Search

KR-20260065552-A - A Novel Nucleic Acid Molecule with Improved Expression Efficiency and Sustainability and Uses Thereof

KR20260065552AKR 20260065552 AKR20260065552 AKR 20260065552AKR-20260065552-A

Abstract

The present invention relates to a modified mRNA molecule capable of efficiently high-expressing a target protein in vivo or in a biological sample. In the RNA molecule of the present invention, the expression amount and expression persistence of the target protein are significantly improved by inserting a virus-derived stem-loop α (SLα) structure or an analogue thereof at an appropriate location within the 3’ UTR to maintain an optimal spatial distance from the poly A tail. Accordingly, the present invention can be usefully utilized as an excellent nucleic acid therapeutic composition capable of stably and continuously producing a therapeutically effective amount of the target protein even at a low dosage.

Inventors

  • 정재성
  • 최정윤
  • 이윤석
  • 권효경
  • 우지수

Assignees

  • 주식회사 녹십자

Dates

Publication Date
20260508
Application Date
20251030
Priority Date
20241030

Claims (9)

  1. RNA molecules containing the following: (1) Open reading frame (ORF) encoding the target protein; (2) A 3' untranslated region (UTR) coupled to the 3' end of the open reading frame and including a stem-loop structure; and (3) A polyadenyl sequence attached to the 3' end of the 3'UTR.
  2. An RNA molecule according to claim 1, wherein the stem-loop structure comprises (i) any first nucleotide sequence; (ii) a second nucleotide sequence having a reverse complementary sequence to the first nucleotide sequence; and (iii) a loop sequence comprising 4 to 7 bases located between the first nucleotide sequence and the second nucleotide sequence.
  3. In claim 2, the RNA molecule is characterized in that the loop sequence is represented by the following general formula 1: General formula 1 CX 1 -GGX 2 In the above general formula 1, X 1 is A, U, G, or C, and X 2 is A, U, C, or does not exist.
  4. An RNA molecule according to claim 1, characterized in that the stem-loop structure is located at a distance of 10 to 40 nt in the 5' direction from the starting point of the polyadenine sequence.
  5. An RNA molecule according to claim 1, characterized in that the stem-loop structure is located at a distance corresponding to 9 to 36% of the total length of the 3' UTR region in the 5' direction from the starting point of the polyadenine sequence.
  6. An RNA molecule according to claim 1, characterized in that all or part of the uracil (U) in the RNA molecule is substituted with a modified U represented by the following chemical formula 1: Chemical formula 1 In the above chemical formula, R1 and R2 are each independently hydrogen, C1 - C3 alkyl, or C1 - C3 alkoxy, and X and A are carbon or nitrogen and are different from each other, and represents a single bond or a double bond.
  7. A DNA molecule encoding an RNA molecule of any one of claims 1 to 6.
  8. A gene delivery vehicle comprising an RNA molecule according to any one of claims 1 to 6.
  9. Cell into which the gene delivery vehicle of claim 8 has been introduced.

Description

A Novel Nucleic Acid Molecule with Improved Expression Efficiency and Sustainability and Uses Thereof The present invention relates to a recombinant mRNA molecule in which a virus-derived stem-loop α (SLα) sequence is artificially introduced at a specific location to dramatically improve the expression amount and expression persistence of a target protein. Although DNA is known to be relatively more stable and easier to handle than RNA in applications such as gene therapy, it has disadvantages. When delivered into a target genome, DNA can be inserted at unintended locations, potentially damaging the host's genes. Furthermore, it can be damaged by anti-DNA antibodies generated by the host's immune response, and the expression level of the target antigen protein is limited by various variables affecting transcription. Additionally, because DNA must pass through the cell's plasma membrane and nuclear membrane for protein expression, it is difficult to achieve high protein expression. In contrast, mRNA synthesizes proteins directly within the cytoplasm without the need for transcription in the nucleus. It poses no risk of damaging the host cell's genetic structure and has a short half-life, which prevents long-term genetic modification. Consequently, it is safer than DNA and offers the advantage of being easy to mass-produce. Building on the achievements of mRNA-based vaccine technology during the COVID-19 pandemic, many global pharmaceutical companies are utilizing mRNA platforms to expand basic research and clinical trials into various fields, including not only vaccines for infectious diseases but also immuno-oncology drugs, treatments for rare diseases, and therapies for autoimmune diseases. However, mRNA has a relatively short half-life within cells, which acts as a major factor limiting the effectiveness of mRNA-based therapeutics. One of the mechanisms that promotes mRNA degradation is the deadenylation of the 3’ polyadenine tail of mRNA, in which the polyadenine tail is completely removed by the PAN2/3 complex and the CNOT complex, followed by the removal of the 5’ cap by the DCP1/2 complex, and finally, the mRNA is completely degraded by enzymes such as Xrn1 and exosome complexes. Meanwhile, it has recently been reported that certain viruses extend their half-life and increase their stability within the host by using terminal nucleotidyl transferase (TENT4) in host cells to add a mixed tail to the 3' end of their RNA, thereby slowing down the rate of RNA degradation. In this mechanism, a stem-loop structure located at the viral post-transcriptional regulatory element (PRE) plays an important role. Accordingly, the inventors intended to improve the production efficiency of the target protein and maximize the efficacy of mRNA-based therapeutics by inserting the viral stem-loop structure into the 3' UTR sequence of the mRNA intended for target protein production to enhance the stability and translation efficiency of the mRNA. Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention. Figure 1 is a schematic diagram of the structure of stem-loop α (SLα) located in the post-transcriptional regulatory element (PRE) of HBV (Hepatitis B virus). Figure 2 is a schematic diagram showing hAG (human alpha globin) 3' UTR variants in which SLα structures are introduced at various positions. Figure 3 shows the nucleotide sequences of hAG 3' UTR variants and control groups with SLα and SLα mutations introduced. Figure 4 shows the results of electrophoretic analysis for each hAG 3' UTR variant with the SLα construct introduced (M: RiboRuler High Range RNA Ladder, Lane 1: HBV PREα, Lane 2: hAG+SLα-0, Lane 3: hAG+SLα-10, Lane 4: hAG+SLα-20, Lane 5: hAG+SLα-30, Lane 6: hAG+SLα-30-mut, Lane 7: hAG+SLα-40, Lane 8: hAG+SLα-50, Lane 9: AES/mtRNR1, Lane 10: hAG) Figure 5 shows images (Figure 5a) of luminescence measured after administering an hAG 3' UTR variant with an SLα structure and a control to BALB/c mice, and the results of quantifying the luminescence (Figure 5b). Figure 6 shows the results of measuring changes in luciferase activity (Figure 6a) and AUC values (Figure 6b) over time, respectively, after transfecting HEK293T cells with an hAG 3' UTR variant and a control group with an SLα structure introduced. Figure 7 shows the nucleotide sequences of each variant to which a variation was applied to the loop region within the SLα structure. Figure 8 is a figure showing the results of measuring changes in luciferase activity and AUC values over time after transfecting HEK293T cells with mRNA containing hAG 3' UTR to which each loop variant sequence shown in Figure 7 was applied. Figure 9 is a figure showing the results of confirming the