBE with SYBR Safe for 10 min before imaging.
  • NEB urea loading buffer
  • Group I introns utilize internal guide sequences (IGS) to form P1 and P10 structures with flanking exons, bringing the exons into proximity to facilitate splicing ( Figure 2A).
  • IGS internal guide sequences
  • TER trans excision ribozyme
  • TER trans excision ribozyme
  • the inventors utilised a group I intron which included an IGS sequence (the cyanobacterium Anabaena tRNA Leu intron, referred to hereafter as “Ana”).
  • IGS sequence the cyanobacterium Anabaena tRNA Leu intron, referred to hereafter as “Ana”.
  • the inventors relocated a 3’ portion of the intron to the 5’ end of the gene of interest ( Figure 2B).
  • This 3’ portion also called a 3’ bridge sequence
  • this 3’ portion would form the P9.0 region with the trans excision ribozyme, thus improving the interaction between ribozyme and gene of interest to facilitate splicing.
  • EGS extended guide sequences
  • eACA extended anticodon arm
  • TER V1 (comprising nucleotides 1 to 241 of Ana, SEQ ID NO: 13) and TER V2 (comprising nucleotides 1 to 226 of Ana, SEQ ID NO: 12)
  • Figure 2B Precursors of the corresponding genes of interest (pGs) were also generated, using a 3’ bridge sequence of nucleotides 242-249 of Ana (SEQ ID NO: 14) for TER V1 , and nucleotides 227-249 of Ana (SEQ ID NO: 15) for TER V2 ( Figure 2B). These 3’ bridge sequences were joined to the 5’ end of a gene of interest encoding EGFP and including other sequence elements such as CVB3 (an IRES), stop and start codons, and polyAC.
  • CVB3 an IRES
  • stop and start codons and polyAC.
  • protocol B TSRs and pGs were individually refolded in splicing buffer, followed by mixing and heating at 55°C for 20 minutes. The final concentration of pGs was 200 nM. Splicing reactions were halted using 2 pL of 100 mM EDTA and loaded onto a 0.8% native agarose gel.
  • TERIC V2 exhibited the highest splicing efficiency at 2 mM GTP when protocol A was employed, although TER V1 also achieved efficient splicing with protocol A. Splicing was also achieved with protocol B, but was less efficient compared to protocol A.
  • the bands marked by empty circles represent circCVB3-EGFP.
  • the inventors optimized the ratio between TERs and pGs. Initially, the inventors maintained the concentration of pGs at 200 nM and gradually increased the concentration of TERs from 200 nM to 1600 nM.
  • Figure 4A illustrates that within the TER/pG ratio range of 1-4, an increase in TER concentration resulted in improved circularization efficiency. However, when the TER/pG ratio exceeded 4, the circularization efficiency did not exhibit significant changes.
  • the inventors fixed the ratio between TERs and pGs at 4 and investigated the effect of pG concentration and circularization time. As depicted in Figures 4B and 4C, pGs concentrations ranging from 200 nM to 400 nM, along with circularization times of 20-40 minutes, yielded the highest circularization efficiency while maintaining a relatively low level of nicking.
  • unmodified PIE and TRIC precursors efficiently convert to circular RNAs, while no circular RNA is observed for the modified PIE or TRIC variants.
  • the inventors synthesized modified precursors while keeping the ribozyme unmodified. Consistent with the previous observations, unmodified TERIC V2 successfully circularized the unmodified pG. However, surprisingly, no circular RNAs were observed for the modified pGs. The inventors identified two potential reasons for the failure to circularize modified pGs.
  • the 3’ bridge sequence located at the 5’ end of the pGs would be modified, and this 3’ bridge sequence spans nucleotides 227-249 of the Ana intron. It is possible that m6A or N1 1 modifications within this short 3’ bridge sequence abolishes TERIC activity.
  • the IGS in TER V2, UUGAG is an AU-rich sequence, and m6A or N1 T modifications in the corresponding eACA could weaken the P1 and P10 structures, thus disrupting TER activity.
  • the purpose of relocating the 3’ portion of the ribozyme to the 5’ end of GOIs is to reconstitute the ribozyme structure between the ribozyme and the GOI. It is the structure, rather than the sequence or length, that is crucial for reconstitution to be functional. To confirm this, we introduced mutations in the 3’ portion of the ribozyme that was moved to the 5’ end of the GOI (SEQ ID NOs: 53 and 54).
  • TERIC can also be used for circularization of modified RNAs where previous approaches such as PIE and TRIC are unsuitable.

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Abstract

La présente invention concerne des procédés de production d'un gène circulaire d'intérêt. L'invention concerne une molécule d'acide nucléique qui comprend, dans la direction 5' vers 3', une première séquence de pontage et un gène d'intérêt. La première séquence de pontage comprend une séquence correspondant à une partie 3' d'un ribozyme. L'invention concerne également un ribozyme modifié comportant un ribozyme tronqué qui ne contient pas une partie 3' d'un ribozyme de type sauvage correspondant. La molécule d'acide nucléique et le ribozyme modifié sont combinés dans des conditions appropriées pour que la circularisation se produise. L'invention concerne également des ribozymes modifiés, des molécules d'acide nucléique et des kits.
PCT/EP2024/074233 2023-09-01 2024-08-29 Procédés de fabrication d'arn circulaires Pending WO2025046039A1 (fr)

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