EP-4735601-A1 - ENGINEERED RNA POLYNUCLEOTIDE AND PREPARATION METHOD THEREOF

Abstract

Provided is an engineered RNA polynucleotide and preparation method thereof, wherein the engineered RNA polynucleotide has reduced immunogenicity as well as improved stability. Also provided are a precursor and a vector for the preparation of the engineered RNA polynucleotide, and the use thereof.

Inventors

• LI, SIQI
• ZHANG, GUIPING
• LUO, ZHENG
• Zhang, Weiyi

Assignees

• Ribox Therapeutics HK Limited

Dates

Publication Date

20260506

Application Date

20240628

Claims (20)

1. An isolated engineered RNA polynucleotide comprising: (a) a 5’ terminal hydroxyl group, (b) a nucleotide sequence of interest, and (c) a 3’ terminal 2’, 3’-cyclic phosphate group.
2. The engineered RNA polynucleotide of claim 1, wherein said engineered RNA polynucleotide has reduced immunogenicity and/or improved stability.
3. The engineered RNA polynucleotide of claim 2 or 3, wherein the nucleotide sequence of interest comprises one or more coding or non-coding sequences.
4. The engineered RNA polynucleotide of claim 3, wherein the nucleotide sequence of interest further comprises a translation initiation element (TIE) , or a non-TIE functional element.
5. The engineered RNA polynucleotide of any one of claims 1 to 4, which is generated via (i) intramolecular self-cleavage of ribozyme, (ii) chemical synthesis, or (iii) in vitro translation from a DNA template followed by terminal modification.
6. An isolated RNA polynucleotide precursor, comprising: (a) a self-cleaving ribozyme I or a fragment thereof, (b) a polynucleotide sequence comprising from 5’ to 3’ direction, a 5’ ribozyme I recognition site, a nucleotide sequence of interest, and a 3’ ribozyme II recognition site, and (c) a self-cleaving ribozyme II or a fragment thereof, wherein said self-cleaving ribozyme I or fragment thereof and said self-cleaving ribozyme II or fragment thereof are capable of catalyzing intramolecular self-cleavage from said RNA polynucleotide precursor, thereby forming an engineered RNA polynucleotide in linear form.
7. An isolated RNA polynucleotide precursor, comprising: (a) a self-cleaving ribozyme I fragment, (b) a polynucleotide sequence comprising from 5’ to 3’ direction, a 5’ ribozyme I recognition site, a nucleotide sequence of interest, and a 3’ ribozyme II recognition site, and (c) a self-cleaving ribozyme II fragment, wherein said self-cleaving ribozyme I fragment and said self-cleaving ribozyme II fragment have a sequence identity of at least 85%to the catalytic domain of said ribozyme I and ribozyme II respectively and are capable of catalyzing the intramolecular self-cleavage from said RNA polynucleotide precursor, thereby forming an engineered RNA polynucleotide in linear form.
8. The RNA polynucleotide precursor of claim 6 or 7, wherein the self-cleaving ribozyme I and self-cleaving ribozyme II are the same or different ribozymes.
9. The RNA polynucleotide precursor of any one of claims 6 to 8, wherein the self-cleaving ribozyme I and self-cleaving ribozyme II are selected from Table 1.
10. The RNA polynucleotide precursor of any one of claims 6 to 9, wherein the engineered RNA polynucleotide in linear form comprises a 5’ end hydroxyl group, the polynucleotide sequence comprising from 5’ to 3’ direction, the 5’ ribozyme I recognition site, the nucleotide sequence of interest, and the 3’ ribozyme II recognition site, and a 3’ end 2’, 3’-cyclic phosphate group.
11. The RNA polynucleotide precursor of any one of claims 6 to 10, further comprising a first linker at 5' upstream of the 5' ribozyme I and/or a second linker at 3' downstream of the 3' ribozyme II.
12. The RNA polynucleotide precursor of claim 11, wherein the first linker comprises a first ligand sequence and/or the second linker comprises a second ligand sequence.
13. An isolated engineered RNA polynucleotide comprising: (a) a 5’ terminal hydroxyl group, (b) a polynucleotide sequence comprising from 5’ to 3’ direction, a 5’ ribozyme I recognition site, a nucleotide sequence of interest, and a 3’ ribozyme II recognition site, and (c) a 3’ terminal 2’, 3’-cyclic phosphate group.
14. The engineered RNA polynucleotide of claim 13, wherein said engineered RNA polynucleotide has reduced immunogenicity and/or improved stability.
15. The engineered RNA polynucleotide of claim 13 or 14, wherein the nucleotide sequence of interest comprises one or more coding or non-coding sequences.
16. The engineered RNA polynucleotide of claim 15, wherein the nucleotide sequence of interest further comprises a translation initiation element (TIE) , or a non-TIE functional element.
17. The engineered RNA polynucleotide of any one of claims 13 to 16, which is generated via intramolecular self-cleavage of the RNA polynucleotide precursor of any one of claims 6 to 12.
18. A vector comprising a DNA sequence encoding the RNA polynucleotide precursor of any one of claims 6 to 12.
19. A cell comprising the vector of claim 18.
20. A method of providing a polypeptide to a subject, comprising (a) delivering to the subject the engineered RNA polynucleotide of any one of claims 1 to 5 and 13 to 17, wherein the sequence of interest comprises (i) one or more coding sequences of the polypeptide, for example a therapeutic protein or an enzyme, and (ii) one or more translation initiation elements, for example, an IRES, and (b) expressing the nucleotide sequence of interest.

Description

Engineered RNA polynucleotide and preparation method thereof Technical field The present invention relates to an engineered RNA polynucleotide and preparation method thereof, wherein the engineered RNA polynucleotide has reduced immunogenicity as well as improved stability. The present invention also relates to a precursor and a vector for the preparation of the engineered RNA polynucleotide, and the use thereof. Background The increasing number of approved RNA-based therapeutics has fueled great hope as an effective treatment for a wide range of incurable diseases, as nucleic acid therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing. In recent years, platform technologies that have enabled the clinical translation of nucleic acid therapeutics, including but not limited to technologies such as mRNA, engineered RNA polynucleotide, small interfering RNA. However, several challenges remain unsolved and need to be overcome to enable RNA molecules to be utilized more effectively. For example, RNA molecules can be recognized by the host’s innate immune system and provoke an undesirable inflammatory response. A variety of RNA species or analogs have also been reported to be immunogenic. Previous studies have identified the immunological targets for these special RNA molecules, including cell surface or endosome Toll-like receptors (TLRs; TLR-3, -7, -8, and -9) and cytosolic sensors (protein kinase R, RIG-I, and MDA-5) . Recognition of these specific RNA sequences (SEQs) or structures by their corresponding immune receptors will trigger a series of immune events that are part of the host’s natural defense, including cytokine secretion, immune cell proliferation and survival, or activation of adaptive immunity, and thus limiting the use of RNA therapeutics. Summary of the invention In one aspect, the present invention provides an RNA polynucleotide precursor comprising: a) a self-cleaving ribozyme I or a fragment thereof, b) a polynucleotide sequence comprising from 5’ to 3’ direction, a 5’ ribozyme I recognition site, a nucleotide sequence of interest, a 3’ ribozyme II recognition site, c) a self-cleaving ribozyme II or a fragment thereof. wherein said self-cleaving ribozyme I or fragment thereof and said self-cleaving ribozyme II or a fragment thereof catalyze intramolecular self-cleavage from said RNA polynucleotide precursor and forms an engineered RNA polynucleotide in linear form. In another aspect, it is an object of the present invention to provide an RNA polynucleotide precursor comprising: (a) a self-cleaving ribozyme I or a fragment thereof, (b) a polynucleotide sequence comprising from 5’ to 3’ direction, a 5’ ribozyme I recognition site, a nucleotide sequence of interest, a 3’ ribozyme II recognition site, (c) a self-cleaving ribozyme II or a fragment thereof, wherein said self-cleaving ribozyme I or fragment thereof and said self-cleaving ribozyme II or a fragment thereof catalyze intramolecular self-cleavage from said RNA polynucleotide precursor, and forms an engineered RNA polynucleotide in linear form, and wherein said ribozyme I and ribozyme II can be the same or different ribozymes, for example, said ribozyme I and ribozyme II are selected from the group including but not limited to: naturally-occurring ribozymes and modified ribozymes, e.g., ribozymes containing one or more modifications, which can be addition, deletion, substitution, and/or alteration of at least one nucleotide. In another aspect, it is an object of the present invention to provide an RNA polynucleotide precursor comprising: (a) a self-cleaving ribozyme I fragment, (b) a polynucleotide sequence comprising from 5’ to 3’ direction, a 5’ ribozyme I recognition site, a nucleotide sequence of interest, a 3’ ribozyme II recognition site, (c) a self-cleaving ribozyme II fragment, wherein said self-cleaving ribozyme I fragment and said self-cleaving ribozyme II fragment each has a sequence identity of at least 85%to the catalytic domain of said ribozyme I and ribozyme II respectively, and catalyze the intramolecular self-cleavage from said RNA polynucleotide precursor to form an engineered RNA polynucleotide in linear form, and wherein said ribozyme I and ribozyme II can be the same or different ribozymes, for example, said ribozyme I and ribozyme II are selected from the group including but not limited to: naturally-occurring ribozymes and modified ribozymes, e.g., ribozymes containing one or more modifications, which can be addition, deletion, substitution, and/or alteration of at least one nucleotide. In one aspect, the present invention provides an engineered RNA polynucleotide comprising: (a) a 5’ terminal hydroxyl group, (b) a polynucleotide sequence comprising from 5’ to 3’ direction, a 5’ ribozyme I recognition site, a nucleotide sequence of interest, a 3’ ribozyme II recognition site, and (c) a 3’ terminal phosphate-containing group, wherein said engineered RNA po