Modified RNA molecules with adjustable expression

Abstract

This application relates to engineered RNA molecules, DNA molecules encoding engineered RNA molecules, and the use of engineered RNA molecules. The engineered RNA molecule comprises a poly(A) tail sequence containing a miRNA binding site. The poly(A) tail enables the precise expression of target genes in organs, tissues, and / or cells.

Description

Technical Field

[0001] (Cross - reference to Related Applications) This application claims priority to Chinese Patent Application No. PCT / CN2023 / 099512 filed on June 9, 2023, Chinese Patent Application No. PCT / CN2023 / 121779 filed on September 26, 2023, and Chinese Patent Application No. 202410711025.6 filed on June 3, 2024. These prior applications are hereby incorporated by reference in their entirety into this specification.

[0002] This application relates to the technical field of biotechnology, and particularly to engineered RNA molecules containing a polyadenylic acid (poly(A)) tail. The poly(A) tail enables the precise expression of engineered RNA molecules in specific organs, tissues, and / or cells.

Background Art

[0003] Deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) are the main components of biological genetic material, and their primary function is to carry and transmit genetic information. In eukaryotic cells, complete mRNA mainly consists of a 5' cap structure, a 5' untranslated region (5'UTR), a coding region (ORF), a 3' untranslated region (3'UTR), and a 3' poly(A) tail structure. The 5' cap structure primarily plays a role in regulating mRNA stability and the initiation of mRNA translation. This cap structure shields the 5' end of mRNA, preventing hydrolysis of mRNA by nucleic acid exonucleases. Furthermore, this cap structure is recognized and bound by cap-binding proteins (eIF-4E), which regulate the binding of mRNA to ribosomes, thereby initiating the translation process. The 5'UTR is a short sequence between the cap and the start codon; if the 5'UTR is too short or too long, it is detrimental to mRNA translation initiation. Generally, the 3'UTR is thought to be primarily involved in post-transcriptional regulation, including the regulation of mRNA's in vivo half-life. For example, many microRNAs (miRNAs) can bind to the 3'UTR of target gene mRNA, reducing the expression of the target gene through degradation or inhibition of binding. The 3'UTR is followed by a poly(A) sequence that can prevent degradation by exonucleases. Furthermore, the poly(A) tail sequence can also bind to poly(A)-binding proteins (PABPs), recruiting many other proteins such as eIF4G, eIF4B, and Paip-1 to form a complex, which is involved in regulating mRNA stability and initiating translation. The poly(A) tail of native mRNA in eukaryotic cells is added post-transcriptionally by poly(A) polymerase. This polymerase has substrate specificity for adenylate during the evolution from fungi and plants to animals. To date, it is not clear whether complex non-A motifs with specific regulatory functions can be formed within native poly(A) tail structures.

[0004] mRNA therapy has a wide range of applications, and depending on the different applications of mRNA therapy, it is mainly directed towards vaccines for infectious diseases, tumor immunotherapy, monoclonal antibody drugs and other protein drug substitutes, as well as gene editing. During the COVID-19 pandemic, the success of mRNA vaccines also underscored the great value of mRNA platforms. mRNA therapy typically delivers mRNA by lipid-based carrier systems (including liposomes and lipid nanoparticles (LNPs)). These lipid carriers generally encapsulate mRNA, improving its intracellular delivery and efficacy. LNP formulations represent innovation in the field of nucleic acid delivery technology. LNPs typically contain one or more cationic lipids and / or amino (ionizable) lipids, phospholipids, structural lipids (such as cholesterol), and / or polyethylene glycol-containing lipids (PEGylated lipids). Cationic lipids and / or ionizable lipids include, for example, amine-containing lipids that can be readily protonated.

[0005] While significant progress has been made in LNP-mediated nucleic acid delivery, most current delivery systems exhibit strong hepatotropism. The lack of clarity regarding the structure-effect relationship of LNP delivery systems makes it difficult to rationally design delivery vectors that are highly specific to organs and tissues, particularly different cell types within the same organ or tissue. As a result, mRNA-LNPs accumulate broadly in multiple organs, tissues, and / or cell types. How to deliver mRNA to specific organs, tissues, and / or cells to achieve specific expression of the delivered mRNA within those specific organs, tissues, and / or cells, while mitigating side effects on other organs, tissues, and / or cells, is a major challenge in this type of therapy.

[0006] miRNAs are small, single-stranded, non-coding RNA molecules, 19–25 nucleotides in length, found in plants, animals, and some viruses. They primarily play a role in mRNA degradation or silencing, and post-transcriptional regulation of gene expression. They become complementary to a double-stranded sequence with a specific sequence in the 3'UTR of a target mRNA molecule via Watson-Crick base pairing, forming that double-stranded sequence and thus causing silencing of the mRNA molecule. miRNA expression is highly specific in organs, tissues, and cells. Examples of tissue-specific miRNA expression include the liver (miRNA-122) and spleen (miRNA-142). The miRNA binding site is usually located in the 3'UTR of mRNA. In the art, it has been reported that miRNA binding sites are incorporated into the 3'UTR to improve off-target expression.

[0007] In the field of mRNA, the first step in preparing mRNA drugs in vitro is to synthesize the mRNA drug by in vitro transcription (IVT) using a linear plasmid containing the designed product sequence as a template, with the poly(A) tail typically added downstream of the 3' UTR via co-transcription. To add poly(A) via co-transcription, the template plasmid must contain a corresponding poly(dA:dT) sequence. However, poly(dA:dT) repeat sequences in plasmids are unstable during the replication process in E. coli, leading to frequent deletion mutations and subsequent shortening of the poly(dA:dT) tail. This phenomenon is detrimental to the preparation process for large-scale production of template plasmids for in vitro transcription by fermentation. Poly(A) shortening significantly impacts the in vivo stability and biological activity of mRNA. Therefore, plasmid replication stability of mRNA is a crucial factor in the investigation of mRNA drugs / vaccines.

[0008] In the field of mRNA technology, there remains an urgent need for the development of mRNA vaccines or drugs that can be delivered to specific organs, tissues, and / or cells, are suitable for industrial production, and have controllable quality and activity, as well as tools and methods for regulating the expression of mRNA delivered to specific organs, tissues, and / or cells. [Overview of the Initiative]

[0009] Natural poly(A) does not have a miRNA binding site. In this application, the inventors have creatively incorporated a miRNA binding site into the poly(A) tail, thereby enhancing not only the specificity of mRNA expression in specific organs, tissues, and / or cells, but also the stability of mRNA plasmid replication. Thus, the quality of the produced mRNA becomes controllable, and the effect is stable.

[0010] Specifically, this application provides the following:

[0011] 1. An engineered poly(A) tail containing a miRNA binding site. The engineered poly(A) tail may contain one, two, or more miRNA binding sites.

[0012] In some embodiments, the sequences of two or more miRNA binding sites are adjacent to each other or separated by one or more bases.

[0013] 2. Includes the structure of the following formula (I), nA-miRNA binding position-mA formula (I), The miRNA binding site includes one or more miRNA binding sites. nA represents n consecutive adenylic acid (A) units located near the 5' end of the miRNA binding site. mA represents m consecutive adenylic acid (A) units located near the 3' end of the miRNA binding site. The operated poly(A)tail described in Solution 1, where m and n are natural numbers such that m+n≦150, m+n≦120, m+n≦100, m+n≦80, m+n≦60, m+n≦30, m+n≦19, or m+n≦14. In some embodiments, the sum of m and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 4 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 ,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150.

[0014] In some embodiments, one or more miRNA binding sites at the miRNA binding site are either directly linked or linked by a spacer sequence. The spacer sequence consists of one or more nucleotides. In some embodiments, the spacer sequence consists of one or more non-A nucleotides.

[0015] In some embodiments, the miRNA binding site in formula (I) consists of 1, 2, 3, 4, 5, or 6 miRNA binding sites. In some embodiments, the miRNA binding site in formula (I) consists of 1 miRNA binding site. In some embodiments, the miRNA binding site in formula (I) includes 2 or more miRNA binding sites that are neighboring or linked by 1 or more nucleotides. In some embodiments, the miRNA binding site in formula (I) includes 3 or more miRNA binding sites that are neighboring or linked by 1 or more nucleotides. In some embodiments, the poly(A) tail has only one structure of formula (I). In some embodiments, the poly(A) tail is the structure of formula (I).

[0016] 3. The operated poly(A) tail according to solution 2, wherein n=0 or n≧1.

[0017] 4. The manipulated poly(A) tail described in solution 2, where m=0.

[0018] 5. An operated poly(A) tail according to Solution 2, where n ≤ 80, n ≤ 60, n ≤ 30, n ≤ 19, n ≤ 14, or n ≤ 10. In some embodiments, this is 14 ≤ n ≤ 30, 14 ≤ n ≤ 19, or 19 ≤ n ≤ 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80.

[0019] 6. The length of the poly(A) tail is 80-240 nt, for example, 100-200 nt, 101-150 nt, 120-150 nt, 130-140 nt, 123-135 nt, or 125-139 nt, as described in any one of Solutions 1-5.In some embodiments, the length of the poly(A) tail is 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 92nt, 93nt, 94nt, 95nt, 96nt, 97nt, 98nt, 99nt, 100nt, 101nt, 102nt, 103nt, 104nt, 105nt, 106nt, 107nt, 108nt, 109nt, 110nt, 111nt, 112nt, 113nt, 114nt, 115nt, 116nt, 117nt, 118nt, 119nt, 1 20nt, 121nt, 122nt, 123nt, 124nt, 125nt, 126nt, 127nt, 128nt, 129nt, 130nt, 131nt, 132nt, 133nt, 134nt, 135nt, 136nt, 137nt, 138nt, 139nt, 14 0nt, 141nt, 142nt, 143nt, 144nt, 145nt, 146nt, 147nt, 148nt, 149nt, 150nt, 151nt, 152nt, 153nt, 154nt, 155nt, 156nt, 157nt, 158nt, 159nt, 160 nt, 161nt, 162nt, 163nt, 164nt, 165nt, 166nt, 167nt, 168nt, 169nt, 170nt, 171nt, 172nt, 173nt, 174nt, 175nt, 176nt, 177nt, 178nt, 179nt, 180 nt, 181nt, 182nt, 183nt, 184nt, 185nt, 186nt, 187nt, 188nt, 189nt, 190nt, 191nt, 192nt, 193nt, 194nt, 195nt, 196nt, 197nt, 198nt, 199nt, 200n t, 201nt, 202nt, 203nt, 204nt, 205nt, 206nt, 207nt, 208nt, 209nt, 210nt, 211nt, 212nt, 213nt, 214nt, 215nt, 216nt, 217nt, 218nt, 219nt, 220nt, 221nt, 222nt, 223nt, 224nt, 225nt, 226nt, 227nt, 228nt, 229nt, 230nt, 231nt, 232nt, 233nt, 234nt, 235nt, 236nt, 237nt, 238nt, or 239nt.

[0020] On the 3'-side of the structure of formula (I), it further comprises a tail fragment directly linked to the 3'-end of the structure of formula (I), preferably, the 5'-terminal nucleotide of the tail fragment is not A, and / or On the 5'-side of the structure of formula (I), it further comprises a head fragment directly linked to the 5'-end of the structure of formula (I), preferably, the 3'-terminal nucleotide of the head fragment is not A, the engineered poly(A) tail according to any one of Solutions 1 to 6.

[0021] In some embodiments, the tail fragment or the head fragment consists of one or more non-A nucleotides and a plurality of A (e.g., including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-A nucleotides).

[0022] In some embodiments, the tail fragment or the head fragment contains p nucleotides, where p ≤ 80, p ≤ 60, p ≤ 30, p ≤ 19, or p ≤ 14. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2�, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, (66), 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79.

[0023] In some embodiments, the tail fragment or the head fragment consists of one or more element c and / or one or more element d and one or more A.

[0024] Element c is a non-A nucleotide, e.g., T, U, C, G.

[0025] Element d consists of any two or more consecutive nucleotides, and the 5' and 3' terminal nucleotides of element d are non-A nucleotides.

[0026] The length of element d is within the range of 2nt ≤ d ≤ 30nt, preferably 6nt ≤ d ≤ 20nt, more preferably 6nt ≤ d ≤ 12nt.

[0027] Element c and element d are not adjacent.

[0028] In some embodiments, the length of element d is 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, or 30nt.

[0029] <000 / 0102>In some embodiments, element d consists of any two or more consecutive nucleotides, and element d does not contain more than three consecutive A's. The nucleotides are selected from A, U, C, and G nucleotides. The 5' and 3' terminal nucleotides of element d are non-A nucleotides. Preferably, the length of element d is within the range of 2nt ≤ d ≤ 30nt, preferably 6nt ≤ d ≤ 20nt, more preferably 6nt ≤ d ≤ 12nt.

[0030] In some embodiments, the number of element c is 0, 1, 2 - 10, 3 - 8, 4 - 6, or 2 - 5, such as 7. In some embodiments, the number of element c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. ]

[0031] In some embodiments, the number of element d is 0 - 5, preferably 1 - 3, such as 2 or 4. In some embodiments, the number of element d is 0, 1, 2, 3, 4, or 5.

[0032] In some embodiments, when both element c and element d are present, the total number of elements c and d is 2 to 15, preferably 3 to 5, and more preferably 3. In some embodiments, the total number of elements c and d is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0033] In some embodiments, the poly(A) tail contains one or more non-A nucleotides in the half portion near the 3' end.

[0034] In some embodiments, the poly(A) tail contains one or more non-A nucleotides in the 1 / 3 portion near the 3' end.

[0035] In some embodiments, the poly(A) tail contains one or more non-A nucleotides in the quarter portion near the 3' end.

[0036] The manipulated poly(A) tail according to any one of the preceding solutions has 0-60, 0-30, 0-20, 0-14, or 0-10 nucleotides at the 5' end of the miRNA binding site, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides.

[0037] 8. The poly(A) tail structure shown below: Element a - Element c - Element b - Element c - Element b - Element c - Element b - Element c - Element b, Element b-Element c-Element b-Element c-Element a-Element d-Element b-Element c-Element b-Element c-Element b, Element b - Element c - Element b - Element c - Element b - Element d - Element a - Element c, Element a - Element d - Element b - Element c - Element b - Element c - Element b, or Element b - Element c - Element b - Element c - Element b - Element d - Element a, Element a - Element d - Element b, Element a - Element d - Element a, Element b - Element d - Element a, Element b - Element d - Element b, Element a - Element dA, An engineered poly(A) tail formed by inserting a miRNA binding site between element a and element cA, Selectively, 1) The miRNA binding site replaces one, two, three, four, five or more elements, 2) The miRNA binding site is inserted into any two elements, or 3) The manipulated poly(A) tail according to any one of solutions 1 to 7, wherein the miRNA binding site is inserted into any two A's in element a or element b.

[0038] In the poly(A)tail structure, element a consists of multiple consecutive adenylate(A) nucleotides, and the length of element a is in the range of 100nt≧a>30nt, preferably 80nt≧a≧60nt.

[0039] Element b consists of multiple consecutive A nucleotides, and the length of element b is in the range of 10nt ≤ b ≤ 30nt, preferably 14nt ≤ b ≤ 20nt.

[0040] Element c consists of non-A nucleotides.

[0041] Element d consists of any two or more consecutive nucleotides and does not contain more than three consecutive A nucleotides. The nucleotides are selected from A, T, C, or G nucleotides. The 5' and 3' terminal nucleotides of element d are non-A nucleotides. Preferably, the length of element d is in the range of 2nt ≤ d ≤ 30nt, preferably 6nt ≤ d ≤ 20nt, and more preferably 6nt ≤ d ≤ 12nt.

[0042] In some embodiments, element a is 31nt, 32nt, 33nt, 34nt, 35nt, 36nt, 37nt, 38nt, 39nt, 40nt, 41nt, 42nt, 43nt, 44nt, 45nt, 46nt, 47nt, 48nt, 49nt, 50nt, 51nt, 52nt, 53nt, 54nt, 55nt, 56nt, 57nt, 58nt, 59nt, 60nt, 61nt, 62nt, 63nt, 64nt , 65nt, 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 92nt, 93nt, 94nt, 95nt, 96nt, 97nt, 98nt, 99nt, or 100nt.

[0043] In some configurations, element b is 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, or 30nt.

[0044] In some embodiments, element d is 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, or 30nt.

[0045] 9. An manipulated poly(A) tail according to any one of solutions 1 to 7, formed by inserting a miRNA binding site into a poly(A) tail structure as described in WO2020074642A1, WO2022028559A1, and US_10717982.

[0046] 10. Element c is G, an operated poly(A) tail as described in any one of solutions 1 to 9.

[0047] 11. Element d is an operated poly(A)tail according to any one of solutions 1 to 10, wherein element d includes a palindrome sequence.

[0048] 12. An operated poly(A) tail according to any one of solutions 1 to 11, wherein the length of element d is 6 nt.

[0049] 13. An operated poly(A) tail according to any one of solutions 1 to 12, wherein element d is one or more selected from GATATC (sequence number 60), GTATAC (sequence number 61), GAATCT (sequence number 62), GCATATGACT (sequence number 63), and GATATCGTATAC (sequence number 64).

[0050] 14. Element d is an operated poly(A) tail as described in any one of solutions 1 to 12, as shown in GATATC (Sequence ID 60).

[0051] 15. An engineered poly(A) tail according to any one of solutions 1 to 14, wherein the ratio of the number of nucleotides in the poly(A) tail on the 5' side of the miRNA binding site to the number of nucleotides in the poly(A) tail on the 3' side of the miRNA binding site is less than 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10.

[0052] 16. The manipulated poly(A) tail according to any one of solutions 6 to 15, wherein the tail fragment has the structure of element d-element b-element c-element b-element c-element b or element c-element b-element c-element b, preferably the tail fragment has the structure of element d-19A-element c-19A-element c-17A or element c-19A-element c-17A, and more preferably the tail fragment has the structure GATATC-19A-G-19A-G-17A. In some embodiments, the manipulated poly(A) tail does not include a head fragment. In some embodiments, the manipulated poly(A) tail includes a tail fragment and the structure of formula (I). In some embodiments, the manipulated poly(A) tail includes a head fragment, a tail fragment, and the structure of formula (I).

[0053] 1) nA-miRNA binding site - mA (150 ≥ m+n ≥ 50, preferably m+n = 120 or m+n = 60), 2) nA-miRNA binding site - mA - element d - element b - element c - element b - element c - element b (80 ≥ m + n ≥ 60, preferably m + n = 60), 3) Element a - Element d - nA - miRNA binding site - mA - Element c - Element b - Element c - Element b (30 ≥ m + n ≥ 10, preferably m + n = 19), 4) nA-miRNA binding site - mA - element c - element b - element c - element b (110 ≥ m + n ≥ 70, preferably m + n = 79), 5) An operated poly(A) tail according to any one of solutions 1 to 15, having any structure selected from nA-miRNA binding site-mA-element c-element b-element c-element b-element d-element a or element b-element c-nA-miRNA binding site-mA-element c-element b-element d-element a (19≧m+n≧14, preferably m+n=19).

[0054] An operated poly(A) tail according to any one of solutions 1 to 15, having the structure nA-miRNA binding site-mA-element d-element b-element c-element b-element c-element b (80≧m+n≧60, preferably m+n=60, more preferably 30≧n≧14, even more preferably 19≧n≧14).

[0055] 1) nA-miRNA binding site - mA (m+n=120, m+n=100, or m+n=60), 2) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (m+n=60), 3) 60A-element d-nA-miRNA binding site-mA-G-19A-G-17A (m+n=19), 4) nA-miRNA binding site - mA-G-19A-G-17A (m+n=79), and 5) An operated poly(A) tail according to any one of solutions 1 to 15, having any structure selected from nA-miRNA binding site-mA-G-19A-G-19A-element d-60A or 19A-G-nA-miRNA binding site-mA-G-19A-element d-60A (m+n=19).

[0056] An operated poly(A) tail according to any one of solutions 1 to 15, having the structure nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (m+n=60, 30≧n≧14, preferably 19≧n≧14).

[0057] 1) nA-miRNA binding site - mA (m+n=120 or m+n=100), 2) nA-miRNA binding site-mA-sequence number 60-19A-G-19A-G-17A (m+n=60), 3) 60A - Sequence ID 60 - nA - miRNA binding site - mA - G - 19A - G - 17A (m+n=19) 4) nA-miRNA binding site - mA-G-19A-G-17A (m+n=79), and 5) An operated poly(A) tail according to any one of solutions 1 to 15, having any structure selected from nA-miRNA binding site-mA-G-19A-G-19A-sequence number 60-60A or 19A-G-nA-miRNA binding site-mA-G-19A-element d-60A (m+n=19).

[0058] An operated poly(A) tail according to any one of solutions 1 to 15, having the structure nA-miRNA binding site-mA-sequence number 60-19A-G-19A-G-17A (m+n=60, 30≧n≧14, preferably 19≧n≧14).

[0059] 17. miRNAs that bind to the miRNA binding site are An operated poly(A) tail according to any one of the solutions 1 to 16, which is one or more selected from miR-142, miR-122, miR-126, miR-148a, miR-133, miR-206, miR-208, miR-17-92, miR-16, miR-21, miR-223, miR-24, miR-27, let-7, miR-30c, miR-1d, miR-149, miR-192, miR-194, and miR-204.

[0060] In some embodiments, the miRNA is one or more selected from miR-142, miR-122, miR-126, and miR-148a.

[0061] In some embodiments, the miRNA is one or more selected from miR-142-3p, miR-122-5p, miR-126-3p, and miR-148a-3p.

[0062] In some embodiments, the miRNA includes or consists of miR142 and miR-122, miR142 and miR-126, miR-142 and miR-148a, miR-122 and miR-126, miR-122 and miR-148a, or miR-126 and miR-148a.

[0063] In some embodiments, the miRNA includes or consists of miR-142, miR-122 and miR-126, miR-142, miR-122 and miR-148a, miR-142, miR-126 and miR-148a, or miR-122, miR-126 and miR-148a.

[0064] In some embodiments, the miRNA includes or consists of miR-142, miR-122, miR-126, and miR-148a.

[0065] In some embodiments, the miRNA includes miR142 and miR-122, or consists of miR142 and miR-122. At the miRNA binding site of poly(A), the miR142 binding site is located 5' to the miR-122 binding site, or the miR142 binding site is located 3' to the miR-122 binding site, and the miR142 binding site and the miR-122 binding site are adjacent to each other.

[0066] In some embodiments, the miRNA includes miR142 and miR-122. At the binding site of poly(A), the miR142 binding site is located 5' to the miR-122 binding site, or the miR142 site is located 3' to the miR-122 site, and the miR142 and miR-122 binding sites are separated by one or more nucleotides.

[0067] In some embodiments, the miRNA contains miR142 and miR-126, or consists of miR142 and miR-126. At the binding site of poly(A), the miR142 binding site is located 5' to the miR-126 binding site, or the miR142 site is located 3' to the miR-126 site, and the miR142 and miR-126 binding sites are adjacent to each other.

[0068] In some embodiments, the miRNA includes miR142 and miR-126. At the binding site of poly(A), the miR142 binding site is located 5' to the miR-126 binding site, or the miR142 site is located 3' to the miR-126 site, and the miR142 and miR-126 binding sites are separated by one or more nucleotides.

[0069] In some embodiments, the miRNA contains miR-142 and miR-148a, or consists of miR-142 and miR-148a. At the poly(A) binding site, the miR-142 binding site is located 5' to the miR-148a binding site, or the miR-142 binding site is located 3' to the miR-148a binding site, and the miR-142 and miR-148a binding sites are adjacent to each other.

[0070] In some embodiments, the miRNA includes miR-142 and miR-148a. At the poly(A) binding site, the miR-142 binding site is located 5' to the miR-148a binding site, or the miR-142 binding site is located 3' to the miR-148a binding site, and the miR-142 and miR-148a binding sites are separated by one or more nucleotides.

[0071] In some embodiments, the miRNA includes miR-122 and miR-126, or consists of miR-122 and miR-126. At the binding site of poly(A), the miR-122 binding site is located 5' to the miR-126 binding site, or the miR-122 binding site is located 3' to the miR-126 binding site, and the miR-122 and miR-126 binding sites are adjacent to each other.

[0072] In some embodiments, the miRNA comprises miR-122 and miR-126. At the binding site of poly(A), the miR-122 binding site is located 5' to the miR-126 binding site, or the miR-122 binding site is located 3' to the miR-126 binding site, and the miR-122 and miR-126 binding sites are separated by one or more nucleotides.

[0073] In some embodiments, the miRNA contains miR-122 and miR-148a, or consists of miR-122 and miR-148a. At the poly(A) binding site, the miR-122 binding site is located 5' to the miR-148a binding site, or the miR-122 binding site is located 3' to the miR-148a binding site, and the miR-122 and miR-148a binding sites are adjacent to each other.

[0074] In some embodiments, the miRNA includes miR-122 and miR-148a. At the binding site of poly(A), the miR-122 binding site is located 5' to the miR-148a binding site, or the miR-122 binding site is located 3' to the miR-148a binding site, and the miR-122 and miR-148a binding sites are separated by one or more nucleotides.

[0075] In some embodiments, the miRNA contains miR-126 and miR-148a, or consists of miR-126 and miR-148a. At the poly(A) binding site, the miR-126 binding site is located 5' to the miR-148a binding site, or the miR-126 binding site is located 3' to the miR-148a binding site, and the miR-126 and miR-148a binding sites are adjacent to each other.

[0076] In some embodiments, the miRNA includes miR-126 and miR-148a. At the poly(A) binding site, the miR-126 binding site is located 5' to the miR-148a binding site, or the miR-126 binding site is located 3' to the miR-148a binding site, and the miR-126 and miR-148a binding sites are separated by one or more nucleotides.

[0077] In some embodiments, the miRNAs include miRNA-142, miR-148a, and miR-126. At the miRNA binding site of poly(A), the miRNA binding site is located from 5' to 3'. miR-126 binding site, miR-148a binding site, and miR-142 binding site, miR-148a binding site, miR-142 binding site, and miR-126 binding site, miR-142 binding site, miR-148a binding site, and miR-126 binding site, miR-126 binding site, miR-142 binding site, and miR-148a binding site, miR-148a binding site, miR-126 binding site, and miR-142 binding site, or The miR-142 binding site, the miR-126 binding site, and the miR-148a binding site are arranged in that order. The miR-126 binding site, the miR-148a binding site, and the miR-142 binding site are adjacent to each other.

[0078] In some embodiments, the miRNAs include miRNA-142, miR-148a, and miR-126. At the miRNA binding site of poly(A), the miRNA binding site is located from 5' to 3'. miR-126 binding site, miR-148a binding site, and miR-142 binding site, miR-148a binding site, miR-142 binding site, and miR-126 binding site, miR-142 binding site, miR-148a binding site, and miR-126 binding site, miR-126 binding site, miR-142 binding site, and miR-148a binding site, miR-148a binding site, miR-126 binding site, and miR-142 binding site, or The miR-142 binding site, the miR-126 binding site, and the miR-148a binding site are arranged in that order. The miR-126 binding site, the miR-148a binding site, and the miR-142 binding site are sequestered by one or more nucleotides.

[0079] In some embodiments, the miRNAs include miRNA-142, miR-148a, and miR-126. At the miRNA binding site of poly(A), the miRNA binding site is located from 5' to 3'. miR-126 binding site, miR-148a binding site, and miR-142 binding site, miR-148a binding site, miR-142 binding site, and miR-126 binding site, miR-142 binding site, miR-148a binding site, and miR-126 binding site, miR-126 binding site, miR-142 binding site, and miR-148a binding site, miR-148a binding site, miR-126 binding site, and miR-142 binding site, or The miR-142 binding site, the miR-126 binding site, and the miR-148a binding site are arranged in that order. The two adjacent miRNA binding sites, miR-126, miR-148a, and miR-142, are neighbors to each other, while the other two miRNA binding sites are isolated by one or more nucleotides.

[0080] 1) nA-miRNA binding site - mA (n=0, m=120, m=100, or m=60), 2) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (m+n=60, n≦20, n≦14, or n≦10), 3) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (n=0, m=60), 4) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (m+n=60, 14≦n≦30, preferably 14≦n≦19), 5) An engineered poly(A) tail having any structure selected from nA-miRNA binding site-mA-G-19A-G-17A (n=60, m=19), Preferably, element d is a palindromic sequence, and more preferably, element d has a polynucleotide sequence represented by any one of sequence numbers 60 to 64, the manipulated poly(A) tail according to any one of the prior solutions.

[0081] 1) nA-miRNA binding site-mA (n=0, m=120, m=100, or m=60, and the miRNA binding site is the miR-122, miR-126, or miR-142 binding site), 2) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (n=14, m=46, and the miRNA binding site is miR-122, miR-126, or miR-142 binding site), 3) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (n=19, m=41, and the miRNA binding site is miR-122, miR-126, or miR-142 binding site), 4) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (n=30, m=30, and the miRNA binding site is miR-122, miR-126, or miR-142 binding site), 5) nA-miRNA binding site-mA-G-19A-G-17A (n=60, m=19, and the miRNA binding site is miR-122, miR-126, or miR142 binding site), 6) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A (n=0, m=60, and the miRNA binding sites are miR-142, miR-122, miR-126, miR-148a, miR-142-miR122, miR142-miR126-miR148a, miR142-miR148a-miR126, miR126-miR142-miR148a, miR126-miR148a-miR142, miR148a-miR142-miR126, or miR148a-miR126-miR142 binding sites), 7) nA-miR-122 coupling position-mA-element d-19A-G-19A-G-17A (n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n+m=60), 8) nA-miR-142 coupling position-mA-element d-19A-G-19A-G-17A (n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n+m=60), 9) nA-miR-126 coupling position-mA-element d-19A-G-19A-G-17A (n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n+m=60), or 10) An operated poly(A) tail having a structure selected from nA-miR-148a bonding position-mA-element d-19A-G-19A-G-17A (n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n+m=60), Preferably, element d is a palindromic sequence, and more preferably, element d is represented by one of sequence numbers 60 to 64, and more preferably, has a polynucleotide sequence represented by sequence number 60, according to any one of the preceding solutions.

[0082] In some embodiments, the miRNA binding sites of the manipulated poly(A) tail include one, two, three, four, or more miRNA binding site sequences selected from SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 32, or SEQ ID NO: 53. In some embodiments, the miRNA binding sites of the manipulated poly(A) tail include one, two, three, four, or more miRNA binding site sequences selected from SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 32, or SEQ ID NO: 53, and zero, one, or more nucleotides between the miRNA binding sites.

[0083] 18. The manipulated poly(A) tail according to any one of solutions 1 to 17, wherein the poly(A) tail sequence includes or is selected from the polynucleotide sequences of SEQ ID NOs. 6-16, SEQ ID NOs. 19-31, SEQ ID NOs. 33-52, SEQ ID NOs. 54-59, and SEQ ID NOs. 65-71.

[0084] 19. An engineered RNA molecule which is mRNA or non-coding RNA and comprises a poly(A) tail as described in any one of solutions 1 to 18 and a target gene sequence located on the 5' side of the poly(A) tail. In some embodiments, the nA in the structure of formula (I) of the poly(A) tail is directly ligated to the 3'UTR.

[0085] 20. An engineered DNA molecule that encodes an engineered poly(A) tail as described in any one of solutions 1 to 18, or an engineered RNA sequence as described in solution 19.

[0086] 21. The manipulated DNA molecule described in solution 20, which is a plasmid or viral vector.

[0087] 22. A DNA-RNA hybrid molecule that carries the same genetic information as the manipulated poly(A) tail described in any one of solutions 1 to 18, the manipulated RNA molecule described in solution 19, or the manipulated DNA molecule described in solution 20 or 21.

[0088] 23. An engineered cell comprising an engineered poly(A) tail as described in any one of solutions 1 to 18, an engineered RNA molecule as described in solution 19, an engineered DNA molecule as described in solution 20 or 21, or a hybrid molecule as described in solution 22, wherein the cell is a eukaryotic cell or a prokaryotic cell.

[0089] 24. The prokaryotic cell is Escherichia coli, the manipulated cell as described in solution 23.

[0090] 25. Solution in enabling low expression of target genes in specific organs, tissues, and / or cells, the use of an engineered poly(A)tail as described in any one of paragraphs 1 to 18, wherein the specific organs, tissues, and / or cells highly express miRNAs, or The use of an engineered poly(A)tail according to any one of solutions 1 to 18 in enabling the specific expression of a target gene in a specific organ, tissue, and / or cell, wherein the organ or tissue is the liver or spleen organ or tissue, and more preferably the cell is a hepatocyte.

[0091] 26. Use of the manipulated DNA molecule described in solution 20 or 21, or the hybrid molecule described in solution 22, to enable more conserved replication of the poly(A) tail DNA coding sequence within a host cell.

[0092] 27. The host cell is a prokaryotic cell, preferably Escherichia coli, as described in solution 26.

[0093] 28. Lipid nanoparticles comprising an engineered poly(A) tail as described in any one of solutions 1 to 18, an engineered RNA molecule as described in solution 19, an engineered DNA molecule as described in solution 20 or 21, or a hybrid molecule as described in solution 22.

[0094] 29. A virion comprising an engineered poly(A) tail as described in any one of solutions 1 to 18, an engineered RNA molecule as described in solution 19, an engineered DNA molecule as described in solution 20 or 21, or a hybrid molecule as described in solution 22.

[0095] It is known to those skilled in the art that the 5' terminal nucleotide of the miRNA binding site may contain zero, one, or more non-A nucleotides further to the 5' end. This technical solution is also included within the scope of this application or is deemed to be an equivalent solution of this application.

[0096] It should be understood that the aspects and embodiments of this application described herein include those “including,” “consisting of,” and “essentially consisting of.” Preferred embodiments of this application are described in detail above; however, this application is not limited thereto. Various simple modifications (including combinations of various technical features by any other suitable method) can be made to the technical solution of this application in the technical idea of ​​this application. These simple modifications and combinations should also be considered as disclosures in this application and are all included within the scope of protection of this application. [Brief explanation of the drawing]

[0097] [Figure 1A] This figure shows the compositions of five poly(A) compounds containing the miR-142 / miR-122 binding site, which was uniquely designed in this invention. [Figure 1B] This figure shows the compositions of five poly(A) compounds containing the miR-142 / miR-122 binding site, which was uniquely designed in this invention. [Figure 2A] This figure shows the absolute numerical results of fluorescence imaging of the spleen / liver of dissected mice after expressing luciferase mRNA containing five poly(A) groups, including a miR-142 binding site, as uniquely designed in this invention. [Figure 2B]This figure shows the absolute numerical results of fluorescence imaging of the spleen / liver of dissected mice after expressing luciferase mRNA containing five poly(A) groups, including a miR-142 binding site, as uniquely designed in this invention. [Figure 3A] This figure shows the absolute numerical results of fluorescence imaging of the spleen / liver of dissected mice after expressing luciferase mRNA containing five poly(A) groups, including a miR-122 binding site, as uniquely designed in this invention. [Figure 3B] This figure shows the absolute numerical results of fluorescence imaging of the spleen / liver of dissected mice after expressing luciferase mRNA containing five poly(A) groups, including a miR-122 binding site, as uniquely designed in this invention. [Figure 4] This figure shows the statistical results of missing bases in *E. coli* DH5α for luciferase DNA plasmids having various poly(A) groups, including the miRNA binding sites miR-142 / miR-122, which were uniquely designed in this invention. [Figure 5] This figure shows the absolute numerical results of enzyme activity detection in the mammalian cell line Raw264.7 for luciferase mRNA having eight poly(A) groups including the miR-142 binding site uniquely designed in this invention. [Figure 6] This figure shows the absolute numerical results of enzyme activity detection in the mammalian cell line Raw264.7 for luciferase mRNA containing poly(A) with miR-142 and miR-122 binding sites. [Figure 7A] This figure shows the absolute numerical results of enzyme activity detection for luciferase mRNA having a poly(A) variant uniquely designed in this invention, within the mammalian cell line Raw264.7 / LSEC / HSC / AML. [Figure 7B] This figure shows the absolute numerical results of enzyme activity detection for luciferase mRNA having a poly(A) variant uniquely designed in this invention, within the mammalian cell line Raw264.7 / LSEC / HSC / AML. [Figure 7C]This figure shows the absolute numerical results of enzyme activity detection for luciferase mRNA having a poly(A) variant uniquely designed in this invention, within the mammalian cell line Raw264.7 / LSEC / HSC / AML. [Figure 7D] This figure shows the absolute numerical results of enzyme activity detection for luciferase mRNA having a poly(A) variant uniquely designed in this invention, within the mammalian cell line Raw264.7 / LSEC / HSC / AML. [Figure 8A] This figure shows the decrease in expression of luciferase mRNA having a poly(A) variant uniquely designed in the present invention, compared to its expression in hepatocyte AML, within Raw264.7 / LSEC / HSC cells. [Figure 8B] This figure shows the decrease in expression of luciferase mRNA having a poly(A) variant uniquely designed in the present invention, compared to its expression in hepatocyte AML, within Raw264.7 / LSEC / HSC cells. [Figure 8C] This figure shows the decrease in expression of luciferase mRNA having a poly(A) variant uniquely designed in the present invention, compared to its expression in hepatocyte AML, within Raw264.7 / LSEC / HSC cells. [Modes for carrying out the invention]

[0098] This application provides a tool for achieving specific expression in specific organs, tissues, and / or cells. Specifically, by incorporating a miRNA binding site into a poly(A) tail, mRNA expression in organs, tissues, and cells where mRNA expression is undesirable can be specifically turned off, thereby reducing off-target toxic side effects caused by systematic mRNA expression.

[0099] This application further provides a method for stably amplifying template DNA for poly(A) tail transcription in vitro, thereby reducing the mutation frequency in the template sequence for poly(A) tail transcription when DNA is replicated in large quantities within cells. In this way, a large amount of RNA containing a definitively sequenced poly(A) tail can be obtained based on the DNA. Based on this, RNA such as mRNA manipulated to have a poly(A) tail with a specific function can be fermented in vitro to achieve large-scale production.

[0100] term In this application, “element c,” “element d,” nA, mA, tail fragment, head fragment, miRNA binding site, and miRNA binding site within the poly(A) tail may all be referred to as “element.” “miRNA binding site,” “nA,” “mA,” “element c,” and “element d” do not overlap or overlap with each other. “miRNA binding site,” “nA,” “mA,” “element c,” and “element d” may be determined as follows:

[0101] The miRNA binding sites in the poly(A) tail are determined by sequence alignment. N consecutive A's are determined as nA by determining the A closest to the 5' end of the miRNA binding site among all miRNA binding sites closest to the 5' end of the poly(A) tail as the 3' end of nA. M consecutive A's are determined as mA by determining the A closest to the 3' end of the miRNA binding site among all miRNA binding sites closest to the 3' end of the poly(A) tail as the 5' end of mA. The portion between nA and mA is the miRNA binding site. If the poly(A) tail has a polynucleotide or polynucleotide sequence on the 5' side of nA, the polynucleotide or polynucleotide sequence is the head fragment, and if the poly(A) tail has a polynucleotide or polynucleotide sequence on the 3' side of mA, the polynucleotide or polynucleotide sequence is the tail fragment. "Element d" and "Element c" are determined as follows: Element d is determined from the target sequence. Element d is a fragment containing a non-A base in the target sequence and consists of any two or more consecutive nucleotides. The 5' and 3' terminal nucleotides of element d are non-A nucleotides, element d does not contain more than three consecutive A's, and both the 5' and 3' ends of element d are neighbors of at least two A's. After element d is determined, all non-A bases are determined to be "element C" from the portion of the target sequence other than element D. In some embodiments, the poly(A) tail contains multiple miRNA binding sites, and the miRNA binding sites at each binding site are either directly linked or linked by non-A bases. In some embodiments, the poly(A) tail contains multiple miRNA binding sites, and the miRNA binding sites at each binding site are either directly linked or linked by nucleic acids, and the nucleic acid sequence does not contain more than three consecutive A's.

[0102] As used herein, “miRNA” or microRNA is generally a short non-coding RNA having a length of less than 22 bp. It binds to a target sequence on mRNA (i.e., a miRNA binding site) and exerts a post-transcriptional regulatory effect. As used herein, preferably, “miRNA binding site” is a miRNA binding site that downregulates mRNA expression after binding to the miRNA.

[0103] In this specification, “code” means i) a DNA sequence containing genetic information transcribed into an RNA molecule, and / or ii) an RNA molecule containing genetic information translatable into an amino acid sequence. Therefore, as used herein, “coding sequence” may be used to refer to a ribonucleic acid (RNA) sequence or fragment thereof in a protein-translatable mRNA precursor or mature mRNA, or a complementary sequence or fragment of a deoxyribonucleic acid (DNA) sequence used as a transcribed template to an mRNA precursor or mature mRNA. Furthermore, “coding sequence” in this application may further include polynucleotide sequences encoding proteins, functional nucleic acids, or fragments thereof, such as miRNA, shRNA, dsRNA, guide RNA, poly(A) tail, 5'UTR, or 3'UTR. A DNA molecule containing genetic information transcribed into an RNA molecule is referred to as the “coding nucleic acid” of an RNA molecule. An RNA molecule containing genetic information translatable into an amino acid sequence is referred to as the “coding nucleic acid” of an amino acid sequence.

[0104] In this application, all nucleotides in a polynucleotide sequence are numbered from the 5' end to the 3' end, i.e., the 5' terminal nucleotide is the first nucleotide and the 3' terminal nucleotide is the last nucleotide. Unless otherwise specified, “5' end” and “5' end” are interchangeable, and “3' end” and “3' end” are interchangeable. Specifically, “5' end” and “3' end” may be used to describe the position of the first and last nucleotides in a nucleic acid sequence, or the position of a fragment of a nucleic acid sequence, respectively. Furthermore, “5'” and “3'” are used in particular to describe the relative positional relationship between nucleotides, between nucleotide fragments, or between nucleotides and nucleotide fragments within the same nucleic acid sequence. For example, “5' side” is used to describe the relative positional relationship between two non-overlapping sequences within the same polynucleotide sequence. When one sequence is described as being located on the 5' side of the other sequence, this means that one sequence is closer to the “5' end” of the polynucleotide sequence than the other sequence. Similarly, when one sequence is described as being located 3' to the other, this means that the one sequence is closer to the "3' end" of the polynucleotide sequence than the other sequence, and that there is no overlap between the two sequences. Furthermore, as used herein, the "5' portion" refers to the half of the polynucleotide sequence closer to the 5' end, divided at the "central position" of the polynucleotide sequence. The "3' portion" refers to the half of the polynucleotide sequence closer to the 3' end, divided at the "central position" of the polynucleotide sequence. In this application, the number of nucleotides from the "central position" to the 5' end and the number of nucleotides from the 5' end to the 3' end are equal.

[0105] As used herein, “neighbors” means that there is no insertion of a nucleotide or base between two elements of a nucleic acid molecule (such as a poly(A) tail) (unless otherwise specified, the terms “nucleotide” and “base” are used interchangeably in this application). That is, the first nucleotide on the 3' end of the polynucleotide sequence at the 3' end of one of the two elements is the 5' terminal nucleotide of the other element. For example, “n consecutive A's in the neighborhood of the 5' end of the miRNA binding site” means that n consecutive A's are located on the 5' end of the miRNA binding site and that no other nucleotide or base is inserted between the 5' terminal nucleotide of the miRNA binding site and the n A's. For example, “neighbors of the 3' end of the miRNA binding site” means that n consecutive A's are located on the 3' end of the miRNA binding site and that no other nucleotide or base is inserted between the 3' terminal nucleotide of the miRNA binding site and the n A's. Furthermore, "neighborhood" is also used to describe the positional relationship between multiple elements, meaning that each element of a group is neighbor to any other element of that group, and that no other element or nucleotide is located between any two elements of that group.

[0106] In this application, when the positional relationship between two or more elements is described as "non-adjacent," it means that the two or more elements are not neighbors to each other. In other words, there is at least one nucleotide or base between the two or more elements other than the nucleotides or bases of those two or more elements.

[0107] As used herein, the term “conservative” means that, when used to describe the replication of nucleic acid molecules, the probability of mutation in the replication process is low. In this context, “conservative” is a relative concept. For example, the statement “Poly(A)-tail DNA coding sequences are used to make the replication of RNA-coding DNA molecules in host cells more conservative” means that, after a parental DNA molecule encoding RNA is replicated to a daughter DNA molecule, if the RNA molecule contains a poly(A)-tail, the daughter DNA molecule is more likely to have 100% sequence identity with the parental DNA molecule, or the average sequence identity of multiple daughter DNA molecules obtained by the replication of the parental DNA molecule to the parental DNA molecule is higher, compared to the coding DNA of an RNA molecule without a poly(A)-tail (e.g., an RNA molecule containing several other poly(A)-tails).

[0108] In this application, when describing the expression of an RNA molecule as “regulating,” “regulating” means increasing or decreasing the total amount of protein or functional RNA expressed by the RNA molecule within the same period of time, or enabling the RNA molecule to express protein or functional RNA over a longer or shorter period of time, where the increase, decrease, or longer or shorter period of time is relative to another RNA molecule expressing the same protein or functional RNA. When describing the expression of a protein as “regulating,” it means regulating the expression of an RNA molecule containing a protein coding sequence. The regulation described herein can be achieved by ligating the poly(A) tail of this application to the 3' end of an RNA molecule that does not have a poly(A) tail, or by replacing the original poly(A) tail of the RNA with the poly(A) tail of this application.

[0109] As used herein, the percentage of "identity" (such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identity) refers to the degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment, and is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, after introducing gaps and other means to ensure that two sequences have the same residue at as many positions as possible, the ratio of the number of positions with the same base or amino acid residue to the total number of positions can be determined. The percentage of "identity" can be determined by software programs known in the art. Default parameters are preferably used for alignment. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following internet address:ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0110] As used herein, “complementarity” of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing. The complementarity percentage means the proportion of residues in one nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (for example, about 5, 6, 7, 8, 9, or 10 residues out of 10 residues mean about 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). “Perfect complementarity” means that all consecutive residues in a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second sequence. As used herein, “substantial complementarity” refers to at least one level of complementarity among at least about 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in a region having about 40, 50, 60, 70, 80, 100, 150, 200, or 250 or more nucleotides, or two nucleic acids hybridized under stringent conditions. According to the Watson-Crick base pairing principle, for a single base or single nucleotide, if A pairs with T or U, and C pairs with G or I, it is said to be complementary or identical, and vice versa. Any other base pairings are said to be non-complementary. In this application, “complementary polynucleotide sequence” of a polynucleotide sequence refers to a polynucleotide sequence that is perfectly complementary to the polynucleotide sequence.

[0111] As used herein, a “conservative substitution variant” of a protein, polypeptide, or amino acid sequence means that one or more amino acid residues undergo an amino acid substitution without altering the overall morphology and function of the protein or enzyme, and such amino acid substitutions include, but are not limited to, amino acid substitutions in the amino acid sequence of the parent protein by the method described above for “conservative substitutions.” Therefore, the similarity between two proteins or amino acid sequences having similar functions may vary. For example, the similarity (identity) based on the MEGALIGN algorithm is 70% to 99%. “Conservative substitution variants” also include polypeptides or enzymes having an amino acid identity of 60% or more, preferably 75% or more, more preferably 85% or more, and most preferably 90% or more, as determined by the BLAST or FASTA algorithm, and having properties or functions identical or substantially similar to the natural or parent protein or enzyme.

[0112] In the context of this application, the terms “DNA” and “RNA” refer to single-stranded or double-stranded DNA molecules or RNA molecules. Unless otherwise indicated, the terms “DNA” and “DNA molecule” refer to a double-stranded DNA molecule formed from A, C, G, and / or T nucleotides. The terms “RNA” and “RNA molecule” refer to a single-stranded RNA molecule formed from A, C, G, and / or U nucleotides. In this specification, A, C, G, T, and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine, and uracil as their respective nitrogen bases.

[0113] RNA molecules include coding RNA, or non-coding RNA (ncRNA), such as pre-mRNA, mature mRNA, or long non-coding RNA (lncRNA).

[0114] As used herein, a "DNA-RNA hybrid molecule" is a molecule containing a polynucleotide sequence consisting of deoxyribonucleotides and ribonucleotides. Replacing one or more deoxyribonucleotides in DNA with ribonucleotides. Replacing one or more rinucleotides in RNA with deoxyribonucleotides, or These can be obtained by biological or chemical synthesis using deoxyribonucleotides and rinucleotides as raw materials for de novo synthesis. However, the methods for obtaining DNA-RNA hybrid molecules are not limited to these, and any DNA-RNA hybrid molecule obtained by any method falls within the scope of "DNA-RNA hybrid molecule" as defined in this application.

[0115] When used herein, if two nucleic acid molecules are described as having “the same genetic information,” this means that the two nucleic acid molecules are complementary, have identical base sequences, or differ from the other nucleic acid molecule only in that one or more bases in its base sequence are replaced by other bases having the same biological function. Same biological function means that the bases of one and the other can undergo conventional Watson-Crick base pairing with the same base. For example, both thymine (T) and uracil (U) can undergo Watson-Crick base pairing with adenine. Both hypoxanthine (I) and cytosine (C) can undergo Watson-Crick base pairing with guanine (G). Therefore, any two of DNA, RNA, and DNA-RNA hybrid molecules can also have the same genetic information. The term “base sequence” refers to the order of arrangement of bases in a polynucleotide molecule. Those skilled in the art will know, unless otherwise indicated, when a base sequence or polynucleotide sequence described in this application is used to describe a DNA sequence, “T” refers to thymine. It is known that when a base sequence or polynucleotide sequence is used to describe RNA (e.g., mRNA), "T" is replaced with "U" (uracil). Therefore, when any DNA is disclosed herein by a specific sequence number (SEQ ID NO), a complementary or corresponding RNA (e.g., mRNA or poly(A)tail) sequence is also disclosed, in which each "T" in the DNA sequence is replaced with "U".

[0116] As used herein, “DNA-RNA hybrid molecule” refers to a nucleic acid molecule containing both deoxyribonucleotides and ribonucleotides. A “DNA-RNA hybrid molecule” may be one in which one or more deoxyribonucleotides in a DNA molecule are replaced by ribonucleotides, or one or more rinucleotides in an RNA molecule are replaced by deoxyribonucleotides.

[0117] Poly(A) tail and its use This application provides an engineered poly(A) tail containing one, two, three, or more miRNA binding sites. Preferably, the engineered poly(A) tail of the present invention contains one, two, three, four, five, six, seven, eight, nine, ten, or more miRNA binding sites. One, two, or more miRNA binding sites may bind to the same or different miRNAs. In some embodiments, the poly(A) tails provided in this application are obtained by inserting one, two, three, or more miRNA binding sites into any poly(A) tail that does not have miRNA binding sites. In some embodiments, the poly(A) tails provided in this application are obtained by inserting one or more miRNA binding sites into any poly(A) tail that does not have miRNA binding sites. In some embodiments, the poly(A) tails provided in this application are obtained by replacing one or more non-A bases or elements structurally equivalent to element d in any poly(A) tail that does not have miRNA binding sites with one or more miRNA binding sites. In some embodiments, the poly(A) tails provided in this application are obtained by replacing one or more non-A bases or one or more elements structurally equivalent to element d in any poly(A) tail that does not have a miRNA binding site, at the miRNA binding site. "Element structurally equivalent to element d" refers to a polynucleotide fragment in a poly(A) tail that does not have a miRNA binding site, consisting of any two or more consecutive nucleotides, where the 5' and 3' terminal nucleotides are not A, and which does not contain more than three consecutive A nucleotides, for example, element d in PCT application number PCT / CN2023 / 079037. "Poly(A) tails that do not have a miRNA binding site" may be any poly(A) tails described in PCT / CN2023 / 079037, WO2022028559A1, WO2020 / 074642, and US10717982B2, and poly(A) tails that exclusively contain A.

[0118] As used herein, the terms “polyA tail” or “polyA sequence” generally refer to a discontinuous or uninterrupted sequence of adenylic acid residues located at the 3' end of an RNA molecule. In RNA, if a 3'-UTR is present, the polyA sequence is ligated to the 3' end of the 3'-UT. A discontinuous polyA tail is characterized by consecutive adenylic acid residues. A polyA tail can be of any length. In some embodiments, a polyA tail contains or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 adenylic acid (A) residues, particularly about 120 A residues. Generally, the majority of nucleotides in a poly(A) tail are adenylates, meaning at least 75%, 80%, 85%, or 90% of the nucleotides, while the remaining nucleotides may be non-A nucleotides such as U (uridylic acid), G (guanylic acid), and C (cytidylic acid).

[0119] In some embodiments, the in vitro preparation process of RNA is a prokaryotic fermentation process, i.e., the coding nucleic acid of an RNA molecule containing a poly(A) tail is introduced into prokaryotic cells, the prokaryotic cells are amplified to amplify the coding nucleic acid, and then the amplified coding nucleic acid is transcribed into RNA. In some embodiments, the in vitro preparation process of RNA is for ligating an RNA fragment containing a protein coding sequence to a poly(A) tail by homologous recombination and enzymatic cleavage, or other non-homologous recombination methods. The poly(A) tail is produced by prokaryotic fermentation. During the prokaryotic fermentation process, the coding nucleic acid containing the poly(A) tail is introduced into prokaryotic cells, the prokaryotic cells are amplified to amplify the coding nucleic acid, and then the amplified coding nucleic acid is transcribed into RNA containing the tail RNA. In some embodiments, the coding nucleic acid is linear. In some embodiments, the coding nucleic acid is circular. In some embodiments, the coding nucleic acid is a plasmid. In some embodiments, the coding nucleic acid is single-stranded or double-stranded. In some embodiments, the coding nucleic acid is chemically modified before introduction into prokaryotic cells. In some embodiments, the coding nucleic acid is chemically synthesized before introduction into prokaryotic cells. In some embodiments, the coding nucleic acid is inserted into the nucleoid genomic DNA of the prokaryotic cell. In some embodiments, the coding nucleic acid exists in a free state within the cytoplasm or outside the nucleoid of the prokaryotic cell. In some embodiments, the prokaryotic cell is E. coli.

[0120] Based on this, the present application provides a series of manipulated poly(A) tails. The manipulated poly(A) tails include a miRNA binding site. In some embodiments, all bases of the manipulated poly(A) tail are A, except for the miRNA binding site. In some embodiments, the manipulated poly(A) tail is more highly conserved during the in vitro preparation of RNA. In some embodiments, the manipulated poly(A) tail can improve the stability of mRNA. In some embodiments, the manipulated poly(A) tail is formed by inserting a miRNA binding site between any two nucleotides of a conserved poly(A) tail. In some embodiments, the manipulated poly(A) tail is formed by directly ligating (conjugating) the miRNA binding site to the 5' terminal nucleotide of the conserved poly(A) tail at the 5' end of the conserved poly(A) tail. In some embodiments, the manipulated poly(A) tail is formed by directly ligating (conjugating) the miRNA binding site to the 3' terminal nucleotide of the conserved poly(A) tail at the 3' end of the conserved poly(A) tail. In some embodiments, the manipulated poly(A) tail is formed by replacing one or more elements in the conserved poly(A) tail with one or more miRNA binding sites.

[0121] As used herein, the term “conserved poly(A) tail” means a class of poly(A) tails containing at least one non-A base that are highly conserved in the in vitro preparation process of RNA and / or can improve the stability of mRNA compared to poly(A) tails whose bases are exclusively A. Conserved poly(A) may be a natural poly(A) tail or a synthetic poly(A) tail and includes, but is not limited to, the various poly(A) tails described in PCT application number PCT / CN2023 / 079037, Chinese Patent Application No. CN112805386A, and U.S. Patent No. 10717982B2 (these are incorporated herein by reference as a whole).

[0122] As used herein, two elements connected by a hyphen ("-") are directly connected. "Directly connected" means that there is no nucleotide between the two elements; therefore, "directly connected" can be connected by any permitted method of connection between nucleotides. In some embodiments, "directly connected" can be connected by a chemical bond. In some embodiments, "directly connected" can be connected by a phosphate diester bond.

[0123] Furthermore, this application also provides the use of poly(A)tails in enabling the low expression of target genes in specific organs, tissues, and / or cells, where specific organs highly express miRNAs.

[0124] Manipulated DNA molecules This application provides an in-cellularly replicable engineered DNA molecule comprising an engineered poly(A) tail coding sequence or a complementary sequence thereof. In some embodiments, the engineered DNA molecule is replicable in a cell. In some embodiments, the engineered DNA molecule can express an engineered poly(A) tail. In some embodiments, the engineered DNA molecule can express an engineered poly(A) tail and is replicable in a cell. In some embodiments, the cell is a prokaryotic or eukaryotic cell. In some embodiments, the prokaryotic cell is Escherichia coli. It is known to those skilled in the art that, in addition to the engineered poly(A) tail coding sequence, the engineered DNA molecule further comprises structural elements essential for the replication or efficient replication of the DNA molecule in a cell. Structural elements essential for the replication or efficient replication of the engineered DNA molecule in a cell are known in the art and include, for example, origins of replication (ORIs). In some embodiments, the manipulated DNA molecule further comprises a marker gene or a fragment thereof, and / or a reporter gene or a fragment thereof, as well as a unique restriction endonuclease site, preferably in the form of a multicloning site (MCS), that enables the insertion of DNA elements. The marker gene facilitates the identification of cells containing the plasmid containing the marker gene and may be selected, for example, from an antibiotic resistance gene. Each restriction endonuclease site within the MCS may be specifically recognized by a different restriction endonuclease.

[0125] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term “DNA plasmid” refers to a plasmid formed from a double-stranded DNA molecule. In some embodiments, the “plasmid” is a circular DNA molecule. In some embodiments, the “plasmid” may further encompass linear DNA molecules. Specifically, the term “plasmid” also encompasses molecules obtained by cleaving a circular plasmid with a restriction endonuclease to convert the circular plasmid into a linear molecule, and linear molecules that can replicate in prokaryotes. Plasmids can replicate, i.e., amplify, in cells independently of the genomic genetic information stored in the nucleoid in prokaryotic cells and can be used for cloning, i.e., amplification, of genetic information in bacterial cells. Preferably, the DNA plasmid according to the present invention is a medium-copy plasmid or a high-copy plasmid, more preferably a high-copy plasmid. Examples of such high-copy plasmids include pUC, pTZ plasmids, or vectors based on any other plasmid (e.g., pMB1, pCoIE1) containing an ORI that supports high-copy plasmids.

[0126] In some embodiments, the DNA molecule is a DNA molecule or fragment thereof that constitutes the nucleoid of a prokaryotic organism, i.e., a coding sequence or complementary sequence containing an engineered poly(A) tail can be replicated together with the prokaryotic genome.

[0127] In some embodiments, the DNA molecule is genomic DNA, such as viral genomic DNA or eukaryotic genomic DNA. In some embodiments, the DNA is mitochondrial DNA. In some embodiments, the DNA becomes free DNA when introduced into a eukaryotic cell. In some embodiments, the DNA is a viral vector.

[0128] In some embodiments, the DNA molecule is further ligated with the target gene fragment at the 5' end of the coding sequence of the engineered poly(A) tail, and the target gene fragment and the coding sequence of the engineered poly(A) tail jointly encode RNA. In some embodiments, the target gene fragment and the coding sequence of the engineered poly(A) tail jointly encode mRNA. The target gene fragment comprises a coding sequence for a protein, polypeptide, or fragment thereof. The coding sequence for a protein, polypeptide, or fragment thereof can ultimately be translated into one or more proteins or one or more polypeptides (such as short-chain peptides, oligopeptides, polypeptides, fusion proteins, proteins and fragments thereof, e.g., parts of known proteins such as functional parts). The functional part may be, for example, a bioactive part of a protein or an antigenic part such as an epitope that can effectively produce antibodies. The coding sequence for a protein, polypeptide, or fragment thereof comprises a start codon (5' end) and a stop codon (3' end) at each end, which are the first three nucleotides and the last three nucleotides of a translatable mRNA molecule.

[0129] In some embodiments, the mRNA of the present invention further comprises a 5'UTR and a 3'UTR, etc. In some embodiments, the mRNA of the present invention comprises, from 5' to 3', at least a 5'UTR sequence, a target gene sequence, a 3'UTR sequence, and the poly(A) tail sequence of the present invention.

[0130] The 5'UTR typically contains at least one ribosome-binding site (RBS), such as the Shine-Dalgarno sequence in prokaryotes, or at least one translation initiation site, such as the Kozak sequence in eukaryotes. RBS facilitate the efficient and accurate translation of mRNA molecules by recruiting ribosomes at translation initiation. The activity of a given RBS or translation initiation site can be optimized by modifying its length, sequence, and distance from the start codon. Selectively, the 5'UTR contains an internal ribosome entry site, or IRES. The 3'UTR may contain one or more regulatory sequences, such as amino acid sequence binding sites to enhance the stability of mRNA molecules, binding sites to regulate RNA molecules (such as miRNA molecules), and / or signal sequences involved in the intracellular transport of mRNA molecules.

[0131] The coding sequences of proteins, polypeptides, or fragments thereof contain codons that can be translated into amino acid sequences. All codons in the coding sequence may be naturally occurring codons that code for amino acids, or they may be partially or entirely composed of synthetic codons. In some embodiments, some or all of the codons undergo codon optimization. In some embodiments, some or all of the codons code for non-natural amino acids.

[0132] In some embodiments, the DNA molecule further includes structural elements essential for the initiation or regulation of RNA transcription at the 5' end of the target gene fragment, and these structural elements are known in the art. In some embodiments, the structural elements include at least a promoter. Promoters and their sequences are known in the art and include weak promoters, moderately strong promoters, strong promoters, mini-promoters, or core promoters. In some specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter can initiate transcription of the target gene fragment and / or the engineered poly(A) tail in prokaryotic cells. In some embodiments, the promoter can initiate transcription of the target gene fragment and / or the engineered poly(A) tail in eukaryotic cells. The “promoter” includes at least one transcription recognition site followed by a transcription factor binding site. The recognition and binding sites can interact with amino acid sequences that mediate or regulate transcription. The binding site is closer to the target gene fragment than the recognition site. The binding site may be, for example, a Pribno box in prokaryotes and a TATA box in eukaryotes. For example, in some embodiments, when a Pribno box is used, the transcription recognition site may be located about 35 bp upstream of the transcription start site, and the transcription factor binding site may be located about 10 bp upstream of the transcription start site. In some embodiments, the promoter includes at least one additional regulatory element, such as an AT-rich upstream element located about 40 and / or 60 nucleotides prior to the transcription start site, and / or an additional regulatory element located between the recognition site and the binding site to enhance promoter activity. In some embodiments, the promoter is a strong promoter; that is, the promoter includes a sequence that promotes the transcription of the RNA coding sequence. Strong promoters are known to those skilled in the art, such as the OXB18, OXB19, and OXB20 promoters derived from the RecA promoter from E. coli, or can be identified or synthesized by routine laboratory procedures. In some embodiments, the promoter is a T7 promoter.In some embodiments, the promoter further includes additional regulatory elements, such as enhancers contained in a DNA plasmid, which can promote the transcription of RNA coding sequences.

[0133] Furthermore, this application further provides the use of engineered DNA molecules in the stable amplification of engineered poly(A) tail coding sequences or RNA coding sequences having engineered poly(A) tails.

[0134] Manipulated RNA This application provides an engineered RNA or engineered RNA molecule comprising the engineered poly(A) tail described above and a target gene fragment at the 5' end of the coding sequence of the poly(A) tail. In some embodiments, the RNA is mRNA.

[0135] In some embodiments, the mRNA molecule in this application includes the 5'UTR and / or 3'UTR.

[0136] In some embodiments, the mRNA molecule in this application further includes a 5' cap. In preferred embodiments, the 5' cap is m7G(5')ppp(5')(2'-OMeA)pG.

[0137] In some embodiments, the mRNA molecule in this application further includes chemical modifications, for example, all or some of the uridines in the polynucleotide sequence are modified to N1-methylpseudridine.

[0138] As used herein, “mRNA” (messenger RNA) is any RNA, naturally occurring, unnaturally occurring, or modified, that codes for at least one protein, polypeptide, or fragment thereof. mRNA has the ability to be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein, polypeptide, or fragment thereof. Thus, mRNA may be mature mRNA or premature mRNA, and its essential or selective elements or structure are known in the art. In some embodiments, mRNA contains coding sequences for several essential functional components for expressing, regulating, or enhancing the expression levels of a protein, polypeptide, or fragment thereof. Functional components include, but are not limited to, a 5' hat, a 5' UTR, or a 3' UTR. Both the 5' UTR and the 3' UTR are typically transcribed from genomic DNA and are elements of premature mRNA.

[0139] In mature mRNA, the term "5' hat" typically refers to a structure located at the 5' end of the mRNA that contains methylated guanylate. This methylated guanylate is mediated by pyrophosphates to the 5' end of the mRNA, forming a 5',5'-triphosphate linkage with the adjacent nucleotide. There are typically three types of 5' hat structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as type O, type I, and type II, respectively. Type O means that the ribose of the terminal nucleotide is not methylated, type I means that the ribose of one terminal nucleotide is methylated, and type II means that the ribose of both terminal nucleotides is methylated. In some embodiments, the 5' hat can be produced by capping the 5' end of a polynucleotide with a chemical RNA cap analog (3'-O-Me-m7G(5')ppp(5')G[ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, Massachusetts), or m7G(5')ppp(5')(2'-OMeA)pG(CleanCapAG)) during in vitro transcription to produce a 5'-guanosine cap structure, as per the manufacturer's instructions. For example, in some embodiments, the 5' capping of modified RNA can be completed after transcription using a vaxinia capping enzyme to produce a type O hat structure m7G(5')ppp(5')G (New England BioLabs, Ipswich, Massachusetts). Vaccinia capping enzymes and 2'-O-methyltransferases can be used to produce a type I hat structure, namely m7G(5')ppp(5')(2'-OMeA)pG. A type II hat structure can then be produced from the type I hat structure by 2'-O-methylation of the third nucleotide from the 5' end using 2'-O-methyltransferase.The type III hat structure can then be produced from the type II hat structure by 2'-O-methylation of the fourth nucleotide from the 5' end using a 2'-O-methyltransferase.

[0140] In some embodiments, the mRNA further comprises stabilizing elements. These stabilizing elements may include, for example, histone stem loops. In some embodiments, the mRNA comprises a coding region, at least one histone stem loop, and selectively a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally enhances the expression level of the encoded protein. In some embodiments, the mRNA comprises a combination of a poly(A) sequence or polyadenylation signal and at least one histone stem loop. While these two have a substitution mechanism in nature, their synergistic effect can increase protein expression to a level higher than that observed with any single element. The synergistic effect of the poly(A) and at least one histone stem loop combination is independent of the order of the elements or the length of the poly(A) sequence. In some embodiments, the histone stem loop typically originates from a histone gene and comprises two adjacent portions separated by a spacer (formed from a short sequence) to form the loop, or an intramolecular base pairing of completely opposite complementary sequences. Unpaired loop regions typically cannot base pair with any of the stem-loop elements. The stability of the stem-loop structure usually depends on the length of the pairing region, the number of mispairs or protrusions, and the base composition. In some embodiments, fluctuating base pairing (non-Watson-Crick base pairing) may occur. In some embodiments, at least one histone stem-loop sequence has a length of 15 to 45 nucleotides.

[0141] In some embodiments, one or more AU-rich sequences in mRNA may be removed. These sequences are sometimes called AURES, which are destabilizing sequences found within the 3'UTR. AURES may be removed from mRNA, or they may be retained within mRNA.

[0142] cell This application further provides cells containing an engineered DNA molecule, the DNA molecule being stored and / or amplified within the cell. In some embodiments, the cell is a prokaryotic cell, and the DNA molecule can be replicated within the prokaryotic cell. In some embodiments, the cell is a prokaryotic cell, and the DNA molecule can be replicated and / or transcribed within the prokaryotic cell. In some embodiments, the DNA molecule is a eukaryotic cell, and the DNA molecule can be replicated within the cell. In some embodiments, the DNA molecule can be transcribed and / or replicated within the cell.

[0143] In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are bacteria, actinomycetes, cyanobacteria, mycoplasma, rickettsia, and chlamydia. In some embodiments, the cells are selected from Bacillus subtilis, Lactobacillus, Acetobacteria, Corynebacterium, Brevibacterium, Arthrobacter, Pseudomonas, and Micrococcus. In some embodiments, the cells are recA-expressing bacteria. In some embodiments, the cells are Escherichia coli. In some embodiments, the cells are Escherichia coli selected from strain K-12 and its derivatives, as well as strain B and its derivatives. In some embodiments, the E. coli is selected from MG1655, DH5 or DH5α, DH10B, BL21, DB3.1, HB101, JM109, JM110, MC1061, MG1655, Pir1, Stbl2, Stbl3, Top10, XL1Blue, XL10Gold, BLR, HMS174, Tuner, Rostetta2, Lemo21, T7Express, or Origami2. In some embodiments, the cells are selected from Streptomyces, Micromonospora, and Nocardia. In some embodiments, the cells are fungi. In some embodiments, the cells are selected from yeast or mold.

[0144] Lipid nanoparticles This application further provides lipid nanoparticles comprising an engineered poly(A) tail, an engineered RNA molecule, and / or an engineered DNA molecule as described herein.

[0145] In some embodiments, lipids are mixed with RNA that has been manipulated to form lipid nanoparticles. In some embodiments, the manipulated poly(A) tails, manipulated RNA molecules, and / or manipulated DNA molecules according to this application are incorporated into lipid nanoparticles. In some embodiments, with respect to lipid nanoparticles, empty lipid nanoparticles are first formed and then combined with or encapsulated with the manipulated poly(A) tails, manipulated RNA molecules, and / or manipulated DNA molecules immediately before application (e.g., within a few minutes to an hour).

[0146] Lipid nanoparticles typically contain ionized lipids, noncationic lipids, sterols, PEGylated lipid components, and target nucleic acids (engineered poly(A) tails, engineered RNA molecules, and / or engineered DNA molecules as per this application).

[0147] The lipid nanoparticles of this application contain ionized lipids, phospholipids, structural lipids, and PEGylated lipids. Preferably, the molar ratio of ionized lipids, phospholipids, structural lipids, and PEGylated lipids is (20-60):(5-25):(25-55):(0.5-15), and more preferably, (40-55):(5-15):(30-50):(1-3).

[0148] The phospholipid is preferably one or more compounds selected from the following: Dilauroyl-sn-glycerophosphocholine (DLPC), Dimyristoyl-sn-glycerophosphocholine (DMPC), Dioleoyl-sn-glycero-phosphocholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoyl-sn-glycero-3-phosphocholine (DSPC), Diundecanoyl-sn-glycerophosphocholine (DUPC), Palmitoyl oleoyl glycerophosphocholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-Oleoyl-2-Cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Divinyl-sn-glycero-3-phosphocholine, 1,2-Diarylacyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Divinylol-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl-SN-Glycero-3-Phosphoethanolamine, 1,2-diaryl-sn-glycero-3-phosphoethanolamine, 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phosphatidyl-(1-glycerol) sodium salt (DOPG), or sphingomyelin.

[0149] For example, phospholipids are DOPE or DSPC.

[0150] The structural lipid is preferably one or more selected from cholesterol, coprosterol, sitosterol, ergosterol, and stigmasterol. For example, the structural lipid is cholesterol, and / or The PEGylated lipid is preferably one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol, for example, the PEGylated lipid is DMG-PEG2000.

[0151] Components, compositions, and methods commonly known in the art may be used to produce the lipid nanoparticles of this disclosure, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US See PCT / US2016 / 014280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, and PCT / US2016 / 069491 (these are incorporated herein by reference as a whole).

[0152] It should be understood that this application includes various aspects, embodiments, and combinations of such aspects and / or embodiments described herein. The above description and the following examples are intended to be illustrative, not to limit the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be obvious to those skilled in the art to which this application belongs. Therefore, those skilled in the art will recognize that the improvements and modifications described to the described aspects and embodiments are also included within the scope of this application.

[0153] Examples Example 1: Construction of luciferase mRNA containing the polyA variant of the present invention 1) Construction of a vector containing the luciferase protein coding region In the vector, the E. coli cloning vector pUC57 was used as the vector backbone, and the T7 promoter sequence (SEQ ID NO: 2), 5'UTR, Kozak sequence (GCCACC), luciferase protein coding sequence, 3'UTR, and poly(dA:dT) designated as RG008 were sequentially placed between the Xba I restriction site and the Sap I restriction site of its multi-cloning site, and used as a control for the poly(A) variant designed in the present invention.

[0154] 2) Replacement of poly(dA:dT) in general-purpose vectors All the sequences required to construct different poly(A) variants (shown in the sequence listing at the end of this disclosure) were synthesized. The variant poly(dA:dT) designated RG008 was removed from the general-purpose vector constructed in 1) by enzymatic digestion with two restriction endonucleases. Then, the different poly(A) variants of the present invention were ligated to the vectors from which poly(dA:dT) had been removed, respectively, using T4 DNA ligase 1, to complete the replacement of poly(dA:dT) in the general-purpose vector.

[0155] Schematic diagrams are shown in Figures 1A and 1B.

[0156] Example 2: Preparation of mRNA-LNPs according to the present invention Acetic acid solution was added to the luciferase mRNA stock solution so that the final concentration of acetic acid was 20 mmol / L and the final concentration of mRNA was 200 μg / ml. The mixture was then uniformly stirred and mixed to obtain an mRNA dilution standard solution. The mRNA dilution standard solution was mixed with a mixed lipid solution (prepared according to Table 1) at a flow rate of 2:1 to 4:1 using a T-type mixer to prepare LNPs. Subsequently, the LNPs were diluted 2 to 5 times with 2 mmol / L acetic acid solution, and then replaced with 2 mmol / L acetic acid solution at least three times. The solution was concentrated to the target concentration. Sucrose solution was added to adjust the osmotic pressure, and the pH was adjusted to 7.0 to 8.0 with Tris solution to obtain the mRNA-lipid nanoparticles (LNPs) of the present invention.

[0157] [Table 1]

[0158] Example 3: Expression study in mice of luciferase mRNA having five poly(A) groups including the miR-142 binding site. Using the luciferase coding region as the protein coding region, we investigated the expression levels of five poly(A) variants (poly(A)-60A insert miR-142, poly(A)-30A insert miR-142, poly(A)-19A insert miR-142, poly(A)-14A insert miR-142, and poly(A)-0A insert miR-142) in the liver of mice with low miR-142 expression and in the spleen of mice with high miR-142 expression to detect whether insertion of the miR-142 binding site can downregulate mRNA expression levels in organs with high miR-142 expression.

[0159] C57BL / 6 mice were randomly divided into groups according to their body weight, and six animals in each group were reared for 2-3 days. 0.05 mg / kg of mRNA-LNP (1 μg) was administered to the mice via tail vein injection in a 100 μL volume. The same volume of PBS was injected into the blank control animals. Six hours after administration, luciferin (200 μL) was administered intraperitoneally at a dose of 150 mg / kg. Ten minutes later, the mice were anesthetized and imaged using a small animal imager. The liver and spleen of the mice were dissected and imaged again. The experiment was performed once, and histograms were created using the measurements for each mouse. The results are shown in Figures 2A-2B.

[0160] Conclusion: Various poly(A) variants are formed by inserting miR-142 binding sites at positions after 0A (SEQ ID NO: 16), 14A (SEQ ID NO: 9), 19A (SEQ ID NO: 8), 30A (SEQ ID NO: 7), and 60A (SEQ ID NO: 6) in RG008. When mRNA containing such variants is packaged in LNPs and injected into mice via the tail vein, macrophages (highly expressing miR-142) take up and degrade the mRNA in the spleen, inhibiting its expression. Regarding expression levels in the spleen, the reduction in expression is similar whether the miR-142 binding site is inserted at 3'UTR or at positions after 0A, 14A, 19A, or 30A, and significantly better than when inserted after 60A.

[0161] Regarding expression levels in the liver, when the miR-142 binding site is inserted at positions 14A, 19A, 30A, and 60A or later, the expression level in the liver is essentially unaffected. When inserted at positions 14A and 30A or later, the expression level is even better than that of the control group without the miR-142 binding site. When inserted at positions 0A or later, the expression level in the liver is clearly reduced.

[0162] By selecting insertion sites 14A to 30A based on expression levels in the liver and spleen, and using them as the optimal insertion sites for the miR-142 binding site in this invention, it is possible to significantly reduce the expression level in the spleen without affecting the expression level in the liver. Therefore, this application is suitable for applications in treating diseases caused by mRNA expressing proteins in the liver.

[0163] Example 4: Expression test in mice of luciferase mRNA having five poly(A) groups including the miR-122 binding site. The expression levels of five poly(A) variants (poly(A)-60A insert miR-122, poly(A)-30A insert miR-122, poly(A)-19A insert miR-122, poly(A)-14A insert miR-122, and poly(A)-0A insert miR-122) were investigated in the spleen of mice with low miR-122 expression and in the liver of mice with high miR-122 expression.

[0164] C57BL / 6 mice were randomly divided into groups according to their body weight, and six animals in each group were reared for 2-3 days. 0.05 mg / kg of mRNA-LNP (1 μg) was administered to the mice via tail vein injection in a 100 μL volume. The same volume of PBS was injected into the blank control animals. Six hours after administration, luciferin (200 μL) was administered intraperitoneally at a dose of 150 mg / kg. Ten minutes later, the mice were anesthetized and imaged using a small animal imager. The liver and spleen of the mice were dissected and imaged again. The experiment was performed once, and histograms were created using the measurements for each mouse. The absolute values ​​of the fluorescence imaging of the liver and spleen are shown in Figures 3A and 3B, respectively.

[0165] Conclusion: Various poly(A) variants are formed by inserting the miR-122 binding site at positions 0A, 14A, 19A, 30A, and 60A and beyond in RG008, respectively. When mRNA containing such variants is packaged with LNP and injected into mice via the tail vein, the mRNA is clearly taken up into the liver, degraded, and its expression is inhibited. When the miR-122 binding site is inserted at positions 0A, 14A, and 19A and beyond in RG008, the decrease in liver expression is significantly lower than when it is inserted at positions 30A and 60A and beyond. Expression levels in the spleen are decreased in all groups. By selecting insertion positions 0A, 14A, and 19A based on the expression levels in the liver and spleen and using them as the optimal insertion positions for the miR-122 binding site in this invention, the expression level in the spleen is maintained without significantly affecting the expression level in the liver. Therefore, this application is suitable for applications to treat diseases caused by mRNA expressing proteins in the spleen.

[0166] Example 5: Stability testing of replication of plasmids containing different poly(A) variants in E. coli. The effects of a total of 10 poly(A) variants (poly(A)-60A insert miR-142, poly(A)-30A insert miR-142, poly(A)-19A insert miR-142, poly(A)-14A insert miR-142, poly(A)-0A insert miR-142, poly(A)-60A insert miR-122, poly(A)-30A insert miR-122, poly(A)-19A insert miR-122, poly(A)-14A insert miR-122, and poly(A)-0A insert miR-122) within two groups on their stability in E. coli were investigated.

[0167] After confirming the correctness of the vector plasmid constructed according to the method of Example 1 by sequencing, it was transformed into E. coli DH5α. Following transformation, the cells were grown on plates at 30°C. The next day, the plates were sent to a sequencing service company, and 50 clones were randomly selected for sequencing. After sequencing, different poly(A) variants were analyzed and calculated based on the sequencing results.

[0168] The results are shown in Figure 4. The base deletion rate after inserting two different miRNA binding sites after positions 14A–30A of the poly(A) variant is lower than when the insertion is at 0A or after 60A and at 3'UTR. This result indicates that plasmids with miRNA binding sites inserted after positions 14A–30A of the RG008 poly(A) variant are more stable.

[0169] Example 6: Intracellular expression test of mRNA having poly(A) variants containing miR-142 binding sites at different insertion positions. Expression levels of eight poly(A) variants (poly(A)-0A insert miR-142, poly(A)-1A insert miR-142, poly(A)-2A insert miR-142, poly(A)-3A insert miR-142, poly(A)-4A insert miR-142, poly(A)-5A insert miR-142, poly(A)-10A insert miR-142, poly(A)-14A insert miR-142) in mouse monocyte / macrophage leukemia cells RAW264.7 with high miR-142 expression.

[0170] RAW246.7 cells were placed in a 24-well cell culture plate at a rate of 1 × 10⁶ 5Cells were cultured at a cell density of cells / well. 500 ng of mRNA, synthesized in vitro in the presence of Lipofectamine 3000 (purchased from Thermo Fisher Scientific), was transfused into the cells in each well. 24 hours after transfusion, the cell culture medium was aspirated and removed, and 200 μL of cell degradation buffer was added to each well. After 10 minutes, the cell lysate was transferred to a 1.5 mL EP tube and centrifuged at 12000 rpm at 4°C for 5 minutes. Next, 20 μL of the supernatant of the lysate was transferred to a black opaque 96-well plate, 200 μL of fluorescein substrate was added, and the values ​​were read using a fluorescence detector. The results are shown in Figure 5.

[0171] Conclusion: Various poly(A) variants are formed by inserting miR-142 binding sites at positions 0A (SEQ ID NO: 16), 1A (SEQ ID NO: 15), 2A (SEQ ID NO: 14), 3A (SEQ ID NO: 13), 4A (SEQ ID NO: 12), 5A (SEQ ID NO: 11), 10A (SEQ ID NO: 10), 14A (SEQ ID NO: 9) and later in RG008. When mRNA containing these variants is delivered to cell lines that overexpress miR-142, such as Raw264.7 cells, the mRNA is degraded by miR-142, inhibiting their expression.

[0172] Example 7: Study on the interaction of various series-connected miRNA binding sites in a poly(A) tail. We investigated the expression levels of two poly(A) variants (poly(A)-0A insert miR-142+miR-122, and poly(A)-0A insert miR-122+miR-142) in mouse monocyte / macrophage leukemia cells RAW264.7, which have high miR-142 expression, to determine whether the insertion of serial miR-122 and miR-142 binding sites affects the downregulation of mRNA expression levels by the miR-142 binding site.

[0173] RAW246.7 cells were placed in a 24-well cell culture plate at a rate of 1 × 10⁶ 5Cells were cultured at a cell density of cells / well. 500 ng of mRNA, synthesized in vitro in the presence of Lipofectamine 3000 (purchased from Thermo Fisher Scientific), was transfused into the cells in each well. 24 hours after transfusion, the cell culture medium was aspirated and removed, and 200 μL of cell degradation buffer was added to each well. After 10 minutes, the cell lysate was transferred to a 1.5 mL EP tube and centrifuged at 12000 rpm at 4°C for 5 minutes. 20 μL of the supernatant of the lysate was then transferred to a black opaque 96-well plate, 200 μL of fluorescein substrate was added, and the values ​​were read using a fluorescence detector. The results are shown in Figure 6.

[0174] Conclusion: Various poly(A) variants are formed by inserting miR-142+miR-122 or miR-122+miR-142 binding sites at positions post-0A of RG008. When mRNAs containing such variants are delivered via LNP to cell lines overexpressing miR-142, such as Raw264.7 cells, the mRNAs are degraded by miR-142, inhibiting their expression. Furthermore, the order in which miR-142 and miR-122 binding sites are connected in series does not affect the inhibitory effect on expression.

[0175] Example 8: Study on the precise intracellular expression of mRNA containing poly(A) variants with multiple miRNA binding sites. Mouse monocyte / macrophage leukemia cells RAW264.7 highly expressing miR-142, mouse hepatic sinusoidal endothelial cells (LSEC) highly expressing miR-126, mouse hepatic stellate cells (HSC) highly expressing miR-148a, and six poly(A) variants in hepatic parenchymal cells AML (Poly(A)-0A insert miR-142+miR-126+miR-148a, Poly(A)-0A insert miR- The expression levels of 142+miR-148a+miR-126, poly(A)-0A insert miR-126+miR-142+miR-148a, poly(A)-0A insert miR-126+miR-148a+miR-142, poly(A)-0A insert miR-148a+miR-142+miR-126, and poly(A)-0A insert miR-148a+miR-126+miR-142) were investigated.

[0176] RAW246.7, LSEC, HSC, and AML12 cells were placed in a 24-well cell culture plate at a rate of 1 × 10⁶ 5 Cells were cultured at a cell density of cells / well. 500 ng of mRNA synthesized in vitro in the presence of Lipofectamine 3000 (purchased from Thermo Fisher Scientific) was transfused into the cells in each well. 24 hours after transfusion, the cell culture medium was aspirated and 200 μL of cell degradation buffer was added to each well. After 10 minutes, the cell lysate was transferred to a 1.5 mL EP tube and centrifuged at 12000 rpm at 4°C for 5 minutes. Next, 20 μL of the supernatant of the lysate was transferred to a black opaque 96-well plate, 200 μL of fluorescein substrate was added, and the values ​​were read using a fluorescence detector. Calculation of the reduction ratio in expression within hepatocytes: Data from the ts group without miRNA was defined as 1, and the relative expression levels of various groups relative to ts without miRNA in various cell line experiments were calculated to bridge the direct expression data of various cell lines. Based on this, the reduction in expression of each target cell (RAW264.7 cells, hepatic sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC)) against hepatic parenchymal cells (AML12) was calculated using the following formula: (1 - relative expression level in target cells / relative expression level in hepatic parenchymal cells) × 100%.

[0177] Conclusion: When miR-148a, miR-142, and miR-126 are linked in series and mRNA is transfused into RAW264.7 cells, the poly(A) variant contains a miR-142 binding site, and mRNA expression within the cell line is reduced. When mRNA is transfused into LSEC cells, the poly(A) variant contains a miR-126 binding site, and mRNA expression within the cell line is reduced. When mRNA is transfused into HSC cells, the poly(A) variant contains a miR-148a binding site, and mRNA expression within the cell line is reduced. Furthermore, the performance of the miR-126+miR-148a+miR-142 and miR-148a+miR-142+miR-126 combinations is superior to other sequence combinations. See Figures 7A-7D and 8A-8C.

[0178] The liver is primarily composed of hepatic parenchymal cells, hepatic macrophages, hepatic sinusoidal endothelial cells, and hepatic stellate cells. This example selects a combination of microRNA binding sites that exhibit superior specific expression in hepatic parenchymal cells, thereby providing a reliable tool for hepatic parenchymal cell-targeted therapies.

[0179] The results of this invention demonstrate that inserting a miRNA binding site into a poly(A) variant allows for different mRNA expression in different mouse cell lines and organs. Therefore, highly specific expression regulation can be achieved by inserting one or more miRNA binding sites into the same sequence.

[0180] The sequences used in the above embodiments of this application are shown in the following sequence listing. It should be understood that the following sequences are merely illustrative sequences in the embodiments of this application and do not limit the embodiments of this application in any way. Nucleic acid sequences in the following sequence listing represent either DNA sequences or RNA sequences. Where an RNA sequence is represented, "T" represents uridine. Also, in the context of RNA, a single "T" in this application may also refer to uracil or uridine.

[0181] [Table 2]

[0182] Table 3

[0183] Table 4

[0184] Table 5

[0185] Table 6

[0186] Table 7

[0187] Table 8

[0188] Table 9

[0189] Table 10

Claims

1. A modified polyadenylic acid (poly(A)) tail containing a miRNA binding site.

2. The structure includes or is the structure of the following formula (I): nA-miRNA binding position-mA formula (I), The aforementioned miRNA binding site is formed by one or more nucleotides, or by one or more directly linked miRNA binding sites. nA represents n consecutive adenylic acid (A) molecules located near the 5' end of the miRNA binding site. mA represents m consecutive adenylic acid (A) molecules located near the 3' end of the miRNA binding site. m and n are natural numbers such that m+n ≤ 150, m+n ≤ 120, m+n ≤ 100, m+n ≤ 80, m+n ≤ 60, m+n ≤ 30, m+n ≤ 19, or m+n ≤ 14, preferably m+n = 60. The manipulated poly(A) tail according to claim 1, wherein the 3' end and the 5' end of the miRNA binding site do not contain any A other than the A included in the miRNA binding site.

3. The manipulated poly(A) tail according to claim 2, wherein n = 0 or n ≥ 1.

4. The manipulated poly(A) tail according to claim 2, wherein n ≤ 60, n ≤ 30, n ≤ 19, n ≤ 14, or n ≤ 10.

5. The operated poly(A) tail according to claim 2, wherein 14 ≤ n ≤ 30, preferably 19 ≤ n ≤ 30 or 14 ≤ n ≤ 19.

6. An operated poly(A) tail according to any one of claims 1 to 5, having a length of 80 to 240 nt, for example, 100 to 200 nt, 101 to 150 nt, 120 to 150 nt, 130 to 140 nt, 123 to 135 nt, or 125 to 139 nt.

7. The 3' side of the structure of formula (I) further includes a tail fragment directly attached to the 3' end of the structure of formula (I), wherein the 5' terminal nucleotide of the tail fragment is not A, and / or The 5' side of the structure of formula (I) further includes a head fragment directly attached to the 5' end of the structure of formula (I), wherein the 3' terminal nucleotide of the head fragment is not A, according to any one of claims 2 to 6.

8. The tail segment or the head segment comprises one or more elements c and / or one or more elements d and one or more elements A, Element c is a non-A nucleotide, Element d contains any two or more consecutive nucleotides, the 5' and 3' terminal nucleotides of element d are not A, and element d does not contain more than three consecutive A's. The length of element d is within the range of 2nt ≤ d ≤ 20nt. The operated poly(A) tail according to claim 7, wherein elements c and d are not adjacent.

9. The manipulated poly(A) tail according to claim 8, wherein the number of elements c included is 2 to 10, 3 to 8, 4 to 6, or 2 to 5, preferably 2, and / or element c is G.

10. The operated poly(A)tail according to any one of claims 8 to 9, wherein element d is any one or more selected from the sequences shown in sequence numbers 60 to 64, preferably element d is as shown in sequence number 60.

11. The operated poly(A) tail according to any one of claims 8 to 10, wherein the number of elements d included is 0 to 5, preferably 1 to 3, and more preferably 1.

12. An operated poly(A) tail, which does not include a head segment, wherein the tail segment has the structure of element d-19A-element c-19A-element c-17A or element c-19A-element c-17A, according to any one of claims 1 to 11.

13. An engineered poly(A) tail according to any one of claims 1 to 12, having the structure nA-miRNA binding site-mA-element d-19A-element c-19A-element c-17A.

14. An engineered poly(A) tail according to any one of claims 1 to 13, having the structure nA-miRNA binding site-mA-SEQ ID NO: 60-19A-G-19A-G-17A.

15. The aforementioned miRNA is, One or more selected from miR-142, miR-122, miR-126, miR-148a, miR-133, miR-206, miR-208, miR-17-92, miR-16, miR-21, miR-223, miR-24, miR-27, let-7, miR-30c, miR-1d, -149, miR-149, miR-192, miR-194, miR-101, and miR-204, Preferably, the miRNA is one or more selected from miR-142, miR-122, miR-126, and miR-148a. Preferably, the miRNA is one or more selected from the combination of miR-142 and miR-122, the combination of miR-126 and miR-148a, and the combination of miR-142, miR-148a, and miR-126, the manipulated poly(A) tail according to any one of claims 1 to 14.

16. The miRNA comprises miRNA-142, miR-148a, and miR-126, and at the miRNA binding site of the poly(A), the miRNA binding site is positioned from 5' to 3'. miR-126 binding site, miR-148a binding site, and miR-142 binding site, miR-148a binding site, miR-142 binding site, and miR-126 binding site, miR-142 binding site, miR-148a binding site, and miR-126 binding site, miR-126 binding site, miR-142 binding site, and miR-148a binding site, miR-148a binding site, miR-126 binding site, and miR-142 binding site, or The miR-142 binding site, the miR-126 binding site, and the miR-148a binding site are arranged in that order. The manipulated poly(A) tail according to any one of claims 1 to 15, wherein the miR-126 binding site, the miR-148a binding site, and the miR-142 binding site are adjacent to each other.

17. The manipulated poly(A) tail according to any one of claims 1 to 16, wherein n = 0, m = 60, and mA and the tail fragment form the polynucleotide sequence shown in Sequence ID No.

1.

18. The manipulated poly(A) tail according to any one of claims 1 to 16, wherein the poly(A) tail comprises any polynucleotide sequence selected from SEQ ID NOs. 6 to 16, SEQ ID NOs. 19 to 31, SEQ ID NOs. 33 to 52, SEQ ID NOs. 54 to 59, and SEQ ID NOs. 65 to 71.

19. An engineered RNA molecule which is mRNA or non-coding RNA and comprises a poly(A) tail according to any one of claims 1 to 18 and a target gene sequence located on the 5' side of the poly(A) tail, wherein the RNA sequence comprises, in order from the 5' end to the 3' end, at least a 5' UTR sequence, the target gene sequence, a 3' UTR sequence, and the poly(A) tail according to any one of claims 1 to 18, wherein nA in the structure of formula (I) of the poly(A) tail is directly ligated to the 3' UTR, and preferably the RNA molecule has a 5' cap and / or all or part of the uridylic acid is modified to N1-methylpseudridine.

20. An engineered DNA molecule encoding an engineered poly(A) tail according to any one of claims 1 to 18, or an engineered RNA sequence according to claim 19, wherein the DNA molecule is preferably a plasmid or a viral vector.

21. An engineered cell comprising an engineered poly(A) tail according to any one of claims 1 to 18, an engineered RNA molecule according to claim 19, or an engineered DNA molecule according to claim 20, wherein the cell is a eukaryotic cell or a prokaryotic cell, preferably the prokaryotic cell is Escherichia coli.

22. Use of an engineered poly(A)tail according to any one of claims 1 to 18 in regulating the expression of a target gene in a specific organ, tissue, and / or cell, wherein the specific organ, tissue, and / or cell highly expresses the miRNA, preferably the regulation is to enable low expression of the target gene in the specific organ, tissue, and / or cell, and more preferably the specific organ, tissue, and / or cell is a liver or spleen organ, tissue, and / or cell.

23. Use of an engineered poly(A)tail according to any one of claims 1 to 18 in enabling the specific expression of a target gene in a specific organ, tissue, and / or cell, preferably the organ or tissue being the liver or spleen organ or tissue, and more preferably the cell being a hepatocyte.

24. Use of the poly(A) tail according to any one of claims 1 to 18 in which the DNA coding sequence encoding the poly(A) tail is enabled to be replicated more conservedly within a host cell, wherein the host cell is preferably a prokaryotic cell, and more preferably Escherichia coli.

25. Lipid nanoparticles comprising an engineered poly(A) tail according to any one of claims 1 to 18, an engineered RNA molecule according to claim 19, or an engineered DNA molecule according to claim 20.

26. The material comprises ionized lipids, phospholipids, structural lipids, and PEGylated lipids, preferably the molar ratio of the ionized lipids, phospholipids, structural lipids, and PEGylated lipids is (20-60):(5-25):(25-55):(0.5-15), and more preferably (40-55):(5-15):(30-50):(1-3). The phospholipid is preferably one or more compounds selected from the following: Dilauroyl-sn-glycerophosphocholine (DLPC), Dimyristoyl-sn-glycerophosphocholine (DMPC), Dioleoyl-sn-glycero-phosphocholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoyl-sn-glycero-3-phosphocholine (DSPC), Diundecanoyl-sn-glycerophosphocholine (DUPC), Palmitoyl-oleoyl-glycero-phosphocholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Dieter PC), 1-Oleoyl-2-Cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-divinyl-sn-glycero-3-phosphocholine, 1,2-diarylacyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Divinylol-sn-glycero-3-phosphoethanolamine, 1,2-divinyl-sn-glycero-3-phosphoethanolamine, 1,2-diaryl-sn-glycero-3-phosphoethanolamine, 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phosphatidyl-(1-glycerol) sodium salt (DOPG), or sphingomyelin, For example, the phospholipid is DOPE or DSPC. The structural lipid is preferably one or more selected from cholesterol, coprosterol, sitosterol, ergosterol, and stigmasterol, for example, the structural lipid is cholesterol and / or The lipid nanoparticle according to claim 25, wherein the PEGylated lipid is preferably one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol, for example, the PEGylated lipid is DMG-PEG2000.

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