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CN-121991942-A - Preparation method of scar-free annular nucleic acid molecule and application of scar-free annular nucleic acid molecule in targeted delivery

CN121991942ACN 121991942 ACN121991942 ACN 121991942ACN-121991942-A

Abstract

The invention provides a preparation method of a scar-free annular nucleic acid molecule and application thereof in targeted delivery. The precursor nucleic acid molecule for preparing the circular nucleic acid molecule comprises a 3' group I intron or a mutant fragment thereof along the 5' direction, b a first unit fragment II, the 5' end of which comprises a II ribozyme recognition fragment, c a functional unit (comprising IRES, RNA aptamer, protein binding sequence, protein coding region, non-coding region, etc. or a combination thereof), e a first unit fragment I, the 3' end of which comprises a I ribozyme recognition fragment, f.5' group I intron or a mutant fragment thereof. The invention realizes the scar-free cyclization of RNA by using the aptamer sequence and improves the targeting of the circular nucleic acid molecule sequence.

Inventors

  • TAN WEIHONG
  • WANG WENXI
  • XIE SITAO
  • ZHANG YU
  • GAN SHAOJU
  • FU TING
  • LIU XIANGSHENG

Assignees

  • 中国科学院杭州医学研究所

Dates

Publication Date
20260508
Application Date
20241107

Claims (20)

  1. 1. A precursor nucleic acid molecule for preparing a circular nucleic acid molecule, characterized in that the precursor nucleic acid molecule comprises in the 5 'to 3' direction: a 3' group I intron or a mutant fragment thereof, B. a first unit fragment II comprising at its 5' end a recognition fragment of the II-th ribozyme, C. functional units (including IRES, RNA aptamer, protein binding sequences, protein coding regions, non-coding regions, etc., or combinations thereof), E. a first unit fragment I, the 3' end of which comprises a recognition fragment of the I-th ribozyme, F.5' group I intron or mutant fragment thereof; The first unit fragment II is positioned at the 3' end of the first unit fragment I, namely the complete first unit sequence comprises: First unit segment I-first unit segment II; The 3' group I intron or mutant fragment thereof is located 3' to the 5' group I intron or mutant fragment thereof, i.e., the complete group I intron or mutant sequence thereof comprises: Group 5'i introns or mutant fragments thereof-group 3' i introns or mutant fragments thereof; the group I intron or the mutant thereof recognizes and covalently links the I-th ribozyme recognition fragment and the II-th ribozyme recognition fragment to obtain the cyclic nucleic acid molecule, namely the complete first unit sequence in the cyclic molecule comprises the I-th ribozyme recognition fragment-II-th ribozyme recognition fragment, which is also called as a loop-forming fragment; the first unit is provided with a local stem structure, a local double bond structure or a local hairpin structure, and the local stem structure, the local double bond structure or the local hairpin structure is adjacent to or comprises the loop-forming segment, so that the interconnection of the I-th ribozyme recognition segment and the II-th ribozyme recognition segment during loop forming is facilitated, and the cyclization efficiency of the circular nucleic acid molecule is improved; Wherein "-" represents a phosphodiester bond; Preferably, the loop-forming fragment is located in a loop of the stem-loop structure of the first unit or the aptamer sequence, there is a double-stranded structure formed by complementary pairing sequences with each other within 100 bases upstream and downstream of the loop-forming fragment, preferably the double-stranded structure comprises at least 5 consecutive complementary pairing bases, and further preferably the number of complementary pairing bases and non-complementary pairing bases in the stem of the stem-loop structure is more than 20bp.
  2. 2. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule of claim 1, wherein said first unit is a nucleic acid aptamer and said first unit fragment I and said first unit fragment II are nucleic acid aptamer fragment I and nucleic acid aptamer fragment II.
  3. 3. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 2, wherein said I and II ribozyme recognition fragments are exon fragment 1 (E1) and exon fragment 2 (E2), respectively, adjacent to said group I intron, E1 is a 5' immediately adjacent exon fragment of an intron in group I, and the length of the fragment is more than or equal to 4 nucleotides; E2 is a 3' immediately adjacent exon fragment of an intron in group I, and the length of the fragment is more than or equal to 0 nucleotide; wherein the E1 and/or E2 is 0-20 nucleotides in length, preferably 1-10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides.
  4. 4. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 3, wherein said 5' immediately adjacent exon fragment comprises or consists of one or more nucleotides which can be paired with the targeting sequence of the corresponding group I intron to form a P1 duplex region during cyclization; preferably, the I-th ribozyme recognition fragment comprises or consists of about 1 to about 7 consecutive nucleotides of the natural 5 'immediately adjacent to the 5' nucleotide of the exon of group I intron. Preferably, the II ribozyme recognition fragment comprises or consists of 0 to about 4 consecutive nucleotides starting from the 5 'nucleotide of the natural 3' immediately adjacent exon of group I intron.
  5. 5. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule of claim 3, wherein said group I intron is TpaCOX introns and the nucleotide sequence 5 'immediately adjacent to the exon region is 5' -ACGTCTT-3', and the nucleotide sequence 3' immediately adjacent to the exon region is 5'-AACCAA-3'.
  6. 6. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule as claimed in claim 3 wherein said group I intron is Ptu intron and the nucleotide sequence 5 'immediately adjacent to the exon region is 5' -AGGGAT-3 'and the nucleotide sequence 3' immediately adjacent to the exon region is 5'-CA-3'.
  7. 7. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule as claimed in claim 3, wherein said group I intron is a group I intron of Azoarcus sp.BH72 pre-tRNA-Ile gene.
  8. 8. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 3, wherein said 3 'immediately adjacent exon fragment is derived from the 3' natural exon of the group I intron of the anabaena pre-tRNA-Leu gene (Ana ribozyme), wherein said 5 'immediately adjacent exon fragment is derived from the 5' natural exon of the group I intron of the anabaena pre-tRNA-Leu gene, and wherein preferably said I ribozyme recognition fragment comprises or consists of CTC, CTT, ACTT, CGAT, AAGT, CGTT, CCGT, ATGT, AATT, ACGT, AGTT and wherein said II ribozyme recognition fragment comprises or consists of AAAA, AAAC, AA, TTTT, CAAA, GAAA, AAA, AAC, AGA, AAT, CCA, TAC.
  9. 9. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 3, wherein said 3 'immediately adjacent exon fragment is derived from the 3' natural exon of group I intron of the T4 bacteriophage td gene (T4 td ribozyme), said 5 'immediately adjacent exon fragment is derived from the 5' natural exon of group I intron of the T4 bacteriophage td gene, preferably said I ribozyme recognition fragment comprises or consists of TTGGGT, CCAAGT, ATTAAT, AACGGT, CCCAGT, GTGACT, CACGAT, and said II ribozyme recognition fragment comprises CT, AA, GA, TA, TG, GC, TC.
  10. 10. A precursor nucleic acid molecule for use in preparing a circular nucleic acid molecule of claim 3, wherein said I and II ribozyme recognition fragments further comprise sequences that differ in base sequence from the 3 'or 5' natural exon regions, but which retain circular activity.
  11. 11. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 10, wherein said group I intron is an Ana ribozyme or a mutant thereof, a T4td ribozyme or a mutant thereof, tpaCOX introns or a mutant thereof, ptu intron or a mutant thereof, Wherein the mutant comprises a mutation of the guide region of P1/P10.
  12. 12. A precursor nucleic acid molecule for use in preparing a circular nucleic acid molecule of claim 11, wherein the group I intron is a mutant of an Ana ribozyme, the I ribozyme recognition fragment is 5' -N 1 N 2 N 3 T-3' or 5' -N 2 N 3 T-3', the II ribozyme recognition fragment is 5' -N 4 N 5 N 6 -3', the mutant of an Ana ribozyme comprises a mutation region N 6' N 7' ATAAN 5' N 4' GN 3' N 2' in the 5' intron fragment, wherein N is A, U, C, G or T, wherein N 3' N 2' is reverse complementarily paired with N 2 N 3 , N 5' N 4' is reverse complementarily paired with N 4 N 5 , N 6' N 7' is reverse complementarily paired with N 5' N 4' , the base complementation pairing comprising A-U, G-C, G-U, A-T, G-T base pairs; The loop forming fragment in the corresponding first unit was N 1 N 2 N 3 TN 4 N 5 N 6 or N 2 N 3 TN 4 N 5 N 6 and the loop forming site was N 1 N 2 N 3 T/N 4 N 5 N 6 or N 2 N 3 T/N 4 N 5 N 6 (slash stands for splice site).
  13. 13. A precursor nucleic acid molecule for use in preparing a circular nucleic acid molecule of claim 11, wherein said group I intron is a mutant of T4td ribozyme, said I ribozyme recognition fragment is 5' -N 1 N 2 N 3 N 4 N 5 T-3' or 5' -N 2 N 3 N 4 N 5 T-3' or 5' -N 3 N 4 N 5 T-3', and said II ribozyme recognition fragment is 5' -N 6 N 7 -3' or N 6 or is absent, and the T4td ribozyme mutant comprises the following mutation region N 8' AATTGN 7' N 6' GN 5' N 4' N 3' N 2' N 1' in the 5' intron fragment, wherein N is A, U, C (U, C), G or T, N 5' N 4' N 3' N 2' N 1' and N 1 N 2 N 3 N 4 N 5 reverse complement pair, or N 5' N 4' N 3' N 2' and N 2 N 3 N 4 N 5 reverse complement pair, or N 5' N 4' N 3' and N 3 N 4 N 5 reverse complement pair, or N 7' N 6' and N 6 N 7 reverse complement pair, or N 6' and N 6 complement pair, N 8' and N 6' complement pair, the base complement pair includes A-U, G-C, G-U, A-T, G-T base pairs, corresponding to the first unit of the loop fragment N 1 N 2 N 3 N 4 N 5 TN 6 N 7 or N 2 N 3 N 4 N 5 TN 6 or N 3 N 4 N 5 T, the loop site N 1 N 2 N 3 N 4 N 5 T/N 6 N 7 or N 2 N 3 N 4 N 5 T/N 6 or N 3 N 4 N 5 T/; Preferably, in the absence of N 6 N 7 , the 3' base T of the I-th ribozyme recognition fragment is immediately adjacent to the stem structure of the first unit, and the P10 leader sequence N 7' N 6' may not be mutated, and more preferably N 6 is U or C, and N 6' is not mutated.
  14. 14. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 2, wherein said aptamer fragment I and said aptamer fragment II are obtained by resolution of said aptamer sequence.
  15. 15. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule of claim 2, wherein said nucleic acid aptamer comprises a protein-targeting nucleic acid aptamer sequence or a cell-targeting nucleic acid aptamer sequence; Preferably, the cell-targeting aptamer sequences include, but are not limited to, aptamers that target muscle cells, immune cells, tumor cells, vascular endothelial cells, stem cells, etc., preferably, aptamers that target Antigen Presenting Cells (APCs) such as DC cells or macrophages; Preferably, the nucleic acid aptamer sequence of the targeting protein is selected from the group consisting of nucleic acid aptamers targeting transferrin, DEC205, eIF4G proteins, nucleic acid aptamers of serum albumin, nucleolin nucleolin, mannose receptor, CD207, DC-SIGN, clec9a and DCIR 2.
  16. 16. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 1, wherein said functional unit comprises one or more additional aptamer sequences; preferably, the number of the nucleic acid aptamer sequences is one or more, more preferably the number of the nucleic acid aptamers is 1-12, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and even more preferably the number of the nucleic acid aptamers is 9; preferably, the nucleic acid aptamer sequences may be the same or different; Preferably, the arrangement structure of the nucleic acid aptamer comprises a linear arrangement structure, a branched arrangement structure or a combination thereof, more preferably a combination of branched arrangement structures.
  17. 17. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule of claim 16, wherein said first unit or nucleic acid aptamer sequence is used to increase the targeting of the coding sequence or protein encoded thereby, and wherein said first unit or nucleic acid aptamer sequence is used to increase the expression efficiency of the coding sequence or protein encoded thereby.
  18. 18. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 1, said functional unit comprising one or more additional aptamer sequences, IRES, protein coding regions, non-coding sequences; preferably, the protein coding region comprises one or more coding sequences; Preferably, the coding sequence comprises an antigenic peptide, a coding fluorescent molecule, a therapeutic polypeptide, a cytokine, an antibody or antigen binding fragment against a tumor specific antigen, an antibody or antigen binding fragment against a pathogen antigen, a sequence of a Car-T molecule, a coding sequence of a protein having gene editing activity; Preferably, the antigenic peptide is a tumor antigenic peptide, and more preferably, a combination of a plurality of tumor antigenic peptides.
  19. 19. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule of claim 16, wherein said plurality of aptamer sequences are linked end-to-end to one or more functional units.
  20. 20. A precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule according to claim 1, wherein said functional unit has a length of 6nt to 10000nt.

Description

Preparation method of scar-free annular nucleic acid molecule and application of scar-free annular nucleic acid molecule in targeted delivery Technical Field The invention relates to the technical field of gene recombination, in particular to a method for preparing a scar-free annular nucleic acid molecule based on a nucleic acid aptamer and application of the scar-free annular nucleic acid molecule in preparing a targeted delivery function annular nucleic acid molecule. Background MRNA medicine is used as a medicine mode with great potential, can express protein in cells for a long time, and is very suitable for being applied to vaccines and protein supplementation therapy. But present some important challenges as drugs, including poor stability, poor cellular uptake, difficulty in lysosomal escape, etc. mRNA drugs that have been clinically approved today are mainly delivered via lipid nanoparticle LNP, and can achieve systemic distributed expression of mRNA in vivo, where expression of mRNA in specific tissues and organs is critical for development of mRNA drugs. At present, methods have been proposed to achieve specific expression of mRNA, including tissue-specific cationic lipids, LNP surface modification targeting molecules, although these methods can achieve mRNA expression in a large portion of a particular tissue organ, there is an unnecessary tissue distribution. Therefore, it is important to develop safer means of targeted delivery of mRNA. mRNA itself has some immunogenicity, and systemic expression of mRNA drugs can lead to some unwanted toxic side effects. The use of Lipid Nanoparticles (LNP) as drug delivery vehicles, particularly in mRNA vaccines, has been found to have significant success, but there are also some post-injection adverse effects of: (1) LNP vectors are the main contributor to adverse reactions such as pain and inflammation after drug injection. (2) Temperature sensitivity mRNA has a tendency to dissociate from LNP when the temperature is increased, so that such formulations must be stored at low temperatures, which also limits the use of such dosage forms worldwide. (3) Immunogenicity LNP-mRNA preparations may cause immunopathogenicity and hepatosplenic toxicity, which may have an impact on the safety and efficacy of the treatment. Circular RNA is a circular nucleic acid molecule, and can also be used for expressing proteins in cells, and the closed loop structure can enable the RNA molecule to be more stable, so that the circular RNA is a potential substitute molecule of mRNA molecules. Although it increases the stability of RNA molecules, in vivo delivery still suffers from difficulties in crossing cell membranes and targeting. Disclosure of Invention Aiming at the defects in the prior art, the invention provides a preparation method of a scar-free annular nucleic acid molecule and application thereof in targeted delivery. The method solves the problems of difficult mRNA membrane crossing and poor targeting in the prior art. In a first aspect of the invention, there is provided a precursor nucleic acid molecule for use in the preparation of a circular nucleic acid molecule, said precursor nucleic acid molecule comprising in the 5 'to 3' direction: a 3' group I intron or a mutant fragment thereof, B. a first unit fragment II comprising at its 5' end a recognition fragment of the II-th ribozyme, C. functional units (including IRES, RNA aptamer, protein binding sequences, protein coding regions, non-coding regions, etc., or combinations thereof), E. a first unit fragment I, the 3' end of which comprises a recognition fragment of the I-th ribozyme, F.5' group I intron or mutant fragment thereof; The first unit fragment II is positioned at the 3' end of the first unit fragment I, namely the complete first unit sequence comprises: First unit segment I-first unit segment II; The 3' group I intron or mutant fragment thereof is located 3' to the 5' group I intron or mutant fragment thereof, i.e., the complete group I intron or mutant sequence thereof comprises: Group 5'i introns or mutant fragments thereof-group 3' i introns or mutant fragments thereof; The group I intron or the mutant thereof recognizes and covalently links the I-th ribozyme recognition fragment and the II-th ribozyme recognition fragment to obtain the cyclic nucleic acid molecule, namely the complete first unit sequence in the cyclic molecule comprises the I-th ribozyme recognition fragment-II-th ribozyme recognition fragment, which is hereinafter referred to as a "cyclic fragment"; the first unit is provided with a local stem structure, a local double bond structure or a local hairpin structure, and the local stem structure, the local double bond structure or the local hairpin structure is adjacent to or comprises the loop-forming segment, so that the interconnection of the I-th ribozyme recognition segment and the II-th ribozyme recognition segment during loop forming is facilitated, and the cyclization efficienc