Novel artificial nucleic acid molecules

Abstract

To provide a novel artificial nucleic acid molecule useful for enhancing efficacy of therapeutic nucleic acids.SOLUTION: Provided is an artificial nucleic acid molecule comprising: a. at least one 5' untranslated region (5'-UTR) element derived from the 5'-UTR of an ATP5A1 gene, or a corresponding RNA sequence, homolog, fragment, or variant thereof; b. at least one 3' untranslated region (3'-UTR) element derived from the 3'-UTR of an RPS9 gene, or a corresponding RNA sequence, homolog, fragment, or variant thereof; and optionally, c. at least one coding region operably linked to the 5'-UTR and the 3'-UTR, wherein the 5'-UTR and / or the 3'-UTR is heterologous to the coding region.SELECTED DRAWING: None

Claims

1. a. at least one 5'-UTR element derived from the 5'-untranslated region (5'-UTR) of the ATP5A1 gene, or its corresponding RNA sequence, homologue, fragment or variant; b. at least one 3'-UTR element derived from the 3' untranslated region (3'-UTR) of the RPS9 gene, or its corresponding RNA sequence, homolog, fragment or variant; and, optionally, c. at least one coding region operably linked to the 5'-UTR and the 3'-UTR; An artificial nucleic acid molecule comprising: wherein the 5'-UTR and / or the 3'-UTR are heterologous to the coding region; Artificial nucleic acid molecule.

2. - the 5'-UTR element derived from the ATP5A1 gene comprises or consists of the DNA sequence set forth in SEQ ID NO: 5 or a DNA sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity (higher is more preferred) to the nucleic acid sequence set forth in SEQ ID NO: 5, or a fragment or variant thereof, or comprises or consists of the RNA sequence set forth in SEQ ID NO: 6 or an RNA sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity (higher is more preferred) to the nucleic acid sequence set forth in SEQ ID NO: 6, or a fragment or variant thereof, the 3'-UTR element derived from the RPS9 gene comprises or consists of the DNA sequence set forth in SEQ ID NO: 33 or a DNA sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity (higher being preferred) to the nucleic acid sequence set forth in SEQ ID NO: 33, or a fragment or variant thereof; or comprises or consists of the RNA sequence set forth in SEQ ID NO: 34 or an RNA sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity (higher being preferred) to the nucleic acid sequence set forth in SEQ ID NO: 34, or a fragment or variant thereof; The artificial nucleic acid molecule of claim 1.

3. The artificial nucleic acid molecule according to claim 1 or 2, wherein the coding region is located between the 5'-UTR and the 3'-UTR, and preferably downstream of the 5'-UTR and upstream of the 3'-UTR.

4. The artificial nucleic acid molecule according to any one of claims 1 to 3, wherein the at least one coding region encodes at least one (poly)peptide or protein of interest, optionally at least one (poly)peptide or protein of interest selected from antigenic (poly)peptides or proteins, allergenic (poly)peptides or proteins, therapeutic (poly)peptides or proteins, antibodies, or a fragment, variant, or derivative of the (poly)peptide or protein of interest.

5. said at least one antigenic (poly)peptide or protein is selected from a tumor antigen, a pathogenic antigen, an autoantigen, an alloantigen, or an allergenic antigen; 5. The artificial nucleic acid molecule according to claim 4, wherein the at least one pathogenic antigen is preferably selected from a bacterial, viral, fungal or protozoan antigen.

6. The therapeutic (poly)peptide or protein may be - therapeutic (poly)peptides or proteins that replace missing, defective or mutated proteins; - therapeutic (poly)peptides or proteins which are advantageous for treating inherited or acquired diseases, infectious diseases or neoplasias (e.g. cancer or tumor diseases); - adjuvant or immunostimulatory therapeutic (poly)peptides or proteins; - therapeutic antibodies; - peptide hormones; - gene editing agents; - immune checkpoint inhibitors; - T cell receptor; -enzyme; and / or - a variant, fragment or derivative of any of the above therapeutic (poly)peptides or proteins, The artificial nucleic acid molecule according to claim 4, wherein the artificial nucleic acid molecule is selected from the group consisting of:

7. The at least one coding region may further comprise: (a) at least one effector domain; (b) at least one peptide or protein tag; (c) at least one localization signal or sequence; (d) at least one nuclear localization signal (NLS); (e) at least one signal peptide; and / or (f) at least one peptide linker; (g) secretory signal peptide (SSP); (h) a multimerizing factor, including a dimerizing factor, a trimerizing factor, a tetramerizing factor, or an oligomerizing factor; (i) virus-like particle (VLP) forming factors; (j) transmembrane factor; (k) dendritic cell targeting factors; (l) immunological adjuvant factors; (m) antigen presentation promoting factor; (n) 2A peptide; (o) an agent that extends protein half-life; and / or (p) factors for post-translational modification (e.g., glycosylation); Code, The artificial nucleic acid molecule according to any one of claims 3 to 6, wherein the artificial nucleic acid molecule optionally further comprises at least one internal ribosome entry site (IRES) and / or at least one miRNA binding site.

8. The artificial nucleic acid molecule according to any one of claims 1 to 7, wherein the artificial nucleic acid molecule is RNA, preferably mRNA, viral RNA, self-replicating RNA, or replicon RNA.

9. The artificial nucleic acid, preferably RNA, according to any one of claims 1 to 8, wherein the artificial nucleic acid is a modified nucleic acid, preferably a stabilized nucleic acid, or the artificial nucleic acid comprises at least one modified or non-natural nucleotide, backbone modification, sugar modification, or base modification.

10. - the G / C content of said at least one coding region of said artificial nucleic acid is increased compared to the G / C content of the corresponding coding sequence of the corresponding wild-type artificial nucleic acid, and / or - the C content of the at least one coding region of the artificial nucleic acid is increased compared to the C content of the corresponding coding sequence of the corresponding wild-type artificial nucleic acid, and / or the codons in the at least one coding region of the artificial nucleic acid are adapted to the human codon usage, and the codon adaptation index (CAI) is preferably increased or maximized in the at least one coding sequence of the artificial nucleic acid; the amino acid sequence encoded by said artificial nucleic acid is preferably unmodified compared to the amino acid sequence encoded by the corresponding wild-type artificial nucleic acid, An artificial nucleic acid, preferably RNA, according to any one of claims 1 to 9.

11. An artificial nucleic acid, preferably RNA, according to any one of claims 1 to 10, comprising a 5' cap structure, preferably m7GpppN or Cap1 and / or at least one histone stem loop.

12. The at least one histone stem loop has a nucleic acid sequence shown in formula (I), (II), (Ia), or (IIa): Formula (I) (stem-loop sequence without stem boundary elements): 【Chemical Formula 1】 Formula (II) (Stem-loop sequence with stem border elements): 【Chemistry 2】 Formula (Ia) (stem-loop sequence without stem boundary elements): 【Chemistry 3】 Formula (IIa) (stem-loop sequence with stem border elements): 【Chemistry 4】 During the ceremony, Boundary element N of stem 1 or stem 2 １－６ is a sequence of 1 to 6, preferably 2 to 6, more preferably 2 to 5, even more preferably 3 to 5, and most preferably 4 to 5 or 5 consecutive Ns, wherein each N is, independently of the other Ns, selected from a nucleotide selected from A, U, T, G and C, or a nucleotide analog thereof; Stem 1 [N ０－２ GN ３－５ ] is a sequence that is reverse complementary or partially reverse complementary to an element of stem 2 and has a contiguous sequence of 5 to 7 nucleotides; Here, N ０－２ is a sequence of 0 to 2, preferably 0 to 1, more preferably 1, consecutive N, wherein each N is independently selected from nucleotides selected from A, U, T, G and C, or nucleotide analogs thereof; Here, N ３－５ is a sequence of 3 to 5, preferably 4 to 5, more preferably 4 consecutive Ns, wherein each N is selected, independently of the other Ns, from a nucleotide selected from A, U, T, G and C, or a nucleotide analog thereof; and wherein G is guanosine or an analog thereof, and may optionally be substituted by cytidine or an analog thereof, provided that its complementary nucleotide in stem 2, cytidine, is substituted by guanosine; Loop sequence [N ０－４ (U / T)N ０－４ ] is located between the elements of stem 1 and stem 2 and is a sequence of 3 to 5 consecutive nucleotides, more preferably 4 consecutive nucleotides; Here, each N ０－４ is another N ０－４ a contiguous sequence of 0 to 4, preferably 1 to 3, more preferably 1 to 2 N, independently of each other N, wherein each N is selected from a nucleotide selected from A, U, T, G and C, or a nucleotide analog thereof; and where U / T is uridine, or optionally thymidine; Stem 2 [N ３－５ CN ０－２ ] is a sequence that is reverse complementary or partially reverse complementary to an element of stem 1 and is a contiguous sequence of 5 to 7 nucleotides; Here, N ３－５ is a sequence of 3 to 5, preferably 4 to 5, more preferably 4 consecutive Ns, wherein each N is independently selected from the nucleotides A, U, T, G and C, or nucleotide analogs thereof; Here, N ０－２ is a sequence of 0 to 2, preferably 0 to 1, more preferably 1, consecutive N, wherein each N is independently selected from nucleotides selected from A, U, T, G and C, or nucleotide analogs thereof; and wherein C is cytidine or an analog thereof, optionally substituted by guanosine or an analog thereof, provided that its complementary nucleotide in stem 1, guanosine, is replaced by cytidine; where: Stem 1 and stem 2 are capable of forming reverse complementary base pairs with each other, where the base pairing can occur between stem 1 and stem 2, or Stem 1 and stem 2 are capable of forming base pairs with each other in a partially reverse complementary sequence, wherein imperfect base pairing can occur between stem 1 and stem 2; 12. An artificial nucleic acid, preferably RNA, according to claim 11.

13. The artificial nucleic acid, preferably RNA, according to any one of claims 1 to 12, optionally comprising a poly(A) sequence, preferably a poly(A) sequence comprising 10 to 200, 10 to 100, 40 to 80, or 50 to 70 adenosine nucleotides.

14. Preferably in the 5' to 3' direction, the following elements: a) a 5' cap structure, preferably m7GpppN or Cap1; b) a 5'-UTR element comprising or consisting of a nucleic acid sequence derived from a 5'-UTR as defined in claim 1 or 2, preferably comprising a nucleic acid sequence corresponding to the nucleic acid sequence set forth in SEQ ID NOs: 1 to 20 or a homologue, fragment or variant thereof; c) at least one coding sequence as defined in any one of claims 3 to 7; d) a 3'-UTR element comprising or consisting of a nucleic acid sequence derived from a 3'-UTR as defined in claim 1 or 2, preferably comprising a nucleic acid sequence corresponding to the nucleic acid sequence set forth in SEQ ID NOs: 23 to 34 or a homologue, fragment or variant thereof; e) optionally a poly(A) tail, preferably a poly(A) tail consisting of 10-1000, 10-500, 10-300, 10-200, 10-100, 40-80 or 50-70 adenosine nucleotides; f) optionally a poly(C) tail, preferably a poly(C) tail consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides; and g) optionally a histone stem loop; The artificial nucleic acid, preferably RNA, according to any one of claims 1 to 13, comprising:

15. A (pharmaceutical) composition or vaccine comprising at least one or more artificial nucleic acid molecules, preferably RNA, according to any one of claims 1 to 14, and a pharmaceutically acceptable carrier and / or excipient.

16. 16. The (pharmaceutical) composition or vaccine according to claim 15, wherein said artificial nucleic acid molecule, preferably RNA, is complexed with one or more cationic or polycationic compounds, preferably with a cationic or polycationic polymer, a cationic or polycationic peptide or protein, such as protamine, a cationic or polycationic polysaccharide, and / or a cationic or polycationic lipid, or a polymeric carrier.

17. 17. The (pharmaceutical) composition or vaccine according to claim 15 or 16, wherein the artificial nucleic acid molecule, preferably RNA, is complexed with one or more lipids, thereby forming a lipid nanoparticle, a lipoplex and / or preferably a liposome.

18. A (pharmaceutical) composition or vaccine according to any one of claims 15 to 17, further comprising at least one further active agent and / or at least one adjuvant.

19. A kit, preferably a kit of parts, comprising an artificial nucleic acid molecule, preferably RNA, according to any one of claims 1 to 14, or a (pharmaceutical) composition or vaccine according to any one of claims 15 to 18, and optionally a liquid vehicle and / or optionally technical instructions containing information on the use and dosage of said artificial nucleic acid molecule or said (pharmaceutical) composition or vaccine.

20. An artificial nucleic acid molecule, preferably RNA, according to any one of claims 1 to 14, a (pharmaceutical) composition or vaccine according to any one of claims 15 to 18, or a kit according to claim 19, for use as a medicament, preferably as a vaccine.

21. An artificial nucleic acid molecule, preferably RNA, according to any one of claims 1 to 14, a (pharmaceutical) composition or a vaccine according to any one of claims 15 to 18, or a kit according to claim 19 for use in the treatment of genetic diseases, cancer, infectious diseases, inflammatory diseases, (auto)immune diseases, allergies and / or for use in gene therapy and / or immunomodulation.

22. said artificial nucleic acid molecule, preferably RNA, (pharmaceutical) composition or vaccine being adapted for liver-targeted delivery, said artificial nucleic acid molecule, preferably RNA, (pharmaceutical) composition or vaccine is adapted for subcutaneous, intracutaneous, intradermal, topical or transdermal administration, and / or 22. The artificial nucleic acid molecule, preferably RNA, (pharmaceutical) composition or vaccine, or kit for use according to claim 20 or 21, wherein said artificial nucleic acid molecule, preferably RNA, (pharmaceutical) composition or vaccine is adapted for intramuscular administration.

23. A method for increasing the expression efficiency of an artificial nucleic acid molecule, preferably an RNA, comprising at least one coding region, preferably encoding a protein or peptide according to any one of claims 3 to 7, comprising: (a) associating the coding region with at least one 5'-UTR element derived from the 5'-UTR of the ATP5A1 gene, or its corresponding RNA sequence, homolog, fragment, or variant; (b) associating the coding region with at least one 3'-UTR element derived from the 3'-UTR of the RPS9 gene, or its corresponding RNA sequence, homolog, fragment, or variant; and (c) obtaining an artificial nucleic acid molecule, preferably RNA, according to any one of claims 1 to 14.

24. 1. A method for identifying a combination of 5′-UTR and 3′-UTR that can increase expression efficiency in a desired tissue or in a cell derived from a desired tissue, comprising: a) generating a library of artificial nucleic acid molecules ("test constructs"), each comprising a "reporter ORF" encoding a detectable reporter polynucleotide, preferably a selected luciferase or eGFP, operably linked to one of the 5'-UTRs and / or one of the 3'-UTRs as defined in claim 1 or 2; b) providing an artificial nucleic acid molecule comprising said "reporter ORF" operably linked to reference 5'- and 3'-UTRs, preferably RPL32 and ALB7 as "reference constructs"; c) introducing the test construct and the reference construct into the desired tissue or cell under suitable conditions that allow their expression; d) detecting and quantifying expression of the polypeptide from the "reporter ORF" from the test construct and the reference construct; e) comparing polypeptide expression from the test construct and the reference construct; Including, wherein a test construct characterized by increased polypeptide expression relative to a reference construct is identified as being capable of increasing expression efficiency in a desired tissue or cell.

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