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CN-121986162-A - Method for preparing circular RNA and nucleic acid sequence for said method

CN121986162ACN 121986162 ACN121986162 ACN 121986162ACN-121986162-A

Abstract

A method for producing a circular RNA is provided, which comprises inserting a poly (A) tag or a functional variant thereof or other purification tag into both end arms of a linear RNA precursor, and separating the circular RNA obtained by cyclization reaction of the linear RNA precursor from other cyclization reaction byproducts as impurities by using an affinity chromatography medium capable of simultaneously binding to the tag. Also provided are linear RNA precursors having a purification tag inserted into a specific region and DNA encoding the same, wherein insertion of the purification tag into the specific region does not affect the yield of linear RNA precursors produced by In Vitro Transcription (IVT) of the encoding DNA.

Inventors

  • ZHANG WEIGUO
  • DONG YIJIE
  • CHEN HUA

Assignees

  • 仁景(苏州)生物科技有限公司
  • 仁景国际香港有限公司

Dates

Publication Date
20260505
Application Date
20240705
Priority Date
20230707

Claims (20)

  1. A method of making a circular RNA, the method comprising: Step A, generating a linear RNA precursor, wherein the linear RNA precursor sequentially comprises a 5 'end arm, a 3' self-splice site, a cyclization region, a 5 'self-splice site and a 3' end arm in a manner of being operably connected with each other in a 5 'to 3' direction, and a first label is inserted into the 5 'end arm and a second label is inserted into the 3' end arm; Step B, subjecting the linear RNA precursor to conditions suitable for self-splicing of the 3 'self-splice site and the 5' self-splice site to obtain a mixture comprising linear RNA fragments bearing the first tag and/or the second tag and circular RNA cyclized by the cyclized region; Step C of contacting said mixture with an affinity chromatography medium capable of simultaneously binding said first tag and said second tag for a time sufficient for said affinity chromatography medium to bind linear RNA fragments comprising at least one of said tags, and Step D, separating the affinity chromatography medium from the mixture contacted with the affinity chromatography medium, collecting supernatant to obtain circular RNA, Wherein the first tag and/or the second tag is independently selected from a poly (a) tag consisting of 20 to 100, preferably 30 to 90, more preferably 40 to 80, even more preferably 45 to 70, most preferably 50 to 65 consecutive adenine nucleotides or a functional variant thereof, and wherein the first tag and the second tag are not present in both the circularized region and the circular RNA.
  2. The method of claim 1, wherein the functional variant of the poly (a) tag is the insertion of 1 or more non-a bases, preferably 1-20 non-a bases, more preferably 1-10 non-a bases, in the poly (a) tag.
  3. The method of any of the preceding claims, wherein the functional variant of the poly (a) tag comprises (1) A unique element a, at least one element b, and at least one element c, (2) A single element a, at least one element b, and at least one element d, or (3) The sole element a, at least one element b, and at least one element c and at least one element d, Wherein the element a is composed of 20 or more consecutive adenine nucleotides, the element b is composed of 3 or more and less than 20 consecutive adenine nucleotides, the element c is composed of one nucleotide selected from uracil nucleotides, cytosine nucleotides, guanine nucleotides, the element d is composed of 2 or more to 20 or less nucleotides, the nucleotides are arbitrarily selected from adenine nucleotides, uracil nucleotides, cytosine nucleotides, guanine nucleotides, and the element d does not contain 3 or more consecutive adenine nucleotides, and the 5 'and 3' terminal-most nucleotides thereof are not adenine nucleotides, Wherein when more than two of said elements b, c or d are contained in said Poly (A) tag at the same time, the sequences of each two elements b may be the same or different, the sequences of each two elements c may be the same or different, and the sequences of each two elements d may be the same or different, And, the element a and the element b, the element c and the element d, the elements b, the elements c and d are not adjacent to each other.
  4. The method according to any one of the preceding claims, wherein the element a consists of more than 20, less than 80 consecutive adenine nucleotides, preferably of 30 to 70, 35 to 65, 40 to 60, or 45 to 55 consecutive adenine nucleotides, more preferably of 60 consecutive adenine nucleotides.
  5. The method according to any one of the preceding claims, wherein the element b consists of 3 to 10, 10 to 19, 12 to 15, 14 to 17, or 16 to 19, preferably 19 consecutive adenine nucleotides.
  6. A method according to any of the preceding claims, wherein the number of elements b is 2-10, preferably 2-5, further preferably 3.
  7. The method according to any one of the preceding claims, wherein said element c is a guanine nucleotide.
  8. The method according to any of the preceding claims, wherein the number of elements c is 2 to 10, 3 to 8, 4 to 6, or2 to 5, preferably 2.
  9. The method according to any of the preceding claims, wherein the element d consists of 3 to 18, 5 to 16, 4 to 10, or 6 to 12 nucleotides, preferably 6 nucleotides.
  10. The method of any one of the preceding claims, wherein the element d is selected from any one of GAUAUC, GUAUAC, GAAUCU, GCAUAUGACU or GAUAUCGUAUAC.
  11. A method according to any of the preceding claims, wherein the number of elements d is 0-5, preferably 1-3, more preferably 1.
  12. The method according to any of the preceding claims, wherein the sum of the number of elements c and d is 2-15, preferably 3-5, more preferably 3, when elements c and d are present at the same time.
  13. The method of any of the preceding claims, wherein the functional variant of the poly (A) tag has any one of the structures selected from the group consisting of, Element a-element c-element b-element c-element b, Element b-element c-element a-element d-element b-element c-element b, Element b-element c-element b-element d-element a-element c, Element a-element d-element b-element c-element b, or Element b-element c-element b-element d-element a.
  14. The method according to any of the preceding claims, wherein the 5' end arm comprises a 5' outer homology arm and a 3' intron fragment in the 5' to 3' direction, the first tag being inserted in the 5' outer homology arm, or in the 3' intron fragment in the 5' end region near the 5' outer homology arm, preferably 20 nucleotides, more preferably 15 nucleotides, further preferably 10 nucleotides, of the 5' end of the 3' intron fragment, or upstream of the 5' end of the 5' outer homology arm, The 3 'end arm comprises a 5' intron fragment and a 3 'outer homology arm in the 5' to 3 'direction, the second tag being inserted in the 3' outer homology arm or in the 3 'end region of the 5' intron fragment near the 3 'outer homology arm, the 3' end region preferably being 20 nucleotides, more preferably 15 nucleotides, still more preferably 10 nucleotides, at the 3 'end of the 5' intron fragment or downstream of the 3 'end of the 3' outer homology arm, Preferably, the method comprises the steps of, The first tag is inserted in the 5' outer homology arm and the second tag is inserted downstream of the 3' end of the 3' outer homology arm; The first tag is inserted in the 5 'outer homology arm and the second tag is inserted in the 3' outer homology arm, or The first tag is inserted in the 5' outer homology arm and the second tag is inserted in the 3' end region of the 5' intron fragment; more preferably, the first tag is inserted at any position in the 5 'outer homology arm except for the 5' terminal 1 st nucleotide residue, and the second tag is inserted downstream of the 3 'end of the 3' outer homology arm; The first tag is inserted at any position in the 5' outer homology arm except for the 1 st nucleotide residue at the 5' end and the second tag is inserted in the 3' outer homology arm, or The first tag is inserted at any position in the 5 'outer homology arm except for the 5' terminal 1 st nucleotide residue, and the second tag is inserted at the 3 'terminal region of the 5' intron fragment.
  15. The method according to any one of the preceding claims, wherein the cyclisation region comprises, in sequence in the 5 'to 3' direction, a 3 'coding region fragment, a translation initiation element, a 5' coding region fragment, in operable linkage with each other.
  16. The method according to any one of the preceding claims, wherein the cyclisation region comprises a3 'exon fragment, a 5' internal homology arm, an insert, a3 'internal homology arm and a 5' exon fragment, in sequence in 5 'to 3' direction, in an operatively linked manner to each other, optionally the cyclisation region comprises a first spacer between the insert and the 5 'internal homology arm and a second spacer between the insert and the 3' internal homology arm.
  17. The method according to claim 16, wherein the insert comprises a translation initiation element, or comprises a translation initiation element and a coding region, wherein the translation initiation element is preferably an IRES sequence.
  18. The method according to any of the preceding claims, wherein the insert comprises a structural gene or a functional fragment thereof encoding a protein hybrid selected from the group consisting of a polypeptide, a protein subunit, a protein active center, a protein or a non-natural catalytic group, a recombinant protein active subunit or active center, a recombinant artificial enzyme or other biological effect unit consisting essentially of amino acids, or a sequence of a non-coding RNA selected from the group consisting of micrornas (mirnas), small interfering RNAs (sirnas), PIWI protein interacting RNAs (pirnas), transfer RNA derived micrornas (tsrnas), nuclear micrornas (snrnas), nucleolar micrornas (snornas), long-chain non-coding RNAs (lncrnas), pseudogenes, ceRNA (competing endogenous RNAs), microRNA sponges or other types of non-mRNA.
  19. The method according to any of the preceding claims, wherein the length of the 5 'and 3' external homology arms is each independently greater than 5nt, greater than 10nt, greater than 15nt, greater than 20nt, greater than 25nt, greater than 30nt, greater than 40nt, greater than 50nt, greater than 60nt, less than 5nt, less than 10nt, less than 15nt, less than 20nt, less than 25nt, less than 30nt, less than 40nt, less than 50nt, less than 60nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, or 50nt, Optionally the 5 'and 3' intron fragments are each independently greater than 5nt, greater than 10nt, greater than 15nt, greater than 20nt, greater than 25nt, greater than 30nt, greater than 40nt, greater than 50nt, greater than 60nt, less than 5nt, less than 10nt, less than 15nt, less than 20nt, less than 25nt, less than 30nt, less than 40nt, less than 50nt, less than 60nt, less than 70nt, less than 80nt, less than 90nt, less than 100nt, less than 150nt, less than 200nt、5～200nt、10～150nt、50～200nt、50～150nt、5～60nt、10～55nt、15～50nt、20～45nt、25～40nt、30～35nt、10nt、15nt、20nt、25nt、30nt、35nt、40nt、45nt、 or 50nt in length, Optionally the 5 'and 3' exon fragments are each independently greater than 5nt, greater than 10nt, greater than 15nt, greater than 20nt, greater than 25nt, greater than 30nt, greater than 40nt, greater than 50nt, greater than 60nt, less than 5nt, less than 10nt, less than 15nt, less than 20nt, less than 25nt, less than 30nt, less than 40nt, less than 50nt, less than 60nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, or 50nt in length, Optionally, the 5 'and 3' internal homology arms each independently have a length of greater than 5nt, greater than 10nt, greater than 15nt, greater than 20nt, greater than 25nt, greater than 30nt, greater than 40nt, greater than 50nt, greater than 60nt, less than 5nt, less than 10nt, less than 15nt, less than 20nt, less than 25nt, less than 30nt, less than 40nt, less than 50nt, less than 60nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, or 50nt, Optionally the insert has a length of greater than 50nt, greater than 100nt, greater than 150nt, greater than 200nt, greater than 250nt, greater than 300nt, greater than 400nt, greater than 500nt, greater than 600nt, greater than 1k nt, greater than 1.5k nt, greater than 2k nt, greater than 3k nt, less than 50nt, less than 100nt, less than 150nt, less than 200nt, less than 250nt, less than 300nt, less than 400nt, less than 500nt, less than 600nt, less than 1k nt, less than 1.5k nt, less than 2k nt, less than 1.5k nt 3k nt、50～5knt、50～5k nt、50～4k nt、50～3k nt、50～2k nt、50～1.5k nt、50～1k nt、50～600nt、100～550nt、150～500nt、200～450nt、250～400nt、300～350nt.
  20. The method of any preceding claim, wherein the 5 'outer homology arm has a sequence as set forth in SEQ ID No.1 or 56 and the 3' outer homology arm has a sequence as set forth in SEQ ID No. 2 or 57.

Description

Method for preparing circular RNA and nucleic acid sequence for said method Technical Field The present invention relates to methods and nucleic acid sequences for the preparation and purification of circular RNAs. Background In recent years, research and development of mRNA vaccines or mRNA drugs plays a vital role in controlling new coronary epidemic, and simultaneously, research and development of mRNA drugs in other disease treatment fields is greatly accelerated. It is noted that linear mRNA drugs face serious challenges such as low stability and short half-life in vivo, whereas circular RNAs, because they do not contain free ends, can be effectively prevented from being degraded by exonucleases in cells, and thus have significantly higher stability than linear mRNA molecules (see non-patent document 1), providing an effective candidate for solving the above challenges. Circular RNAs (circrnas) are a class of covalently closed circular RNA molecules, hundreds to thousands of natural circular RNAs have been identified in viruses (e.g., hepatitis delta virus HDV) and eukaryotic cells (e.g., mammalian cells) since the first discovery in 1976 that the genome of plant-based viruses (plant viroids) was circular RNA molecules, and these molecules were found to perform a variety of different biological functions in vivo, including, but not limited to, encoding proteins, regulating gene transcription, as microRNA sponges (microRNA sponges), protein scaffolds, and the like (see non-patent document 2). Research analysis of related molecular biology and functions has driven in vitro synthesis techniques of circular RNAs, and chemical synthesis methods, ligase methods (T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2), and ribozyme methods (including type I and type II intron ribozymes) have been reported (see non-patent document 1). Among them, the ribozyme method is one of the main routes for synthesizing circular RNA in the current nucleic acid drug field, which utilizes PIE System (Permuted Intron-Exon System) established by engineering type I or type II self-splicing introns to prepare circular RNA. It has been reported that splicing of type I introns is accomplished by a two-step transesterification reaction in the presence of Mg 2+ and guanosine, independent of any protein, in that in the first step guanosine attacks the 5 'splice site (5' SS) where the 3 'hydroxyl group of guanosine undergoes transesterification allowing cleavage between the 5' exon and the 5 'intron, and in the second step 3' -OH of the intermediate formed after the first step attacks the 3 'splice site (3' SS) where a second transesterification reaction occurs resulting in direct linkage of exons located on both sides of the intron, whereas the 5 'end of the intron itself undergoes cyclization with the 3' end and is cleaved off. According to the above mechanism of action, the natural type I self-splicing intron is divided into two parts, and the arrangement sequence of the "exon 1-intron fragment 2-exon 2" is artificially replaced by the arrangement of the "exon 2-exon 1-intron fragment 1", so that the circular RNA obtained by cyclizing the "exon 2-exon 1" fragment can be prepared in the presence of Mg 2+ and guanosine. For example, non-patent documents 3 and 4 report PIE systems established by modifying the type I self-splicing intron of Anabaena (anabaena) pre-Trna or T4 phage Td gene in the substitution manner described above, respectively, which can successfully cyclize RNAs of unequal lengths of 100nt or 550 nt. PIE systems based on these 2 type I self-splicing intron ribozymes have remained widely used for circular RNA synthesis to date. Non-patent document 5 also reports that optimization of a PIE system based on Anabaena pre-tRNA type I intron ribozyme can significantly improve in vitro cyclization efficiency by adding an external homology arm (external homology arm), an internal homology arm (internal homology arm) and the like, and increases the length of a cyclizable RNA fragment to 5kb, thereby laying a good foundation for synthesis of a circular RNA drug. The synthesis of circular RNA as described above produces a variety of RNA byproducts including, but not limited to, flanking introns that are sheared after the cyclization reaction, linear RNA precursors that have not been cyclized, and linear and circular high molecular weight polymeric RNA produced by polymerization of a plurality of linear precursors. The presence of these by-products has been reported in the prior art to have various adverse effects on the final circular RNA biologic, not only to reduce the purity and potency of the product, but also to significantly increase the immunogenicity in vivo (see non-patent document 2). Therefore, there is an urgent need for a method capable of efficiently producing a high-purity circular RNA molecule, particularly a production method capable of satisfying the industrial production level. In the conventional preparation method