Artificial nucleic acid molecule and application thereof

Abstract

The invention provides an artificial nucleic acid molecule and application thereof. Specifically, the artificial nucleic acid molecule provided by the invention comprises at least one 5 '-non-coding region (5'-UTR) element and / or at least one 3 '-non-coding region (3'-UTR) element, the at least one 5 '-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NO: 1-16, or a homolog or a fragment thereof, and the at least one 5'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NO: 2-16. The at least one 3 '-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NO: 17-29, or a homolog or fragment thereof, or a sequence as shown in any one or more of SEQ ID NO: 17-29, or a sequence as shown in at least 50% sequence consistency and retaining functions thereof. The present disclosure also provides the use of the artificial nucleic acid molecule as a vaccine or for gene therapy.

Description

Technical Field

[0001] This disclosure relates to artificial nucleic acid molecules, carriers comprising said artificial nucleic acid molecules, cells containing said artificial nucleic acid molecules or said carriers; pharmaceutical compositions comprising said artificial nucleic acid molecules or said carriers; kits comprising said artificial nucleic acid molecules, said carriers and / or said pharmaceutical compositions; and applications thereof. Background Technology

[0002] mRNA is composed of elements such as the 5' cap, 5' UTR (5' non-coding region), ORF (open reading frame), 3' UTR (3' non-coding region), and 3' poly(A) tail. The main targets of mRNA sequence design are the 5' UTR, ORF, and 3' UTR. The secondary structure, position, length, and GC content of the 5' UTR all have a significant impact on mRNA translation efficiency and sequence stability. The 3' UTR is a regulatory element that can regulate mRNA translation initiation and efficiency, modulate mRNA stability through rich AU elements, and perform regulatory functions and protein-protein interactions by binding to multiple RNA-binding proteins.

[0003] Gene therapy and mRNA vaccines are rapidly developing modern medical approaches. Billions of people worldwide have been vaccinated with mRNA COVID-19 vaccines, and their safety and efficacy have been widely validated. mRNA vaccine therapy holds enormous potential. Stable and effective expression of mRNA in the body is a fundamental condition for its efficacy.

[0004] Because the ORF sequences to be expressed in different nucleic acid molecules vary greatly, studies have shown that the expression of nucleic acids (such as mRNA molecules) is closely related to their functional elements 5'UTR and 3'UTR. An efficient combination of 5'UTR and 3'UTR improves the sequence stability, half-life, and protein translation level of mRNA vaccines in vivo.

[0005] Therefore, a combination of 5'UTR and 3'UTR sequences that is stable, highly efficient in expression, and universally applicable is the structural basis for the successful development of mRNA drugs. Summary of the Invention

[0006] To address one of the aforementioned technical problems in the prior art, this disclosure provides nucleic acid molecules suitable for gene therapy and / or immunization vaccines. Specifically, this disclosure provides an artificial nucleic acid molecule having a combination of 5'UTR and 3'UTR capable of enhancing the expression of encoded proteins. This disclosure also provides pharmaceutical compositions containing said artificial nucleic acid molecule, and their use in gene therapy and / or immunization vaccines.

[0007] According to one aspect of this disclosure, an artificial nucleic acid molecule is provided, comprising: at least one 5'-untranslated region (5'-UTR) element and at least one 3'-untranslated region (3'-UTR) element, wherein the at least one 5'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NO:1 to 16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function, and the at least one 3'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NO:17 to 29, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0008] In some embodiments, an artificial nucleic acid molecule includes, along the 5' to 3' direction: at least one open reading frame; at least one 5'-uncoding region (5'-UTR) element; and at least one 3'-uncoding region (3'-UTR) element.

[0009] The artificial nucleic acid molecule disclosed herein, by containing a combination of at least one 5'-UTR element and at least one 3'-UTR element, is capable of enhancing the expression of proteins encoded by operatively linked open reading frames.

[0010] In some embodiments, the 5'-UTR element and / or the 3'-UTR element may be derived from animals, bacteria, fungi, or viruses.

[0011] In some embodiments, the 5'-UTR element and / or the 3'-UTR element may be derived from vertebrate genes, preferably from mammals, more preferably from the 5'-UTR and / or 3'-UTR of primate or rodent genes, or from variants that retain their function. In specific embodiments, the 5'-UTR element and / or the 3'-UTR element may be derived from, but is not limited to, the 5'-UTR and / or 3'-UTR of genes from humans, mice, rats, rabbits, dogs, cats, guinea pigs, orangutans, or from variants that retain their function.

[0012] In some embodiments, the 5'-UTR element and / or the 3'-UTR element may be derived from viruses, such as, but not limited to, the 5'-UTR and / or 3'-UTR of genes such as TEV (Tobacco Mosaic Virus), CMV (Cytomegalovirus), DEN (Dengue Virus), BMV (Brome Mosaic Virus), influenza virus, SINV (Simbis virus), HAV (Hepatitis A Virus), HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), TMV (Tobacco Mosaic Virus), etc., or variants that retain their function.

[0013] In some embodiments, the 5'-UTR element and / or the 3'-UTR element may comprise nucleic acid sequences of the 5'-UTR and / or 3'-UTR of transcripts from the following genes, or variants thereof that retain their function: HBB (hemoglobin subunit β), TEV (tobacco aberration virus), Actb (actin β), scrUTR (scrambled short 5'UTR), CYBA (cytochrome b-245α chain), TMV (tobacco mosaic virus), DEN2 (dengue virus type 2), CMV (cytomegalovirus), and DSS (deep sequencing virus). Squence, miniUTR, HBA2 (hemoglobin subunit α2), Rps27a-44 (ribosomal protein S27a), AG (human alpha globin), COL1A2 (type I collagen α-2 chain), HRP (methemoglobin-associated protein), FGA (fibrinogen α chain), FGB (fibrinogen β chain), TOP-HAG (5' end oligopyrimidine-influenza virus hemagglutinin), SINV (Simbis virus), BMV (Brome Mosaic Virus), HBA1 (hemoglobin subunit α1), AAT (α-1 antitrypsin), TF (transcription factor), HCV (hepatitis C virus), AES-mtRNR1, MOD1 (mosaic death 1) and / or MOD2.

[0014] In some embodiments, the 5'-UTR element may comprise 10 to 200 nucleotides, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or any number of nucleotides between these values. In some embodiments, the 5'-UTR element may comprise 40 to 150 nucleotides, preferably 40 to 140 nucleotides, or any number of nucleotides between these values.

[0015] In some embodiments, the 3'-UTR element may comprise 60 to 500 nucleotides, for example, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or any number of nucleotides between these values. In some embodiments, the 3'-UTR element may comprise 100 to 400 nucleotides.

[0016] In some embodiments, the 5'-UTR region element may include a nucleotide sequence as shown in any one of SEQ ID NO:1 to 16, or a homology or fragment thereof, or a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the nucleotide sequence and retaining its function.

[0017] In some embodiments, the 3'-UTR element may include a nucleotide sequence as shown in any one of SEQ ID NO:17-29, or a homology or fragment thereof, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the nucleotide sequence and retaining its function.

[0018] In some embodiments, the at least one 5'-UTR region element comprises a nucleotide sequence as shown in any one or more of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function.

[0019] In some embodiments, the at least one 3'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NO:17-19 and 22-29, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function.

[0020] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:17, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0021] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:18, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0022] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0023] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:22, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0024] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0025] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0026] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0027] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:26, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0028] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:27, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0029] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:28, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0030] In some embodiments, the artificial nucleic acid molecule includes: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NO:1-11 and 13-16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function; and a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO:29, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with the nucleotide sequence and retaining its function.

[0031] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:3, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0032] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:5, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0033] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:6, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0034] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:7, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0035] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:8, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0036] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:10, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0037] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:11, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0038] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:19, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function.

[0039] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:3, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0040] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:5, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0041] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:6, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0042] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:7, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0043] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:8, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0044] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:10, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0045] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:11, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0046] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:23, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function.

[0047] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:3, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0048] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:5, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0049] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:6, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0050] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:7, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0051] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:8, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0052] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:10, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function.

[0053] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:11, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0054] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:24, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0055] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:3, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0056] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:5, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0057] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:6, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0058] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:7, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0059] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:8, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0060] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:10, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0061] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:11, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with respect to its function.

[0062] In some embodiments, the artificial nucleic acid molecule may include: a 5'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:16, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function; and / or a 3'-UTR element comprising a nucleotide sequence as shown in SEQ ID NO:25, or a homology or fragment thereof, or a sequence having at least 50% sequence identity with it and retaining its function.

[0063] In some embodiments, the artificial nucleic acid molecule may also include a polyadenylation sequence and / or a polyadenylation signal.

[0064] In some embodiments, the polyadenylation sequence and / or polyadenylation signal is located on the 3' end side of the at least one 3'-UTR element.

[0065] In some embodiments, the polyadenylated sequence may have about 20 to about 400, preferably about 50 to about 400, about 50 to about 300, more preferably about 50 to about 250, or about 60 to about 250 adenine nucleotides. In some embodiments, the polyadenylated sequence may have about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, or any number of adenine nucleotides between these values.

[0066] In some embodiments, the artificial nucleic acid molecule may further include a 5'-cap structure, a polycytidine sequence, a histone stem-loop, and / or an internal ribosome entry side (IRES) motif.

[0067] In some embodiments, the artificial nucleic acid molecule may further include a promoter. In some embodiments, the promoter may be a constitutive promoter or an inducible promoter. There are no particular limitations on the promoter used, and those skilled in the art can choose conventionally used promoters as needed.

[0068] In some embodiments, the promoter may be an RNA polymerase promoter. Preferably, the promoter is suitable for eukaryotic, prokaryotic, viral, or bacteriophage transcription systems. The promoter sequence is recognized by a polymerase, such as an RNA polymerase, for example, an RNA polymerase of eukaryotic, prokaryotic, viral, or bacteriophage form. In a preferred embodiment, the promoter may include, but is not limited to, the SP6, Ts, or T7 promoter.

[0069] In some implementations, the open reading frame includes a codon-optimized region.

[0070] In some embodiments, the artificial nucleic acid molecule may be a DNA or RNA molecule. In a preferred embodiment, the artificial nucleic acid molecule may be an RNA molecule, preferably an mRNA molecule.

[0071] According to another aspect of this disclosure, a carrier is provided that includes the artificial nucleic acid molecule described above.

[0072] In some embodiments, the vector may include a DNA vector.

[0073] In some embodiments, the vector may include a plasmid vector, a bacteriophage vector, or a viral vector.

[0074] In some embodiments, the carrier may be a cyclic molecule.

[0075] In some embodiments, the vector further includes restriction enzyme sites for linearizing the circular vector molecule.

[0076] According to another aspect of this disclosure, a cell is provided that comprises the artificial nucleic acid molecule or the vector described above.

[0077] In some implementations, the cells can be cells of any origin, such as cells derived from mammals or the subject.

[0078] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising the artificial nucleic acid molecule described above, the carrier described above, or the cell described above.

[0079] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0080] According to another aspect of this disclosure, the artificial nucleic acid molecule, the vector, the cell, or the pharmaceutical composition described herein are provided for use as a drug, or as a vaccine, or for gene therapy.

[0081] According to another aspect of this disclosure, a method for preventing or treating a disease is provided, the method comprising administering to a subject in need the aforementioned artificial nucleic acid molecule, the aforementioned carrier, the aforementioned cell, or the aforementioned pharmaceutical composition of this disclosure.

[0082] In some embodiments, the method may include an immunization method or a gene therapy method. In some embodiments, the vaccine includes a nucleic acid vaccine, preferably an RNA vaccine, and more preferably an mRNA vaccine.

[0083] According to another aspect of this disclosure, a method for improving the production of a target protein is provided, the method comprising the step of associating an open reading frame encoding the target protein with the aforementioned 5'-UTR and / or 3'-UTR sequences to obtain an artificial nucleic acid molecule. Attached Figure Description

[0084] Figure 1 illustrates the expressive effect of 208 combinations of 5'UTR / 3'UTR according to some embodiments of the present disclosure.

[0085] Figure 2 The expression effects of 16 combinations of 5'UTR / 3'UTR according to some embodiments of the present disclosure in HepG2, C2C12, RD and HEK293 cells are shown. Figure 2 A shows the expressive effect of BMV 3'UTR combined with different 5'UTR combinations; Figure 2 B shows the expressive effect of AAT 3'UTR combined with different 5'UTRs.

[0086] Figure 3 The effects of HCV 3'UTR combined with different 5'UTRs on expression in HepG2, C2C12, RD and HEK293 cells were shown.

[0087] Figure 4 The expression effects of the TMV 5'UTR / TF 3'UTR combination in HepG2, C2C12, RD and HEK293 cells were shown. Detailed Implementation

[0088] Stable and effective expression of mRNA vaccines in vivo is a fundamental condition for their efficacy. Studies have shown that the expression of mRNA molecules is closely related to their functional elements 5'UTR and 3'UTR. An efficient combination of 5'UTR and 3'UTR can improve the sequence stability, half-life, and protein translation level of mRNA vaccines in vivo.

[0089] The purpose of this disclosure is to optimize and obtain highly stable, highly expressible, and universally applicable 5'UTR and 3'UTR sequences and combinations for use in vectors expressing mRNA. This disclosure involves screening for native 5'UTRs and 3'UTRs of highly expressed animal, bacterial, and viral proteins, and then constructing expressible EGFP-mRNA sequences through cross-combination. High-throughput screening experiments identified 32 UTR combinations capable of achieving efficient expression. Subsequently, this disclosure examined the expression of these 32 UTRs in different cell lines to evaluate the universality of expression of these UTR sequences in different cell line contexts.

[0090] The results of this disclosure show that the UTRs combinations screened in this disclosure can achieve efficient expression in different cell lines.

[0091] In a specific implementation, the inventors of this disclosure further designed a novel 5'UTR sequence (TOP-hHAG) by comprehensively comparing parameters such as UTR sequence length, secondary structure, and base type ratio, based on the fundamental principles of sequence length between 12-500 nt, absence of special secondary structures affecting transcription and translation, and absence of miRNAs rich in adenosine / uridine elements, cis-regulatory elements, and trans-acting factors. Furthermore, by combining this TOP-hHAG 5'UTR with different 3'UTRs, it was found that it can achieve high efficiency and versatility in expression when combined with different 3'UTRs.

[0092] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0093] definition

[0094] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0095] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0096] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0097] As used herein, the terms “polynucleotide,” “nucleic acid sequence,” or “nucleic acid molecule” are used interchangeably and refer to a polymer of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), their analogues, or mixtures thereof. This term refers to the primary structure of the molecule. Therefore, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid (“DNA”) and triple-stranded, double-stranded, and single-stranded ribonucleic acid (“RNA”). It also includes modified (e.g., by alkylation and / or by capping) and unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose); polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or unspliced; any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base; and other polymers containing a positive nucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholinopolymers; and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, as found in DNA and RNA. Preferably, the nucleic acid molecule is a polymer comprising or composed of nucleotide monomers covalently linked to each other by phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid molecule" also includes modified nucleic acid molecules, such as, but not limited to, DNA or RNA molecules modified by bases, sugars, or backbones.

[0098] Typically, the artificial nucleic acid molecules disclosed herein can contain any naturally occurring nucleotide, such as guanosine, uracil, adenosine, and / or cytosine or analogues thereof. In this regard, nucleotide analogues are defined as naturally occurring variants of the naturally occurring nucleotides adenosine, cytosine, thymidine, guanosine, and uridine. Optionally, nucleotide analogues may also have, for example, non-naturally occurring functional groups, preferably naturally occurring functional groups added to or derived from naturally occurring nucleotides, or nucleotides lacking or replacing them. The various components of the naturally occurring nucleotides can be modified, i.e., the base component, the sugar (ribose) component, and / or the phosphate component forming the backbone of the RNA sequence.Analogs of guanosine, uridine, adenosine, thymidine, and cytosine include, but are not limited to, any naturally occurring or non-natural guanosine, uridine, adenosine, thymidine, or cytosine modified, for example, by acetylation, methylation, hydroxylation, etc., including 1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-hypoxanthin, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2'-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-amino-6-chloropurine riboside, 2-aminopurine-riboside, 2'-arabinoadecanoside, 2'-arabinoadecanoside, 2'-arabino-2'-deoxyadenosine, 2'-azido ... 2'-Deoxycytidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, 2-chloroadenosine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isoopenenyl-adenosine, 2'-O-methyl-2-aminoadenosine, 2'-O-methyl-2'-deoxyadenosine, 2'-O-methyl-2'-deoxycytidine, 2'-O-methyl-2'-deoxyguanosine, 2'-O-methyl-2'-deoxyuridine, 2'-O-methyl-5-methyluridine, 2'-O 2'-Methylhypoxanthin, 2'-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uracil, 5,6-dihydrouridine, 5-aminoallylcytidine, 5-aminoallyl-deoxy-uridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-carboxymethylaminomethyl-uridine, 5-chloro-arabinocytosine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine riboside, 6-mercapto- Guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza-2'-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside, β-D-mannosyl-queosine, dihydro-uracil, hypoxanthin, N1-methyladenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6-isopropenyl-adenosine, N6-methyl-adenosine, N7-methyl-flavin, N-uracil-5-oxyacetic acid methyl ester, puromycin, queeosine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, flavin, and xylose (Xylo)-adenosine.

[0099] As used herein, the term "DNA" or "deoxyribonucleotide" refers to nucleic acid molecules, which are polymers composed of nucleotides. These nucleotides are typically monomers of deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate, which themselves consist of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, polymerized through a characteristic backbone structure. This backbone structure is typically formed by a phosphodiester bond between the sugar moiety (deoxyribose) of the nucleotide of the first adjacent monomer and the phosphate moiety of the second adjacent monomer. The specific sequence of the monomers, i.e., the sequence of bases linked to the sugar / phosphate backbone, is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, for example, through A / T-base pairing and G / C-base pairing.

[0100] The term "RNA" as used in this article is the common abbreviation for ribonucleic acid. RNA belongs to the category of nucleic acid molecules, which are polymers composed of nucleotides. These nucleotides are typically adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers linked together along a backbone. The backbone is formed by phosphodiester bonds between the sugar of the first adjacent monomer, i.e., ribose, and the phosphate moiety of the second adjacent monomer. This specific sequence of monomers is called the RNA sequence. RNA can be obtained, for example, through transcription of a DNA sequence within a cell. In eukaryotic cells, transcription typically takes place in the nucleus or mitochondria. Typically, transcription of DNA usually produces pre-mature RNA (pre-RNA), which needs to be processed into messenger RNA (mRNA). For example, in eukaryotes, the processing of pre-mature RNA involves a variety of different post-transcriptional modifications, such as splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. The sum of these processes is also called RNA maturation. Mature messenger RNA typically provides a nucleotide sequence that can be translated into a specific peptide or protein amino acid sequence. Mature mRNA typically includes a 5' cap, 5' UTR, open reading frame, 3' UTR, and a polyadenylated nucleotide sequence. In addition to messenger RNA, there are several types of non-coding RNAs that can participate in regulating transcription and / or translation.

[0101] As used herein, the term "3'-UTR" or "3'-uncoding region" refers to a portion of an artificial nucleic acid molecule located at the 3' end (i.e., "downstream") of an open reading frame (ORF) and not translated into a protein. A 3'-UTR typically refers to a portion of the sequence between the protein-coding region (ORF) or coding sequence (CDS) on mRNA and the polyadenylated nucleotide sequence. In this disclosure, the 3'-UTR may also include a sequence added post-transcriptionally, such as a polyadenylated nucleotide sequence. The 3'-UTR of mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is typically encoded by a gene that is transcribed into its respective mRNA during gene expression. The genome sequence is first transcribed into pre-mature mRNA, optionally containing introns. The pre-mature mRNA is then further processed into mature mRNA during maturation. The maturation process includes the following steps: 5' capping, splicing, and optional intron removal to obtain pre-mature mRNA, followed by 3' end modification (e.g., polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / exonuclease cleavage). The 3'-UTR is located between the protein-coding region stop codon (preferably immediately following the 3' end of the protein-coding region stop codon) and the polyadenylated sequence of the mRNA. The term "3'-UTR" of a gene includes both the DNA and RNA sequences of the 3'-UTR, and includes both the sense and antisense strands, as well as both mature and immature RNA. In a preferred embodiment, the 3'-UTR may have a length of 20, 30, 40, or 50 or more nucleotides.

[0102] As used herein, the term "5'-UTR" or "5'-uncoding region" refers to a portion of an artificial nucleic acid molecule located at the 5' end (i.e., "upstream") of the open reading frame and not translated into a protein. Typically, the 5'-UTR begins at a transcription start site and terminates one nucleotide before the start codon in the open reading frame. Preferably, the 5'-UTR has a length of 20, 30, 40, or 50 or more nucleotides. The 5'-UTR may contain elements for controlling gene expression, also known as regulatory elements, such as ribosome binding sites. The 5'-UTR may be posttranscribed, for example, by adding a 5'-cap. The 5'-UTR sequence is typically encoded by the gene transcribed into individual mRNAs during gene expression. In this disclosure, the 5'-UTR is located within the mature mRNA sequence between the start codon and, for example, a 5'-cap. Preferably, the 5'-UTR corresponds to the sequence from the nucleotide on the 3' side of the 5' cap (more preferably the nucleotide immediately adjacent to the 5' cap) to the nucleotide on the 5' side of the start codon in the protein-coding region (preferably the nucleotide immediately adjacent to the 5' side of the start codon in the protein-coding region). The nucleotide immediately adjacent to the 3' side of the 5' cap of mature mRNA typically corresponds to the transcription start site. In this disclosure, the term "5'-UTR" of a gene corresponds to the sequence of the 5'-UTR of the mature mRNA derived from that gene, including both the DNA and RNA sequences of the 5'-UTR, and including both sense and antisense strands, as well as both mature and immature RNA.

[0103] The above-mentioned expression "corresponds to" means that the 5'-UTR or 3'-UTR sequence can be an RNA sequence in the mRNA sequence used to define the 5'-UTR or 3'-UTR sequence, or a DNA sequence corresponding to this RNA sequence.

[0104] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0105] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0106] Those skilled in the art will understand that mentioning "at least 50% sequence identity" with a sequence includes any sequence that has more than 50% sequence identity through the sequence alignment method described above. For example, it may include sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0107] As used herein, the term "operably linked" refers to the connection of at least a first element and a second element such that the constituent elements are in a relationship that allows them to function in their intended manner. For example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if a regulatory sequence (e.g., a promoter sequence) and a coding sequence are linked in a manner that allows the expression of the coding sequence to be controlled by the regulatory sequence. In some embodiments, the "operably linked" regulatory sequence is covalently bound to the coding sequence, directly or indirectly (e.g., in a single nucleic acid molecule). In some embodiments, the regulatory sequence controls the expression of the coding sequence in a trans-regulatory manner, and including the regulatory sequence in the same nucleic acid as the coding sequence is not a requirement for operational linking.

[0108] The term "G / C-modified" nucleic acid as used herein can typically refer to nucleic acids, preferably artificial nucleic acid molecules as defined herein, based on a modified wild-type sequence that preferably contains an increased number of guanosine and / or cytosine nucleotides compared to the wild-type sequence. This increased number can be achieved by replacing a codon containing adenosine or thymidine nucleotides with a codon containing a guanosine or cytosine nucleotide. If the enriched G / C content occurs in the coding region of DNA or RNA, it utilizes the degeneracy of the genetic code. Therefore, the codon substitution preferably does not change the encoded amino acid residues but only increases the G / C content of the nucleic acid molecule.

[0109] As used herein, the term "gene therapy" can be understood as treating a patient's body or isolated tissues / cells with nucleic acids encoding peptides or proteins. Gene therapy typically includes at least one of the following steps: a) administering nucleic acids, preferably artificial nucleic acid molecules as disclosed herein, directly to a patient or in vitro to isolated cells / tissues via any route of administration; b) transcribing and / or translating the introduced nucleic acid molecules; and, optionally c) if the nucleic acids are not directly administered to the patient, then re-administering isolated, transfected cells to the patient.

[0110] As used herein, the term "vaccination" refers to nucleic acid molecules and can be understood as vaccination by administering nucleic acid molecules encoding antigens or immunogens or fragments thereof. Nucleic acid molecules can be administered to the body of a subject or to isolated cells. When certain cells in the body are transfected or when isolated cells are transfected, the antigen or immunogen can be expressed by those cells and subsequently presented to the immune system, eliciting an adaptive, i.e., antigen-specific immune response. Vaccination typically involves at least one of the following steps: a) administering nucleic acid, preferably an artificial nucleic acid molecule as disclosed herein, to a subject (e.g., a patient) or to isolated cells; b) transcribing and / or translating the introduced nucleic acid molecule; and, optionally c) if the nucleic acid was not directly administered to the patient, then re-administering isolated, transfected cells to the subject (e.g., the patient).

[0111] Gene vaccines typically consist of genetically modified nucleic acid molecules that allow the expression of characteristic peptides or protein (antigen) fragments of pathogens or tumor antigens. After administration to a patient, the gene vaccine is taken up by target cells to express the target gene carried by the vaccine. The expression of the administered nucleic acid leads to the production of the encoded protein. If these proteins are recognized as foreign by the patient's immune system, an immune response is triggered.

[0112] Gene therapy and gene vaccination methods are essentially based on administering nucleic acid molecules to a patient and subsequently transcribing and / or translating the genetic information encoded therein. Optionally, gene vaccination or gene therapy may also involve isolating specific somatic cells from the patient to be treated, subsequently transfecting these cells in vitro, and then administering the treated cells back to the patient.

[0113] DNA and RNA can be used as nucleic acid molecules in gene therapy or gene-based vaccines.

[0114] The terms “polyadenylated sequence,” “polyadenylated tail,” “3'-polyadenylated tail,” or “PolyA” as used herein are used interchangeably and are understood to refer to an adenosine nucleotide sequence comprising, for example, up to about 400 adenosine nucleotides (also referred to as adenosine nucleotides), for example from about 20 to about 400, preferably about 50 to about 400, more preferably about 50 to about 300, even more preferably about 50 to about 250, and most preferably about 60 to about 250 adenosine nucleotides. The polyadenylated sequence is typically located at the 3' end of mRNA. In this disclosure, the polyadenylated sequence may be located, for example, within mRNA via transcription by a vector or in any other nucleic acid molecule (e.g., a template vector for generating RNA), preferably in mRNA.

[0115] As used herein, the term "polyadenylation" is generally understood to refer to the addition of a polyadenylated sequence to a nucleic acid molecule, such as RNA. Polyadenylation can be induced by a polyadenylation signal. This polyadenylation signal is preferably located within a nucleotide segment at the 3' end of the nucleic acid molecule, such as RNA, to be polyadenylated. The polyadenylation signal typically comprises a hexamer of adenine and uracil / thymine nucleotides, preferably the hexamer sequence AA(U / T)AAA or A(U / T)(U / T)AAA. Polyadenylation typically occurs during the processing of pre-mRNA, usually during the RNA maturation (from pre-mRNA to mature mRNA) step to incorporate the polyadenylation.

[0116] The artificial nucleic acid molecules disclosed herein can be RNA or DNA molecules, preferably RNA molecules. The artificial nucleic acid molecules disclosed herein can be modified by adding a 5'-cap structure. The 5'-cap structure can be formed from modified nucleotides, particularly derivatives of guanine nucleotides. In some embodiments, the 5'-cap can be linked to the 5' end of the nucleic acid molecule via a 5'-5'-triphosphate bond. The 5'-cap can be methylated, for example, m7GpppN, where N is the 5'-terminal nucleotide of the nucleic acid molecule carrying the 5'-cap. Other examples of 5' cap structures may include glycerol (inverted deoxy-debasing residue (partial)), 4',5' methylene nucleotide, 1-(β-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1,5-dehydrated hexitol nucleotide, L-nucleotide, α-nucleotide, modified base nucleotide, threo-pentafuranosyl nucleotide, acyclic 3',4'-open nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentayl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted debasing moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted debasing moiety, 1,4-butanediol phosphate, 3'-aminophosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-thiophosphate, dithiophosphate, or bridged or non-bridged methyl phosphate moiety.

[0117] Preferred modified 5'-cap structures may include CAP1 (ribose methylation of the nucleotide adjacent to m7G), CAP2 (ribose methylation of the second nucleotide downstream of m7G), CAP3 (ribose methylation of the third nucleotide downstream of m7G), CAP4 (ribose methylation of the fourth nucleotide downstream of m7G), ARCA (anti-reactive CAP analogs), modified ARCA (e.g., phosphate thioester modified ARCA), hypoxanthin, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0118] In some embodiments, the artificial nucleic acid molecules of this disclosure may encode pathogenic antigens or fragments, variants, or derivatives thereof. The pathogenic antigens are derived from pathogenic organisms, particularly bacteria, viruses, or protozoan (multicellular) pathogenic organisms, which elicit an immunological response in subjects, particularly mammalian subjects, and more particularly humans. More specifically, the pathogenic antigens are preferably surface antigens, for example, proteins (or fragments of proteins, e.g., the outer portion of a surface antigen) located on the surface of a viral, bacterial, or protozoan organism.

[0119] In some embodiments, the artificial nucleic acid molecules of this disclosure may encode tumor antigens or fragments, variants or derivatives thereof.

[0120] In some embodiments, the artificial nucleic acid molecules of this disclosure may encode therapeutic proteins or fragments, variants, or derivatives thereof. Therapeutic proteins, as defined herein, are peptides or proteins that are beneficial for the treatment of any genetic or acquired disease or for improving the condition of an individual. Specifically, among other functions, therapeutic proteins play an important role in generating therapeutic agents that can modify and repair genetic errors, destroy cancer cells or pathogen-infected cells, treat immune system disorders, or treat metabolic or endocrine disorders. For example, therapeutic proteins encompass adjuvant proteins, therapeutic antibodies, and hormones or alternatives thereof.

[0121] In some implementations, therapeutic proteins can be used for a variety of purposes, including the treatment of a variety of diseases such as, for example, infectious diseases, tumors (e.g., cancer or neoplastic diseases), diseases of the blood and blood-forming organs, endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the reproductive and urinary systems.

[0122] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0123] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0124] Example

[0125] Example 1. Preparation of mRNA

[0126] 1.1 Preparation of DNA Template

[0127] First, the designed 16 5'UTR and 13 3'UTR sequences (sequences shown in Table 1 below) were combined to obtain 208 5'UTR / 3'UTR combinations. Then, the EGFP sequence (nucleotide sequence shown in SEQ ID NO:31) was inserted into the middle of each 5'UTR and 3'UTR element combination. The 208 sequences confirmed by sequencing (containing the T7 promoter-5'UTR-EGFP gene-3'UTR) were synthesized and constructed into a pV5 plasmid backbone to prepare an in vitro transcription DNA plasmid. The in vitro transcription DNA plasmid was linearized by enzyme digestion, and the linearized DNA plasmid was purified and recovered.

[0128] Table 1: 5'UTR Names and Their Sequences

[0129]

[0130]

[0131] Table 2: 3'UTR Names and Sequences

[0132]

[0133]

[0134] The sequence of the T7 promoter is: TAATACGACTCACTATA (SEQ ID NO:30).

[0135] 1.2 In vitro transcription

[0136] Using linearized DNA as a template for in vitro transcription, 208 Cap1-pseudouridine Ψ-mRNA samples were prepared for use.

[0137] Specific experimental method: In vitro transcription reaction was carried out according to Table 3, and the reaction was carried out at 45℃ for 75 min.

[0138] Table 3. In vitro transcription reaction system

[0139] Element Dosage (μL) Linear pDNA (0.5 μg / μD) 100 T7 Reaction Buffer 200 1 M MgCl2(In house) 31 NTP / CleanCap mixture (100mM) (Shanghai Zhaowei) 275 T7 enzyme mixture 126 WFI 268 total 1000

[0140] T7 enzyme mixture: T7 RNA polymerase (3415 U / ml), Promega; T7 RNA preservation solution, Promega; RNase inhibitor (40 U / ml), Promega; inorganic pyrophosphatase (0.1 U / ml), Thermo Fisher.

[0141] After the reaction was completed, the DNA template degradation reaction was carried out according to the system shown in Table 4. After thorough mixing, the mixture was incubated at 37°C for 30±5 min.

[0142] Table 4. DNA template degradation reaction system

[0143] Element Dosage (μL) DnaseI (60U / μL) (Thermo Fisher) 13.3 In vitro transcripts 100

[0144] After the reaction was complete, EDTA was added to terminate the reaction. The in vitro transcribed mRNA product was then purified and recovered using LiCl, NaCl, and anhydrous ethanol, respectively. The purified mRNA was reconstituted with an equal volume of enzyme-free deionized water to obtain the final purified mRNA product. The concentration was determined spectrophotometer-wise, and the integrity was verified by electrophoresis.

[0145] Example 2. Expression of EGFP in HEK293 cells

[0146] Take cultured HEK293 cells and administer at a dose of 1×10⁻⁶. 4 After layering 0.1 mL of 1640 complete medium (Gibco, containing 10% FBS) into each well of a 96-well plate, the plates were incubated for 24 hours. The mRNA transfection was then performed using Lipofectamine mRNA transfection reagent. TM Cell transfection was performed according to the instructions for Messenger MAX (Thermo Fisher Scientific, LMRNA015): (1) Take 3 times the concentration of Lipofectamine for mRNA transfection. TM Messenger MAX transfection reagent, then add a certain amount of serum-free culture medium diluent, mix thoroughly to prepare transfection reagent dilution, and let stand at room temperature for 10 minutes. (2) Dispense 10 μL / tube of transfection reagent dilution into EP tubes containing experimental samples, add 90 μL of serum-free culture medium, and let stand at room temperature for 15 minutes to prepare transfection complex. (3) Take 20 μL of the prepared transfection complex (containing 50 ng mRNA sample) and add it to the wells of the plate containing HEK293 cells, and gently pipette 2-3 times to mix evenly. After completing the cell transfection operation, continue to place the culture plate in the cell culture incubator for static culture, and read the EGFP fluorescence value using an ELISA reader at 6 h, 24 h, 48 h and 72 h after transfection. After standardization, compare with the positive control PC-1 (mRNA-EGFP prepared based on the eTheRNA backbone (WO2015071295A1)).

[0147] Data Analysis:

[0148] (1) Calculate the average fluorescence value of the negative control NC group (untransfected HEK293 cells) in each plate;

[0149] (2) Subtract the average value of the negative control NC group from the fluorescence value X of each experimental group in each plate: X-NC;

[0150] (3) The average value of the positive control PC minus the negative control NC in each plate: PC-NC;

[0151] (4) Divide the average value X-NC calculated in step (2) of each experimental group by the average value PC-NC calculated in step (3) as the normalized value;

[0152] (5) Import the obtained normalized values ​​into Prism, and plot a bar chart with the mean and standard deviation of each data set, using the UTR name as the X-axis and the normalized values ​​as the Y-axis.

[0153] The results are as follows Figures 1A to 1M As shown.

[0154] The results show that, except for combinations using hCOL1A2 as the 5'UTR and some combinations using DEN2 and hHBA1 as the 3'UTR, most of the 3'UTR and 5'UTR combinations used in this experiment achieved higher expression levels than the positive control.

[0155] Example 3. Expression of EGFP in HepG2, A549, C2C12, and RD cells.

[0156] To verify whether candidate UTR sequences can be universally expressed in different cell lines, four human / mouse-derived normal and tumor cell lines—HepG2 (human hepatocellular carcinoma cells), RD (human malignant embryonic rhabdomyosarcoma cells), C2C12 (mouse myoblasts), and HEK293 (human embryonic kidney cells)—were selected. EGFP-mRNA containing candidate UTR combinations was transfected into these four cell lines to verify the expression of EGFP protein mediated by the UTR combinations.

[0157] To demonstrate the universality of the 3'UTR and 5'UTR combination discovered in this disclosure in different cell lines, 16 candidate combinations were first selected from Example 2 (see Table 5), and the cellular universality of these 16 combinations was further studied.

[0158] Experimental steps:

[0159] (1) HepG2, C2C12, RD and HEK293 cells were revived and cultured respectively. After the cells were in stable growth state, they were collected and cultured at 1×10⁻⁶. 4 / Place the cells into 96-well cell culture plates and place the plated cells in a cell culture incubator for 24 hours for further culture.

[0160] (2) After 24 hours, use the mRNA transfection reagent Lipofectamine. TMFollowing the instructions in the Messenger MAX (Thermo Fisher Scientific, LMRNA015) manual, EGFP-mRNA samples of 16 candidate UTRs were transfected into the wells containing HepG2, C2C12, RD, and HEK293 cells at a rate of 50 ng / well. The transfection results were observed under a microscope 24 hours after transfection, and the fluorescence values ​​of the experimental wells were read using a microplate reader.

[0161] (3) The data obtained by detection were normalized in accordance with the method of Example 2, and the expression levels of the same UTR combination in different cell lines were compared.

[0162] Table 5.16 Candidate UTRs Combination Names

[0163] Serial Number 5'UTR 3'UTR 1 TOP-hHAG BMV 2 ScrUTR BMV 3 TMV BMV 4 DEN2 BMV 5 hCMV BMV 6 DSS BMV 7 HBA2 BMV 8 AG+G BMV 9 TOP-hHAG AAT 10 ScrUTR AAT 11 TMV AAT 12 DEN2 AAT 13 hCMV AAT 14 DSS AAT 15 HBA2 AAT 16 AG+G AAT

[0164] The results are shown in Figure 2 From Figure 2 A and Figure 2 The results in B show that, compared with the positive control, all 16 combinations exhibited significantly better expression in four different cell types. This demonstrates that these 16 UTR combinations have the advantage of high universality and are suitable for achieving better expression in different cell types (including normal and tumor cells) from different species (e.g., humans and mice).

[0165] Following the experimental steps above, this embodiment also verified the mediating effect of HCV 3'UTR with DSS 5'UTR and scrUTR 5'UTR, respectively, and TMV 5'UTR with TF 3'UTR, on EGFP protein expression in HepG2, C2C12, RD, and HEK293 cells. The results are shown in [Figures to be inserted]. Figure 3 and Figure 4 From Figure 3 and Figure 4 It can be seen that these three UTR combinations can achieve better expression in different cells.

[0166] In addition, the inventors verified the expression of other UTR combinations in different cells, as shown in Table 6 below. The results showed that these UTR combinations achieved better expression in the verified cells.

[0167] Table 6.

[0168]

[0169]

[0170] Note: "×" indicates that no verification was performed; "√" indicates that verification was performed.

[0171] Example 4. Expression of different proteins in HEK293 cells

[0172] To test whether candidate highly expressed UTR sequences are universally applicable to different protein genes, this embodiment selects the herpes zoster VZV-gE gene (excluding the EGFP encoding gene) and the spike protein gene of the novel coronavirus to test the universality of gene expression of candidate mRNA in vitro transcription elements.

[0173] Following the method described in Example 1, the candidate group 5'UTR×3'UTR sequence was combined with the nucleic acid sequences of the coding regions of the selected VZV-gE (SEQ ID NO:32) and spike protein (SEQ ID NO:33) and then the DNA transcription vector was prepared by Nanjing GenScript through gene synthesis.

[0174] (1) Linearization of DNA template plasmid

[0175] The prepared DNA transcription vector was linearized using the enzyme digestion system shown in Table 7. After purification by salt-ethanol precipitation, a linearized template for in vitro transcription was obtained. The completeness of linearization was verified by agarose gel electrophoresis.

[0176] Table 7. DNA plasmid linearization reaction system

[0177] Element Dosage pUTR-gE / spike (1 μg / μL) 40μL BfuA1 buffer (10X) 16μL BfuA1 (20 U / μL) 8μL WFI 96μL reaction volume 160μL

[0178] After incubation at 37°C for 90 min, the linearized product was purified and recovered according to the system in Table 8.

[0179] Table 8. Salt / ethanol precipitation purification system

[0180] Components Volume (160 μL system) 5M NaCl 3.2μL Anhydrous ethanol 320μL

[0181] a. Let stand in a -20℃ refrigerator for more than 30 minutes, then centrifuge at 9500 rpm for 15 minutes at 4℃, and discard the supernatant;

[0182] b. Add 70% ethanol to each, centrifuge at 9500 rpm for 15 min at 4℃, and discard the supernatant;

[0183] c. Centrifuge at 9500 rpm for 2 min at 4℃, discard the supernatant, air dry, add 80 μL of water for injection to dissolve, and measure the concentration (OD).

[0184] (2) In vitro transcription

[0185] The purified linearized templates were used to perform in vitro mRNA transcription according to the method described in Example 1 to obtain purified VZV-gE-mRNA and spike-mRNA with different candidate UTRs. The in vitro mRNA transcription results were verified by gel electrophoresis.

[0186] (3) Cell transfection

[0187] The purified VZV-gE-mRNA and spike-mRNA were transfected into HEK293 cells to detect and analyze the protein expression levels of VZV-gE-mRNA and spike-mRNA.

[0188] 1. Seed cells 1 day in advance: Seed HEK293 cells in 24-well plates (5×10⁻⁶ cells per well). 5 / well), the total amount of DMEM full medium (Cityva, containing 10% FBS) before transfection was 0.45mL.

[0189] 2. Transfection process: (The volume of each reaction mixture is the volume of a single well; calculate the volume of other wells proportionally.)

[0190] a. Use eTheRNA backbone VZV gE and spike mRNA as positive controls PC, and HEK293 cells without transfection reagent as negative controls NC; then take the candidate VZV-gE-mRNA and spike-mRNA from each group and add them to a certain amount of serum-free culture medium, mix well and prepare RNA dilution solution.

[0191] b. Take 0.5 μL of Lipofectamine TM Messenger MAX TM Lipofectamine is prepared by adding a certain amount of serum-free culture medium and mixing well. TM Messenger MAX TM Diluted solution, let stand at room temperature for 5 minutes;

[0192] c. Lipofectamine TM Messenger MAX TM Mix the diluent and RNA diluent thoroughly and let stand at room temperature for 15 minutes.

[0193] d. Add 50 μL of the transfection complex to HEK293 cells containing 0.45 mL of complete culture medium (which may contain 10% serum and antibiotics) and mix well;

[0194] e. Observe the cell status 6 hours after transfection. If the cell status is good, there is no need to change the culture medium and continue culturing for 24 hours.

[0195] (4) Protein expression detection

[0196] 24 hours after transfection, cells in each well were lysed using Western blotting and IP cell lysis buffer (50 μL per well in a 24-well plate). The lysis buffers were then collected and the protein expression levels were detected using the Jess automated protein expression analysis system.

[0197] Jess equipment testing methods:

[0198] 1. Add PMSF to Western blotting and IP cell lysis buffer (Beyotime P0013) a few minutes before use, to a final concentration of 1 mM;

[0199] 2. Cells were lysed 24 h after transfection using Western blotting and IP cell lysis buffer (50 μL per well in a 24-well plate) and the samples were stored at -80 °C.

[0200] 3. JESS Equipment Preparation:

[0201] a. Open Jess, Computer, and Compass software in sequence;

[0202] b. Gently touch the silver area on the Jess door to open it, clean the bevel with a lint-free paper soaked in anhydrous ethanol, and click Instrument>Self-Test to perform a hardware self-test;

[0203] 4. Sample addition:

[0204] a. Prepare an ice box and crushed ice, and place the relevant reagents on the ice;

[0205] b. Tear off the aluminum foil from all the 0.2mL tubes or poke a hole in the tube with your hand;

[0206] c. Preparation of DTT: Add 40 μL of WFI to the white DTT powder in the transparent tube and mix thoroughly by blowing and stirring.

[0207] d. Prepare 5× Master Mix (i.e. Loading Buffer): Add 20 μL of DTT solution and 20 μL of 10× Sample Buffer to the pink Master Mix powder in the pink transparent tube, and mix well by pipetting.

[0208] e. Preparation of Ladder (marker): Add 20 μL of pink ladder powder to a light blue transparent tube and add WFI, then mix thoroughly by suction and whisking;

[0209] f. Sample preparation: 3 μL of sample is required per well. Denature at 95°C for 5 minutes, cool on ice for 5 minutes, vortex to mix, then briefly centrifuge and place on ice for later use.

[0210] g. Preparation of primary antibodies: Antibody Diluent II 1:200 dilution of VZV gE antibody (Santa Cruz), 1:X (to be determined) dilution of SARS-CoV-2-S2P-BA.4 / 5-5spike antibody (Sino), 1:200 dilution of β-Actin antibody (Santa Cruz) (10 μL of diluted primary antibody is required per well), pAb goat anti-mouse IgG secondary antibody (Abcam) is ready to use and does not require preparation;

[0211] h. Preparation of luminescent solution: Take 200 μL each of Lumino-S and Peroxide, vortex to mix, and place on ice for later use;

[0212] i. Remove the sample plate, add the prepared reagents into the plate in sequence, cover it, balance it, and centrifuge at 2500 rpm (~1000 g) for 5 min at room temperature;

[0213] J. After the plate processing is completed, place the plate into the Jess program to read the protein expression results and perform quantification using grayscale.

[0214] The results of spike-mRNA protein expression level detection are shown in Table 9 below, and the results of VZV-gE-mRNA protein expression level detection are shown in Table 10 below. The results are statistically represented as the ratio of the protein expression level obtained by each combination to the positive control PC-1 (mRNA-EGFP prepared based on the eTheRNA backbone).

[0215] Table 9. Spike protein expression levels.

[0216]

[0217] Note: A<1;1 <B≤1.5;1.5<C≤2.0。

[0218] Table 10. VZV-gE protein expression levels.

[0219]

[0220]

[0221] Note: A<1;1 <B≤1.5;1.5<C≤2.0。

[0222] As can be seen from the experimental results, all 10 UTR combinations detected in this embodiment can achieve high expression of different proteins of VZV-gE and CoV-SARS-Spike.

[0223] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An artificial nucleic acid molecule comprising: at least one 5'-untranslated region (5'-UTR) element and / or at least one 3'-untranslated region (3'-UTR) element, wherein the at least one 5'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1-16, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof, the at least one 3'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NOs: 17-29, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof.

2. The artificial nucleic acid molecule of claim 1, wherein, the at least one 5'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1-16, or a homolog or fragment thereof, or a sequence with at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity thereto and which retains the function thereof, and / or the at least one 3'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NOs: 17-29, or a homolog or fragment thereof, or a sequence with at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity thereto and which retains the function thereof.

3. The artificial nucleic acid molecule of claim 1, wherein, the at least one 5'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1-11 and 13-16, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof; and / or the at least one 3'-UTR element comprises a nucleotide sequence as shown in any one or more of SEQ ID NOs: 17-19 and 22-29, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof.

4. The artificial nucleic acid molecule of claim 1, wherein, the artificial nucleic acid molecule comprises any one or more of: a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NOs: 1-11 and 13-16, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof; and, a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO: 17, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof, a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NOs: 1-11 and 13-16, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof; and, a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO: 18, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof, a 5'-UTR element having a nucleotide sequence as shown in any one of SEQ ID NOs: 1-11 and 13-16, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof; and, a 3'-UTR element having a nucleotide sequence as shown in SEQ ID NO: 18, or a homolog or fragment thereof, or a sequence with at least 50% sequence identity thereto and which retains the function thereof, 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 19, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 22, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 23, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 24, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 25, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 26, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 27, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 28, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, and 5'-UTR region element having a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 11 and 13 to 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and, a 3'-UTR element having a nucleotide sequence as set forth in SEQ ID NO: 29, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof.

5. The artificial nucleic acid molecule of claim 1, wherein, the 5'-UTR region element has a nucleotide sequence as set forth in any one of SEQ ID NOs: 3, 5 to 8, 10, 11, or 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, preferably a nucleotide sequence as set forth in any one of SEQ ID NOs: 3, 5, 7, 8, 10, 11, or 16, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof; and / or the 3'-UTR element has a nucleotide sequence as set forth in any one of SEQ ID NOs: 19 and 23 to 25, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof, preferably a nucleotide sequence as set forth in SEQ ID NOs: 19 and 23 to 24, or a homolog or fragment thereof, or a sequence having at least 50% sequence identity thereto and retaining the function thereof.

6. The artificial nucleic acid molecule of claim 1, wherein, the artificial nucleic acid molecule further comprises a poly(A) sequence and / or a polyadenylation signal, preferably, the poly(A) sequence and / or the polyadenylation signal is located at the 3' end side of the at least one 3'-UTR element, more preferably, the poly(A) sequence has 20 to 400 adenine nucleotides, preferably, the artificial nucleic acid molecule further comprises a promoter, a 5'-cap structure, a poly(C) sequence, a histone stem loop, and / or an internal ribosome entry side (IRES) motif.

7. The artificial nucleic acid molecule of claim 1, wherein, the artificial nucleic acid molecule is a DNA or RNA molecule, preferably an RNA molecule, more preferably an mRNA molecule.

8. A vector comprising the artificial nucleic acid molecule of any one of claims 1 to 7.

9. The carrier of claim 8, wherein, The vector comprises a DNA vector, preferably a plasmid vector, a phage vector or a viral vector, Preferably, the vector is a circular molecule, more preferably the vector further comprises a restriction enzyme site for linearizing the circular vector molecule.

10. A cell comprising the artificial nucleic acid molecule of any one of claims 1 to 7 or the vector of claim 8 or 9, Preferably, the cell is a mammalian or subject-derived cell.

11. A pharmaceutical composition comprising the artificial nucleic acid molecule of any one of claims 1 to 7, the vector of claim 8 or 9 or the cell of claim 10, and a pharmaceutically acceptable carrier.

12. The artificial nucleic acid molecule of any one of claims 1 to 7, the vector of claim 8 or 9, the cell of claim 10 or the pharmaceutical composition of claim 11 for use as a medicament, for use as a vaccine or for use in gene therapy, Preferably, the vaccine comprises a nucleic acid vaccine, preferably an RNA vaccine, more preferably an mRNA vaccine; Preferably, the vaccine or the gene therapy is for treating or preventing a viral infection, a bacterial infection, a tumor, a metabolic disease.

13. A method for increasing the production of a protein of interest, the method comprising the step of associating an open reading frame encoding the protein of interest with the 5'-UTR and / or 3'-UTR sequences described above, to obtain the artificial nucleic acid molecule of any one of claims 1 to 7.

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