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CN-122029278-A - Constructs, methods and uses for the preparation of circular RNAs

CN122029278ACN 122029278 ACN122029278 ACN 122029278ACN-122029278-A

Abstract

Disclosed herein are constructs and methods for making circular RNAs and uses thereof. In particular, provided herein are novel constructs comprising modified group II introns and having group II introns self-splicing activity, and their use in the preparation of circular RNAs. Also provided is the use of the resulting circRNA. Related compositions and systems are also provided herein.

Inventors

  • YANG BIN

Assignees

  • 上海环码生物医药有限公司

Dates

Publication Date
20260512
Application Date
20241009
Priority Date
20231009

Claims (20)

  1. 1. A non-naturally occurring RNA comprising the following operably linked elements from 5 'to 3': (1) A 3' intron fragment; (2) A target sequence consisting of (i) a 3 'target sequence fragment and (ii) a 5' target sequence fragment from 5 'to 3', and (3) A 5' intron fragment; Wherein the RNA has group II intron activity and upon self-splicing a circular RNA (circRNA) is formed, the circular RNA comprising the 5 'target sequence fragment and a 3' target sequence fragment, the 3 'end of the 5' target sequence fragment being linked to the 5 'end of the 3' target sequence fragment.
  2. 2. The RNA of claim 1, wherein the circRNA comprises a translation initiation sequence (TI) and a protein coding sequence (Z1), and wherein the 3 'end of TI is operably linked to the 5' end of Z1.
  3. 3. The RNA of claim 2, wherein Z1 encodes a therapeutic product.
  4. 4. The RNA of claim 2, wherein Z1 is at least 95%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 107-112, 214-221 and 258-259.
  5. 5. The RNA of claim 3, wherein the therapeutic product has an amino acid sequence selected from the group consisting of SEQ ID NOs 113-118.
  6. 6. The RNA according to any one of claims 2 to 5, wherein TI comprises a sequence selected from the group consisting of a spacer sequence of SEQ ID NO 4-6, a polyA sequence, a poly-A-C sequence, a poly-U sequence, an Internal Ribosome Entry Site (IRES), an IRES-like nucleotide sequence, a ribosome binding site, an aptamer sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translational regulatory sequence and a protein binding site.
  7. 7. The RNA of claim 6, wherein TI comprises an IRES, an IRES-like nucleotide sequence, or a combination thereof.
  8. 8. The RNA of claim 6, wherein TI is at least 95%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS 222-225.
  9. 9. The RNA according to any one of claims 2 to 7, wherein the 3 'target sequence fragment comprises Z1 and the 5' target sequence fragment comprises TI.
  10. 10. The RNA of claim 8, wherein the 3' target sequence fragment further comprises two linkers (L) flanking Z1.
  11. 11. The RNA according to any one of claims 2 to 7, wherein the 3 'target sequence fragment comprises TI and the 5' target sequence fragment comprises Z1.
  12. 12. The RNA of claim 11, wherein the 3' target sequence fragment further comprises two linkers (L) flanking the TI.
  13. 13. The RNA of any one of claims 2 to 7, wherein the 3 'target sequence fragment comprises a 3' fragment of TI (TI B ) and Z1 from 5 'to 3', and wherein the 5 'target sequence fragment comprises a 5' fragment of TI (TI A ).
  14. 14. The RNA of claim 13, wherein the 3' target sequence further comprises two linkers (L) flanking Z1.
  15. 15. The RNA according to any one of claims 2 to 7, wherein the 3 'target sequence fragment comprises a 3' fragment of Z1 (Z1 B ), wherein the 5 'target sequence fragment comprises from 5' to 3 'a 5' fragment of TI and Z1 (Z1 A ).
  16. 16. The RNA of claim 15, wherein the 3' target sequence further comprises two linkers (L) flanking the TI.
  17. 17. The RNA of claim 1, having a structure of formulae (I) - (IV) selected from the group consisting of: (I) 5'-(3' IF)-(L) n -Z1-(L) n -TI-(5' IF)-3'; (II) 5'-(3' IF)-(L) n -TI-(L) n -Z1-(5' IF)-3';(III) 5'-(3' IF)-TI B -(L) n -Z1-(L) n -TI A -(5' IF)-3';(IV) 5'-(3' IF)-Z1 B -(L) n -TI-(L) n -Z1 A -(5' IF)-3'; And Wherein 3'IF is a 3' intron fragment, 5'IF is a 5' intron fragment, TI is a translation initiation sequence which may be segmented into a5 'fragment of TI (TI A ) and a 3' fragment of TI (TI B ), Z1 is a protein coding sequence which may be segmented into a5 'fragment of Z1 (Z1 A ) and a 3' fragment of Z1 (Z1 B ), each L is independently a linker sequence, and n=0, 1 or 2.
  18. 18. The RNA according to any one of claims 2 to 14 or 17, having a structure selected from formulae (I) - (III), further comprising (1) an exon fragment 2 (E2) between the 3 'intron fragment and the target sequence, (2) an exon fragment 1 (E1) between the target sequence and the 5' intron fragment, or (3) both (1) and (2).
  19. 19. The RNA according to claim 18, wherein E2 has a nucleotide sequence selected from the group consisting of SEQ ID NOs 53-63.
  20. 20. The RNA according to claim 18 or 19, wherein E1 has a nucleotide sequence selected from SEQ ID NOs 64-74.

Description

Constructs, methods and uses for the preparation of circular RNAs 1. Related applications The present application claims priority and benefit from PCT application number PCT/CN2023/123553 filed on 10/2023/9, 2023, the entire contents of which are incorporated herein by reference in their entirety. 2. Incorporated by reference sequence listing The contents of the electronic sequence list (TFG 00911PCT sequence listing. Xml; size: 341,593 bytes; date of creation: 2024, 10, 8 days) are incorporated herein by reference in their entirety. 3. Field of application The present invention relates to the field of molecular biology, in particular to constructs and methods for the preparation of circular RNAs and the use of circular RNAs, for example, to express a protein of interest in eukaryotic cells or to function as non-coding RNAs. 4. Background Circular RNA (circRNA) is a recently discovered RNA molecule .(Yanget al.,Cell Research, 27(5):626-641 (2017); Abeet al.,Scientific Reports, 5:16435 (2015); Gaoet al.,Nature Cell Biology, 23(3):278-291 (2021); Pamudurtiet al.,Molecular Cell, 66(1):9-21 (2017)). with various biological functions formed by head-to-tail connection, and compared with linear RNA, the circRNA has better stability, so that a novel platform with wide prospect is provided for research of RNA medicaments. Despite the great therapeutic potential and recent advances in this field, there is still a lack of efficient and reliable methods for preparing circular RNAs. The compositions, methods, and systems provided herein address this need and provide related advantages. 5. Summary of the invention The invention provides a non-naturally occurring RNA comprising, from 5 'to 3', operably linked elements (1) a 3 'intron fragment, (2) a target sequence consisting of (i) a 3' target sequence fragment and (II) a 5 'target sequence fragment, from 5' to 3', and (3) a 5' intron fragment, wherein the RNA has group II intron activity and upon self-splicing forms a circular RNA (circRNA), the circular RNA comprising a 5 'target sequence fragment and a 3' target sequence fragment, the 3 'end of the 5' target sequence fragment being linked to the 5 'end of the 3' target sequence fragment. In some embodiments, the circRNA consists of a target sequence. In some embodiments, the circRNA comprises a translation initiation sequence (TI) and a protein coding sequence (Z1), wherein the 3 'end of TI is operably linked to the 5' end of Z1. In some embodiments, Z1 encodes a therapeutic product. In some embodiments, Z1 is at least 95%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS 107-112, 214-221 and 258-259. In some embodiments, the therapeutic product has an amino acid sequence selected from the group consisting of SEQ ID NOS 113-118. In some embodiments, TI comprises a sequence selected from the group consisting of a spacer sequence of SEQ ID NO: 4-6, a polyA sequence, a poly-A-C sequence, a poly-U sequence, an Internal Ribosome Entry Site (IRES), an IRES-like nucleotide sequence, a ribosome binding site, an aptamer sequence, an RNA scaffold, a riboswitch, a ribozyme other than an auto-splice ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translational regulatory sequence, and a protein binding site. In some embodiments, the TI comprises an IRES, an IRES-like nucleotide sequence, or a combination thereof. In some embodiments, TI is at least 95%, at least 98%, at least 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS 222-225. In some embodiments of the RNAs provided herein, the 3 'target sequence fragment comprises Z1 and the 5' target sequence fragment comprises TI. In some embodiments, the 3' target sequence fragment further comprises two linkers (L) flanking Z1. In some embodiments of the RNAs provided herein, the 3 'target sequence fragment comprises TI and the 5' target sequence fragment comprises Z1. In some embodiments, the 3' target sequence fragment further comprises two linkers (L) flanking the TI. In some embodiments of the RNAs provided herein, the 3 'target sequence fragment comprises, from 5' to 3', a 3' fragment of TI (TI B) and Z1, wherein the 5 'target sequence fragment comprises a 5' fragment of TI (TI A). In some embodiments, the 3' target sequence further comprises two linkers (L) flanking Z1. In some embodiments of the RNAs provided herein, the 3 'target sequence fragment comprises a 3' fragment of Z1 (Z1 B), wherein said 5 'target sequence fragment comprises from 5' to 3 'a 5' fragment of TI and Z1 (Z1 A). In some embodiments, the 3' target sequence further comprises two linkers (L) flanking the TI. In some embodiments, the RNAs provided herein have a structure selected from the group consisting of formulas (I) - (IV): (I) 5'-(3' IF)-(L)n-Z1-(L)n-TI-(5' IF)-3'; (II) 5'-(3' IF)-(L)n-TI-(L)n-Z1-(5' IF)-3';(III)5'-(3' IF)-TIB-(L)n-Z1-(L)n-TIA-(5' IF)-3