Engineered RNA molecules with regulatable expression

Abstract

The invention relates to an engineered RNA molecule, a DNA molecule encoding the RNA molecule and application of the RNA molecule. The engineered RNA molecule comprises a Poly (A) tail sequence comprising a miRNA binding site. The Poly (A) tail can be used for enabling a target gene to realize precise expression of organs, tissues and / or cells.

Description

[0001] Related Applications

[0002] This application claims priority to Chinese provisional application PCT / CN2023 / 099512 filed on June 9, 2023, Chinese provisional application PCT / CN2023 / 121779 filed on September 26, 2023, and Chinese provisional application 202410711025.6 filed on June 3, 2024. The entire contents of the prior applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to the field of biotechnology, and in particular to an engineered RNA molecule comprising a Poly(A) tail, wherein the Poly(A) tail enables the engineered RNA molecule to be accurately expressed in specific organs, tissues and / or cells. Background Art

[0004] Deoxyribonucleic acid (DNA) and messenger RNA (mRNA) are the main components of biological genetic material, and their main function is to carry and transmit genetic information. In eukaryotic cells, the complete mRNA mainly includes the 5' cap structure, the 5' untranslated region (5'untranslated region, 5'UTR), the coding region (ORF), the 3' untranslated region (3'untranslated region, 3'UTR) and the 3' polyadenylic acid tail structure poly (A). The 5' cap structure mainly regulates the stability of mRNA and the initiation of mRNA translation. The cap structure blocks the 5' end to prevent mRNA from being hydrolyzed by nuclease exonucleases, and it can be recognized and bound by the cap binding protein (eIF-4E), regulating the binding of mRNA to the ribosome, thereby initiating the translation process. The 5'UTR is a short sequence between the cap and the start codon. Too short or too long is not conducive to the initiation of mRNA translation. It is generally believed that 3'UTR is mainly involved in post-transcriptional regulation, including regulating the in vivo half-life of mRNA. For example, many miRNAs can bind to the 3'UTR of target gene mRNA and reduce the expression of target genes by degradation or binding inhibition. There is also a poly (A) sequence after the 3'UTR, which can prevent exonuclease degradation. In addition, the poly (A) tail sequence can also bind to poly (A) binding protein (PABP) and further recruit eIF4G, eIF4B, Paip-1 and other proteins to form a complex, which participates in the regulation of mRNA stability and the translation initiation process. The poly (A) tail of natural mRNA in eukaryotic cells is added after transcription by poly (A) polymerase. In the evolutionary process from fungi, plants to animals, this polymerase has substrate specificity for adenylate. So far, no clear evidence has been found to show that complex non-adenylate motifs with specific regulatory functions can be formed in the natural poly (A) tail structure.

[0005] mRNA therapy has a wide range of applications. According to the different applications of mRNA therapy, it can be mainly divided into the following directions: infectious disease vaccines, tumor immunotherapy, monoclonal antibody drugs and other protein drug replacements, and gene editing. The success of mRNA vaccines during the COVID-19 epidemic also confirms the great value of the mRNA platform. mRNA therapy usually delivers mRNA through lipid-based carrier systems (including liposomes and lipid nanoparticles). These lipid carriers usually encapsulate mRNA and can improve the intracellular delivery and effectiveness of mRNA. Lipid nanoparticle (LNP) formulations represent a revolution in the field of nucleic acid delivery. LNPs usually include one or more cationic lipids and / or amino (ionizable) lipids, phospholipids, structural lipids (such as cholesterol) and / or lipids containing polyethylene glycol (PEG lipids). Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated.

[0006] Although research related to LNP-mediated nucleic acid delivery has made great progress, almost all current delivery systems have strong hepatotropism. Due to the unclear structure-activity relationship of LNP-delivered lipids, it is difficult to rationally design highly organ- and tissue-specific delivery vectors, especially for different cell types of the same organ and tissue, resulting in mRNA-LNP being widely accumulated 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 in specific organs, tissues and / or cells and reduce side effects in other organs, tissues and / or cells is also a major challenge for this type of therapy.

[0007] miRNA (also known as microRNA) is a small single-stranded, 19-25 nucleotide long non-coding RNA molecule found in plants, animals and some viruses. Its main function is mRNA degradation or silencing and post-transcriptional regulation of gene expression. They complement specific sequences in the 3' untranslated region (3'UTR) of target mRNA molecules through Watson-Crick base pairing, thereby causing these mRNA molecules to be silenced. The expression of miRNA is highly specific in organs, tissues, and cells. Examples of tissue-specific expression of miRNA include liver (miRNA-122) and spleen (miRNA-142). The miRNA binding sequence is usually located in the 3'UTR of mRNA. The integration of miRNA binding sites into the 3'UTR to improve off-target expression has been documented in the art.

[0008] In the field of mRNA, the first step in the in vitro preparation of mRNA drugs is to synthesize through in vitro transcription (IVT) using a linearized plasmid containing a designed product sequence as a template, and the Poly(A) tail is usually added downstream of the 3'UTR in a co-transcriptional manner. In order to achieve co-transcriptional addition of Poly(A), it is necessary to include the corresponding poly(dA:dT) sequence in the template plasmid. However, the poly(dA:dT) repeat sequence in the plasmid is unstable during replication in Escherichia coli, and deletion mutations often occur, causing poly(dA:dT) to become shorter. This phenomenon is not conducive to the preparation process of in vitro transcription template plasmids produced on a large scale through fermentation, and Poly(A) truncation has a significant effect on the in vivo stability and biological activity of mRNA. Therefore, the plasmid replication stability of mRNA is an important consideration factor for mRNA drugs / vaccines.

[0009] The mRNA field is still in urgent need of developing mRNA vaccines or drugs that can be delivered to specific organs, tissues and / or cells with controllable quality and activity and are easy to produce industrially, as well as tools and methods for regulating the expression of the delivered mRNA in specific organs, tissues and / or cells.

[0010] Application Overview

[0011] There is no miRNA binding site in the polyA of natural mRNA. The inventors of the present application creatively integrated the miRNA binding site into the poly (A) tail, thereby enhancing the specificity of mRNA expression in specific organs, tissues and / or cells while taking into account the plasmid replication stability of mRNA, so that the quality of the produced mRNA can be controlled and the efficacy is stable.

[0012] Specifically, the present application provides:

[0013] 1. An engineered Poly(A) tail, wherein the Poly(A) tail comprises a miRNA binding site. The engineered Poly(A) tail may comprise one, two or more miRNA binding sites.

[0014] In some embodiments, the two or more miRNA binding site sequences are adjacent to each other or separated by one or more bases.

[0015] 2. An engineered Poly(A) tail according to Scheme 1, wherein the Poly(A) tail comprises the following formula (I):

[0016] nA-miRNA binding site-mA formula (I);

[0017] The miRNA binding site contains one or more miRNA binding sites.

[0018] nA represents n consecutive adenylate nucleotides (A) adjacent to the 5' end of the miRNA binding site,

[0019] mA represents the m consecutive adenylate nucleotides (A) adjacent to the 3' end of the miRNA binding site,

[0020] m and n are natural numbers, and 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, 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 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, 1 19, 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.

[0021] In some embodiments, one or more miRNA binding sites in the miRNA binding site are directly connected or connected through 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.

[0022] 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 one miRNA binding site. In some embodiments, the miRNA binding site in formula (I) comprises two or more miRNA binding sites that are adjacent to each other or connected by one or more nucleotides. In some embodiments, the miRNA binding site in formula (I) comprises three or more miRNA binding sites that are adjacent to each other or connected by one or more nucleotides. In some embodiments, there is only one formula (I) in the Poly (A) tail. In some embodiments, the Poly (A) tail is formula (I).

[0023] 3. An engineered Poly(A) tail according to Scheme 2, wherein n=0 or n≥1.

[0024] 4. Engineered Poly(A) tail according to Scheme 2, where m=0.

[0025] 5. The engineered Poly(A) tail of Scheme 2, wherein n≤80, n≤60, n≤30, n≤19, n≤14, or n≤10. In some embodiments, 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.

[0026] 6. An engineered Poly(A) tail according to any one of Schemes 1 to 5, wherein the length of the Poly(A) tail is 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 or 125 to 139 nt.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, 120nt, 121nt, 122nt, 123nt, 124nt, 125nt, 126nt, 127nt, 128nt, 129nt, 130nt, 131nt, 132nt, 133nt, 134nt, 135nt, 136nt, 137nt, 138nt, 139nt, 140nt, 141nt, 142nt 20nt, 121nt, 122nt, 123nt, 124nt, 125nt, 126nt, 127nt, 128nt, 129nt, 130nt, 131nt, 132nt, 133nt, 134nt, 135nt, 136nt, 137nt, 138nt, 139nt, 1 40nt, 141nt, 142nt, 143nt, 144nt, 145nt, 146nt, 147nt, 148nt, 149nt, 150nt, 151nt, 152nt, 153nt, 154nt, 155nt, 156nt, 157nt, 158nt, 159nt, 1 60nt, 161nt, 162nt, 163nt, 164nt, 165nt, 166nt, 167nt, 168nt, 169nt, 170nt, 171nt, 172nt, 173nt, 174nt, 175nt, 176nt, 177nt, 178nt, 179nt, 1 80nt,181nt,182nt,183nt,184nt,185nt,186nt,187nt,188nt,189nt,190nt,191nt,192nt,193nt,194nt,195nt,196nt,197nt,198nt,199nt,2 00nt, 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.

[0027] 7. An engineered Poly(A) tail according to any one of Schemes 1 to 6, wherein the Poly(A) tail:

[0028] The 3' side of formula (I) further comprises a tail segment directly connected to the 3' end of the structure of formula (I), and preferably the nucleotide at the 5' end of the tail segment is not A; and / or

[0029] The 5' side of formula (I) further comprises a head fragment directly connected to the 5' end of the structure of formula (I), and preferably the nucleotide at the 3' end of the head fragment is not A.

[0030] In some embodiments, the tail fragment or the head fragment consists of one or more non-A nucleotides and multiple A's, for example, 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.

[0031] In some embodiments, the number of nucleotides in the tail fragment or the head fragment is p, wherein 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, 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.

[0032] In some embodiments, the tail segment or the head segment consists of one or more elements c and / or one or more elements d and one or more A,

[0033] The element c is a non-A nucleotide; for example, T, U, C or G;

[0034] The element d is composed of any two or more consecutive nucleotides, and the nucleotides at the 5' and 3' ends of the element d are not A nucleotides;

[0035] The length of the element d is in the range of 2nt≤d≤30nt; preferably 6nt≤d≤20nt, more preferably 6nt≤d≤12nt;

[0036] The element c and the element d are not adjacent.

[0037] 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.

[0038] In some embodiments, the element d is composed of any two or more consecutive nucleotides, the element d does not contain more than three consecutive A's, and the nucleotides are selected from A, U, C, and G nucleotides, wherein the nucleotides at the 5' and 3' ends of the element d are not A nucleotides, and preferably the length range of the element d is 2nt≤d≤30nt, preferably 6nt≤d≤20nt, and more preferably 6nt≤d≤12nt.

[0039] In some embodiments, the number of the element c is 0, 1, 2 to 10, 3 to 8, or 4 to 6 or 2-5, for example 7. In some embodiments, the number of element c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0040] In some embodiments, the number of the elements d is 0-5, preferably 1-3, such as 2 or 4. In some embodiments, the number of the elements d is 0, 1, 2, 3, 4, or 5.

[0041] In some embodiments, when element c and element d are present at the same time, the sum of the number of elements c and element d is 2-15, preferably 3-5, and more preferably 3. In some embodiments, the sum of the number of elements c and element d is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0042] In some embodiments, the 1 / 2 portion of the Poly(A) tail close to the 3' end comprises one or more non-A nucleotides.

[0043] In some embodiments, the poly(A) tail comprises one or more non-A nucleotides in the 1 / 3 near the 3' end.

[0044] In some embodiments, the Poly(A) tail comprises one or more non-A nucleotides in the 1 / 4 near the 3' end.

[0045] According to the engineered Poly (A) tail described in any of the aforementioned schemes, it comprises 0 to 60, 0 to 30, 0 to 20, 0 to 14, or 0 to 10 nucleotides on the 5' side of the miRNA binding site, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides.

[0046] 8. An engineered Poly(A) tail according to any one of Schemes 1 to 7, wherein the engineered Poly(A) tail is formed by inserting the miRNA binding site into a Poly(A) tail structure as shown below:

[0047] Element a-element c-element b-element c-element b-element c-element b-element c-element b;

[0048] Element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b;

[0049] Element b-element c-element b-element c-element b-element d-element a-element c;

[0050] element a-element d-element b-element c-element b-element c-element b; or

[0051] Element b-element c-element b-element c-element b-element d-element a;

[0052] Component a - component d - component b;

[0053] Element a-element d-element a;

[0054] Element b - element d - element a;

[0055] Element b-element d-element b;

[0056] Element a - Element dA

[0057] Component a-component cA

[0058] Optionally:

[0059] 1) formed by replacing any 1, 2, 3, 4, 5 or more elements at the miRNA binding site;

[0060] 2) formed by inserting the miRNA binding site between any two elements; or

[0061] 3) The miRNA binding site is inserted between any two As in element a or element b.

[0062] In the poly (A) tail structure, the element a is composed of a plurality of consecutive adenine (A) nucleotides, and the length of the element a ranges from 100nt ≥ a > 30nt, preferably 80nt ≥ a ≥ 60nt;

[0063] The element b is composed of a plurality of consecutive A nucleotides, and the length range of the element b is 10nt≤b≤30nt, preferably 14nt≤b≤20nt;

[0064] The element c consists of a non-A nucleotide;

[0065] The element d is composed of any two or more consecutive nucleotides, the element d does not contain more than three consecutive A's, the nucleotides are selected from A, T, C, and G nucleotides, wherein the nucleotides at the 5' and 3' ends of the element d are not A nucleotides, and the length range of the element d is preferably 2nt≤d≤30nt, preferably 6nt≤d≤20nt, and more preferably 6nt≤d≤12nt.

[0066] In some embodiments, the 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 t, 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, 100nt.

[0067] In some embodiments, the 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.

[0068] In some embodiments, the 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.

[0069] 9. An engineered Poly (A) tail according to any one of Schemes 1 to 7, wherein the engineered Poly (A) tail is formed by inserting the miRNA binding site into the Poly (A) tail structure described in WO2020074642A1, WO2022028559A1, and US_10717982.

[0070] 10. An engineered Poly(A) tail according to any one of Schemes 1-9, wherein the element c is G.

[0071] 11. An engineered Poly(A) tail according to any one of Schemes 1-10, wherein the element d comprises a palindromic sequence.

[0072] 12. An engineered Poly(A) tail according to any one of Schemes 1-11, wherein the length of element d is 6 nt.

[0073] 13. An engineered Poly(A) tail according to any one of schemes 1-12, wherein the element d is selected from any one or more of the following sequences: GATATC (SEQ ID NO: 60), GTATAC (SEQ ID NO: 61), GAATCT (SEQ ID NO: 62), GCATATGACT (SEQ ID NO: 63) and GATATCGTATAC (SEQ ID NO: 64).

[0074] 14. An engineered Poly(A) tail according to any one of schemes 1-12, wherein the element d is represented by GATATC (SEQ ID NO: 60).

[0075] 15. An engineered Poly(A) tail according to any one of Schemes 1-14, wherein the ratio of the number of nucleotides contained in the Poly(A) tail on the 5' side of the miRNA binding site to the number of nucleotides contained 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.

[0076] 16. The engineered Poly (A) tail according to any one of schemes 6-15, wherein the structure of the tail fragment is: element d-element b-element c-element b-element c-element b or element c-element b-element c-element b, preferably the structure of the tail fragment is: element d-19A-element c-19A-element c-17A or element c-19A-element c-17A, and more preferably the structure of the tail fragment is: GATATC-19A-G-19A-G-17A. In some embodiments, the engineered Poly (A) tail does not contain a head fragment. In some embodiments, the engineered Poly (A) tail consists of a tail fragment and formula (I). In some embodiments, the engineered Poly (A) tail consists of a head fragment, a tail fragment and formula (I).

[0077] In the engineered Poly (A) tail according to any one of the above 1-15, its structure is selected from any one of the following:

[0078] 1) nA-miRNA binding site-mA, wherein 150≥m+n≥50, preferably, m+n=120 or m+n=60;

[0079] 2) nA-miRNA binding site-mA-element d-element b-element c-element b-element c-element b, wherein 80≥m+n≥60, preferably, m+n=60;

[0080] 3) element a-element d-nA-miRNA binding site-mA-element c-element b-element c-element b, wherein 30≥m+n≥10, preferably, m+n=19;

[0081] 4) nA-miRNA binding site-mA-element c-element b-element c-element b, wherein 110≥m+n≥70, preferably, m+n=79;

[0082] 5) 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, wherein 19≥m+n≥14, preferably, m+n=19.

[0083] In the engineered Poly (A) tail according to any one of 1-15 above, the structure is selected as:

[0084] nA-miRNA binding site-mA-element d-element b-element c-element b-element c-element b, wherein 80≥m+n≥60, preferably, m+n=60; further preferably, 30≥n≥14; more preferably, 19≥n≥14.

[0085] In the engineered Poly (A) tail according to any one of the above 1-15, its structure is selected from any one of the following:

[0086] 1) nA-miRNA binding site-mA, where m+n=120 or m+n=100 or m+n=60;

[0087] 2) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, m+n=60;

[0088] 3) 60A-element d-nA-miRNA binding site-mA-G-19A-G-17A, m+n=19;

[0089] 4) nA-miRNA binding site-mA-G-19A-G-17A, m+n=79;

[0090] 5) 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, wherein m+n=19.

[0091] In the engineered Poly (A) tail according to any one of 1-15 above, its structure is:

[0092] nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, m+n=60 and 30≥n≥14; preferably, 19≥n≥14.

[0093] In the engineered Poly (A) tail according to any one of the above 1-15, its structure is selected from any one of the following:

[0094] 1) nA-miRNA binding site-mA, where m+n=120 or m+n=100;

[0095] 2) nA-miRNA binding site-mA-SEQ ID NO: 60-19A-G-19A-G-17A, m+n=60;

[0096] 3) 60A-SEQ ID NO: 60-nA-miRNA binding site-mA-G-19A-G-17A, m+n=19;

[0097] 4) nA-miRNA binding site-mA-G-19A-G-17A, m+n=79;

[0098] 5) nA-miRNA binding site-mA-G-19A-G-19A-SEQ ID NO: 60-60A or 19A-G-nA-miRNA binding site-mA-G-19A-element d-60A, wherein m+n=19.

[0099] In the engineered Poly (A) tail according to any one of 1-15 above, its structure is:

[0100] nA-miRNA binding site-mA-SEQ ID NO:60-19A-G-19A-G-17A, m+n=60 and 30≥n≥14, preferably, 19≥n≥14.

[0101] 17. An engineered Poly(A) tail according to any one of schemes 1-16, wherein the miRNA bound to the miRNA binding site is selected from one or more of the following:

[0102] 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.

[0103] In some embodiments, the miRNA is selected from one or more of miR-142, miR-122, miR-126, and miR-148a.

[0104] In some embodiments, the miRNA is selected from one or more of miR-142-3p, miR-122-5p, miR-126-3p and miR-148a-3p.

[0105] In some embodiments, the miRNA comprises 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.

[0106] In some embodiments, the miRNA comprises 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.

[0107] In some embodiments, the miRNA comprises or consists of: miR-142, miR-122, miR-126, and miR-148a.

[0108] In some embodiments, the miRNA includes miR142 and miR-122 or consists of miR142 and miR-122, and in the miRNA binding site of the Poly(A), the miR142 binding site is located on the 5' side of the miR-122 binding site or the miR142 binding site is located on the 3' side of the miR-122 binding site, and the miR142 binding site and the miR-122 binding site are adjacent to each other.

[0109] In some embodiments, the miRNA includes miR142 and miR-122, and in the binding site of the Poly(A), the miR142 binding site is located on the 5' side of the miR-122 binding site or the miR142 site is located on the 3' side of the miR-122 site, and the miR142 binding site is separated from the miR-122 binding site by one or more nucleotides.

[0110] In some embodiments, the miRNA includes miR142 and miR-126 or consists of miR142 and miR-126, and in the binding site of the Poly (A), the miR142 binding site is located on the 5' side of the miR-126 binding site or the miR142 site is located on the 3' side of the miR-126 site, and the miR142 binding site and the miR-126 binding site are adjacent to each other.

[0111] In some embodiments, the miRNA includes miR142 and miR-126, and in the binding site of the Poly (A), the miR142 binding site is located on the 5' side of the miR-126 binding site or the miR142 site is located on the 3' side of the miR-126 site, and the miR142 binding site is separated from the miR-126 binding site by one or more nucleotides.

[0112] In some embodiments, the miRNA includes or consists of miR-142 and miR-148a, and in the binding site of the Poly (A), the miR-142 binding site is located on the 5' side of the miR-148a binding site or the miR-142 binding site is located on the 3' side of the miR-148a binding site, and the miR-142 binding site and the miR-148a binding site are adjacent to each other.

[0113] In some embodiments, the miRNA includes miR-142 and miR-148a, and in the binding site of the Poly (A), the miR-142 binding site is located on the 5' side of the miR-148a binding site or the miR-142 binding site is located on the 3' side of the miR-148a binding site, and the miR-142 binding site is separated from the miR-148a binding site by one or more nucleotides.

[0114] In some embodiments, the miRNA includes or consists of miR-122 and miR-126, and in the binding site of the Poly(A), the miR-122 binding site is located on the 5' side of the miR-miR-126 binding site or the miR-122 binding site is located on the 3' side of the miR-126 binding site, and the miR-122 binding site and the miR-126 binding site are adjacent to each other.

[0115] In some embodiments, the miRNA includes miR-122 and miR-126, and in the binding site of the Poly(A), the miR-122 binding site is located on the 5' side of the miR-126 binding site or the miR-122 binding site is located on the 3' side of the miR-126 binding site, and the miR-122 binding site is separated from the miR-126 binding site by one or more nucleotides.

[0116] In some embodiments, the miRNA includes or consists of miR-122 and miR-148a, and in the binding site of the Poly(A), the miR-122 binding site is located on the 5' side of the miR-148a binding site or the miR-122 binding site is located on the 3' side of the miR-148a binding site, and the miR-122 binding site and the miR-148a binding site are adjacent to each other.

[0117] In some embodiments, the miRNA includes miR-122 and miR-148a, and in the binding site of the Poly(A), the miR-122 binding site is located on the 5' side of the miR-148a binding site or the miR-122 binding site is located on the 3' side of the miR-148a binding site, and the miR-122 binding site is separated from the miR-148a binding site by one or more nucleotides.

[0118] In some embodiments, the miRNA includes or consists of miR-126 and miR-148a, and in the binding site of the Poly (A), the miR-126 binding site is located on the 5' side of the miR-148a binding site or the miR-126 binding site is located on the 3' side of the miR-148a binding site, and the miR-126 binding site and the miR-148a binding site are adjacent to each other.

[0119] In some embodiments, the miRNA includes miR-126 and miR-148a, and in the binding site of the Poly (A), the miR-126 binding site is located on the 5' side of the miR-148a binding site or the miR-126 binding site is located on the 3' side of the miR-148a binding site, and the miR-126 binding site is separated from the miR-148a binding site by one or more nucleotides.

[0120] In some embodiments, the miRNA includes miRNA-142, miR-148a and miR-126, and in the miRNA binding site of the Poly(A), the order of arrangement of each miRNA binding site from 5' to 3' is:

[0121] miR-126 binding site, miR-148a binding site, and miR-142 binding site;

[0122] miR-148a binding site, miR-142 binding site, and miR-126 binding site;

[0123] miR-142 binding site, miR-148a binding site, and miR-126 binding site;

[0124] miR-126 binding site, miR-142 binding site, and miR-148a binding site;

[0125] miR-148a binding site, miR-126 binding site and miR-142 binding site; or

[0126] miR-142 binding site, miR-126 binding site, and miR-148a binding site;

[0127] The miR-126 binding site, miR-148a binding site and miR-142 binding site are adjacent to each other.

[0128] In some embodiments, the miRNA includes miRNA-142, miR-148a and miR-126, and in the miRNA binding site of the Poly(A), the arrangement order of each miRNA binding site from 5' to 3' is:

[0129] miR-126 binding site, miR-148a binding site, and miR-142 binding site;

[0130] miR-148a binding site, miR-142 binding site, and miR-126 binding site;

[0131] miR-142 binding site, miR-148a binding site, and miR-126 binding site;

[0132] miR-126 binding site, miR-142 binding site, and miR-148a binding site;

[0133] miR-148a binding site, miR-126 binding site and miR-142 binding site; or

[0134] miR-142 binding site, miR-126 binding site, and miR-148a binding site;

[0135] The miR-126 binding site, miR-148a binding site and miR-142 binding site are separated from each other by one or more nucleotides.

[0136] In some embodiments, the miRNA includes miRNA-142, miR-148a and miR-126, and in the miRNA binding site of the Poly(A), the arrangement order of each miRNA binding site from 5' to 3' is:

[0137] miR-126 binding site, miR-148a binding site, and miR-142 binding site;

[0138] miR-148a binding site, miR-142 binding site, and miR-126 binding site;

[0139] miR-142 binding site, miR-148a binding site, and miR-126 binding site;

[0140] miR-126 binding site, miR-142 binding site, and miR-148a binding site;

[0141] miR-148a binding site, miR-126 binding site and miR-142 binding site; or

[0142] miR-142 binding site, miR-126 binding site, and miR-148a binding site;

[0143] Among the miR-126 binding site, miR-148a binding site and miR-142 binding site, two adjacent miRNA binding sites are adjacent to each other, and the other two adjacent miRNA binding sites are separated by one or more nucleotides.

[0144] In the engineered Poly(A) tail according to any of the foregoing items, the structure thereof is selected from any of the following: 1) nA-miRNA binding site-mA, wherein n=0, m=120 or m=100 or m=60;

[0145] 2) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, wherein m+n=60, n≤20 or n≤14 or n≤10;

[0146] 3) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, where n=0, m=60;

[0147] 4) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, wherein m+n=60, 14≤n≤30; preferably 14≤n≤19;

[0148] 5) nA-miRNA binding site-mA-G-19A-G-17A, where n=60, m=19;

[0149] Preferably, the element d is a palindromic sequence, and more preferably, the polynucleotide sequence of the element d is shown in any one of SEQ ID NOs: 60 to 64.

[0150] In the engineered Poly(A) tail according to any of the preceding items, the structure is selected from any of the following:

[0151] 1) nA-miRNA binding site-mA, wherein n=0, m=120 or m=100 or m=60, and the miRNA binding site is the binding site of miR-122, miR-126 or miR-142;

[0152] 2) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, wherein n=14, m=46, and the miRNA binding site is the binding site of miR-122, miR-126 or miR-142;

[0153] 3) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, wherein n=19, m=41, and the miRNA binding site is the binding site of miR-122, miR-126 or miR-142;

[0154] 4) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, wherein n=30, m=30, and the miRNA binding site is the binding site of miR-122, miR-126 or miR-142;

[0155] 5) nA-miRNA binding site-mA-G-19A-G-17A, wherein n=60, m=19, and the miRNA binding site is the binding site of miR-122, miR-126 or miR142;

[0156] 6) nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, wherein n=0, m=60, and the miRNA binding site is the binding site of 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; or

[0157] 7) nA-miR-122 binding site-mA-element d-19A-G-19A-G-17A, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n+m=60,

[0158] 8) nA-miR-142 binding site-mA-element d-19A-G-19A-G-17A, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n+m=60,

[0159] 9) nA-miR-126 binding site-mA-element d-19A-G-19A-G-17A, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n+m=60,

[0160] 10) nA-miR-148a binding site-mA-element d-19A-G-19A-G-17A, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n+m=60,

[0161] Preferably, the element d is a palindromic sequence, and more preferably, the polynucleotide sequence of the element d is as shown in any one of SEQ ID NOs: 60 to 64, preferably SEQ ID NO: 60.

[0162] In some embodiments, the miRNA binding site of the engineered Poly (A) tail consists of one, two, three, four or more miRNA binding site sequences selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 32 or SEQ ID NO: 53. In some embodiments, the miRNA binding site of the engineered Poly (A) tail consists of one, two, three, four or more miRNA binding site sequences selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 32 or SEQ ID NO: 53 and 0, 1 or more nucleotides between the miRNA binding sites.

[0163] 18. An engineered Poly(A) tail according to any one of Schemes 1-17, wherein the sequence of the Poly(A) tail comprises a polynucleotide sequence selected from the following or is any one of the following polynucleotide sequences: SEQ ID NO: 6-16, SEQ ID NO: 19-31, SEQ ID NO: 33-52, SEQ ID NO: 54-59, SEQ ID NO: 65-71.

[0164] 19. An engineered RNA molecule, which is an mRNA or a non-coding RNA, comprising a Poly(A) tail according to any one of Schemes 1 to 18 and a target gene sequence located on the 5' side of the Poly(A) tail. In some embodiments, nA in formula (I) in the Poly(A) tail is directly connected to the 3'UTR.

[0165] 20. An engineered DNA molecule encoding an engineered Poly(A) tail according to any one of Schemes 1-18, or an engineered RNA sequence according to Scheme 19.

[0166] 21. According to the engineered DNA molecule described in Scheme 20, the DNA molecule is a plasmid or a viral vector.

[0167] 22. A hybrid molecule of DNA and RNA carrying the same genetic information as the engineered Poly(A) tail according to any one of Schemes 1-18, the engineered RNA molecule according to Scheme 19, or the engineered DNA molecule according to Scheme 20 or 21.

[0168] 23. An engineered cell comprising an engineered Poly(A) tail according to any one of Schemes 1-18, an engineered RNA molecule according to Scheme 19, an engineered DNA molecule according to Scheme 20 or 21, or a hybrid molecule according to Scheme 22, wherein the cell is a eukaryotic or prokaryotic cell.

[0169] 24. An engineered cell according to Scheme 23, wherein the prokaryotic cell is Escherichia coli.

[0170] 25. Use of the engineered Poly(A) tail according to any one of schemes 1-18 to reduce 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 or

[0171] The engineered Poly (A) tail according to any one of Schemes 1-18 is used to specifically express a target gene in a specific organ, tissue and / or cell. Preferably, the organ or tissue is preferably liver or spleen organ tissue; more preferably, the cell is a hepatocyte.

[0172] 26. Use of the engineered DNA molecule according to Scheme 20 or 21, or the hybrid molecule according to Scheme 22, to make the replication of the DNA coding sequence encoding the Poly(A) tail in a host cell more conservative.

[0173] 27. According to the use of scheme 26, the host cell is a prokaryotic cell, preferably Escherichia coli.

[0174] 28. A lipid nanoparticle comprising an engineered Poly(A) tail according to any one of Schemes 1-18, an engineered RNA molecule according to Scheme 19, an engineered DNA molecule according to Scheme 20 or 21, or a hybrid molecule according to Scheme 22.

[0175] 29. A viral particle comprising an engineered Poly(A) tail according to any one of Schemes 1-18, an engineered RNA molecule according to Scheme 19, an engineered DNA molecule according to Scheme 20 or 21, or a hybrid molecule according to Scheme 22.

[0176] Those skilled in the art should be aware that the 5' side of the 5' terminal nucleotide at the miRNA binding site may further include 0, 1 or more non-A nucleotides, and this technical solution should also fall within the scope of this application, or be an equivalent solution to the solution of this application.

[0177] It should be understood that the aspects and embodiments of the present application described herein include aspects and embodiments of "comprising", "consisting of" and "consisting essentially of...". The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, a variety of simple modifications can be made to the technical solution of the present application, including combining various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed in the present application, and all belong to the protection scope of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0178] Figure 1A-1B The figure shows the composition diagram of 5 kinds of Poly(A) containing miR-142 / miR-122 binding sites designed independently by the present invention;

[0179] Figure 2A-2B The absolute numerical results of fluorescence imaging of spleen / liver of mice after expressing luciferase mRNA containing five poly(A)s independently designed by the present invention and containing miR-142 binding sites in mice are shown.

[0180] Figure 3A-3B The absolute numerical results of fluorescence imaging of liver / spleen obtained by dissecting mice after expressing luciferase mRNA containing five poly(A)s containing miR-122 binding sites independently designed by the present invention in mice are shown.

[0181] Figure 4 The base deletion statistics of luciferase DNA plasmids with different poly(A) containing miRNA binding sites miR-142 / miR-122 independently designed by the present invention in Escherichia coli DH5α are shown.

[0182] Figure 5 The absolute numerical results of the enzyme activity detection of luciferase mRNA containing 8 poly(A)s independently designed by the present invention and containing miR-142 binding sites in the mammalian cell line Raw 264.7 are shown.

[0183] Figure 6 The absolute numerical results of the enzyme activity detection of luciferase mRNA with miR-142 binding site and miR-122 binding site poly (A) in the mammalian cell line Raw 264.7 are shown.

[0184] Figures 7A-7D The absolute numerical results of the enzyme activity detection of luciferase mRNA with the independently designed poly (A) variant of the present invention in the mammalian cell line Raw 264.7 / LSEC / HSC / AML are shown.

[0185] Figures 8A-8C The graph shows the expression ratio of luciferase mRNA with the independently designed poly(A) variant of the present invention in Raw 264.7 / LSEC / HSC relative to the reduced expression in hepatocyte AML. DETAILED DESCRIPTION OF THE INVENTION

[0187] The present application first provides a tool for achieving specific expression in specific organs, tissues and / or cells, namely, by integrating miRNA binding sites into the Poly (A) tail to specifically shut down mRNA expression in organs, tissues and / or cells where mRNA expression is not desired, so as to reduce off-target toxic side effects of mRNA systemic expression.

[0188] The present application also provides a method for stably amplifying a poly (A) tail transcription template DNA in vitro to reduce the mutation frequency of the poly (A) tail transcription template sequence when the DNA is replicated in large quantities in cells. Thus, a large number of RNAs containing poly (A) tails with determined sequences are obtained based on the DNA. On this basis, RNAs, such as mRNAs, with poly (A) tails that have been engineered to have certain specific functions can be produced on a large scale through in vitro fermentation.

[0189] the term

[0190] In the present application, "element c", "element d", nA, mA, tail fragment, head fragment, miRNA binding site and miRNA binding site in the poly (A) tail can all be referred to by the term "element". Among them, "miRNA binding site", "nA", "mA", "element c" and "element d" do not overlap or contain each other, and the method of determining "miRNA binding site", "nA", "mA", "element c" and "element d" can be:

[0191] First, the miRNA binding site is determined in the Poly(A) tail by sequence alignment, and then the A closest to the 5' side of the miRNA binding site closest to the 5' end of the Poly(A) tail among all miRNA binding sites is used as the 3' end of nA, thereby determining that the continuous n A is nA; and the A closest to the 3' side of the miRNA binding site closest to the 3' end of the Poly(A) tail among all miRNA binding sites is used as the 5' end of mA, thereby determining that the continuous m A is mA; the part 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 a head fragment; if the Poly(A) tail has a polynucleotide or polynucleotide sequence on the 3' side of mA, the polynucleotide or polynucleotide sequence is a tail fragment. The method of determining "element d" and "element c" is to first determine element d from the target sequence, wherein element d is a segment of the target sequence containing non-A bases, which is composed of any two or more consecutive nucleotides, the nucleotides at the 5' and 3' ends of element d are not A nucleotides, element d does not contain more than three consecutive A's, and the 5' and 3' ends of element d are both adjacent to at least 2 A's. After element d is determined, all non-A bases in the part other than element d in the target sequence are determined to be "element c". In some embodiments, the Poly (A) tail comprises a plurality of miRNA binding sites, and the miRNA binding sites in each binding site are directly connected or connected through non-A bases. In some embodiments, the Poly (A) tail comprises a plurality of miRNA binding sites, and the miRNA binding sites in each binding site are directly connected or connected through a nucleic acid sequence, and the nucleic acid sequence does not contain more than three consecutive A's.

[0192] As used herein, "miRNA", i.e., microRNA or microRNA, is a type of short non-coding RNA, usually less than 22 bp in length. It exerts a post-transcriptional regulatory effect by binding to a target sequence (i.e., miRNA binding site) on mRNA. As used herein, preferably, "miRNA binding site" is a miRNA binding site that downregulates mRNA expression after binding to miRNA.

[0193] In this article, "coding" refers to i) genetic information contained in a DNA sequence that can be transcribed into an RNA molecule, and / or ii) genetic information contained in an RNA molecule that can be translated into an amino acid sequence. Therefore, as used herein, "coding sequence" can be used to refer to a ribonucleotide (RNA) sequence or a fragment thereof that can be translated into a protein in an mRNA precursor or mature mRNA, and can also refer to a complementary sequence or a fragment thereof of a deoxyribonucleotide (DNA) sequence that is used as a template to transcribe the mRNA precursor or mature mRNA. In addition, the "coding sequence" of the present 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, 3'UTR, etc. Among them, a DNA molecule containing genetic information that can be transcribed into an RNA molecule is called the "coding nucleic acid" of the RNA molecule; an RNA molecule containing genetic information that can be translated into an amino acid sequence is called the "coding nucleic acid" of the amino acid sequence.

[0194] In the present application, the nucleotides in all polynucleotide sequences are numbered from the 5' end to the 3' end, that is, the nucleotide at the 5' end is the first nucleotide, and the nucleotide at the 3' end is the last nucleotide. Unless otherwise specified, "5' end" and "5' end" can be used interchangeably, and "3' end" and "3' end" can be used interchangeably. In particular, "5' end" and "3' end" can be used to describe the positions of the first and last nucleotides of a nucleic acid sequence or a segment of a nucleic acid sequence, respectively. In addition, "5'" and "3'" are particularly used to describe the relative positional relationship between nucleotides, between nucleotide sequence segments, or between nucleotides and nucleotide sequence segments in the same nucleic acid sequence; for example, "5' side" is used to describe the relative positional relationship between two sequences with no overlapping parts in the same polynucleotide sequence. When describing a sequence segment being located on the 5' side of another sequence segment, it means that the sequence segment is closer to the "5' end" of the polynucleotide sequence relative to the other sequence segment. Similarly, when describing a sequence as being located on the 3' side of another sequence, it means that one sequence is closer to the "3' end" of the polynucleotide sequence relative to the other sequence, and the one sequence and the other sequence do not contain any overlapping parts. In addition, as used herein, the "5' part" refers to the half of the polynucleotide sequence that is bounded by the "center position" of the polynucleotide sequence and is close to the 5' end. The "3' part" refers to the half of the polynucleotide sequence that is bounded by the center position of the polynucleotide sequence and is close to the 3' end. The number of nucleotides from the "center position" to the 5' end and to the 3' end described in this application is equal.

[0195] As used herein, "adjacent" means that there is no nucleotide or base inserted between two elements of a nucleic acid molecule (e.g., a Poly (A) tail) (unless otherwise specified, the term "nucleotide" and the term "base" are used interchangeably in this application), that is, the first nucleotide on the 3' side of the 3' end of the polynucleotide sequence of one of the two elements is the 5' terminal nucleotide of the other element. For example, "n consecutive A's adjacent to the 5' end of the miRNA binding site" means that the consecutive n A's are located on the 5' side of the miRNA binding site, and no other nucleotides or bases are inserted between the 5' terminal nucleotide of the miRNA binding site and the n A's. For example, "adjacent to the 3' end of the miRNA binding site" means that the consecutive n A's are located on the 3' side of the miRNA binding site, and no other nucleotides or bases are inserted between the 3' terminal nucleotide of the miRNA binding site and the n A's. In addition, "adjacent" can also be used to describe the positional relationship between multiple elements, to indicate that each of the multiple elements is adjacent to any other element of the multiple elements, and no other elements or nucleotides other than the multiple elements are included between any two elements of the multiple elements.

[0196] In this application, when describing the positional relationship of two or more elements as "non-adjacent", it means that the two or more elements are not adjacent to each other. In other words, the two or more elements contain at least one or more other nucleotides or bases other than the nucleotides of the two elements.

[0197] As used herein, when the term "conservative" is used to describe the replication of a nucleic acid molecule, it means that the probability of mutations occurring during the replication process is low. In this context, "conservative" is a relative concept. For example, when describing that "a DNA coding sequence of a poly (A) tail is used to make the replication of a DNA molecule encoding RNA more conservative in a host cell", it means that after a parent DNA molecule encoding RNA is replicated into a daughter DNA molecule, if the RNA molecule comprises the poly (A) tail, then relative to the coding DNA of an RNA molecule that does not comprise the poly (A) tail (e.g., an RNA molecule comprising some other poly (A) tail), the probability that the daughter DNA molecule has 100% sequence identity with the parent DNA molecule is higher; or the average sequence identity between the multiple daughter DNA molecules obtained by replication of the parent DNA molecule and the parent DNA molecule is higher.

[0198] In the present application, when describing "regulating" the expression of RNA molecules, the "regulation" refers to: increasing or decreasing the total amount of protein or functional RNA expressed by the RNA molecule within the same time period; or allowing the RNA to express protein or functional RNA within a longer or shorter time frame, and the increase, decrease, or longer or shorter time frame is relative to another RNA molecule expressing the same protein or functional RNA. When describing "regulating" protein expression, it refers to regulating the expression of RNA molecules containing the protein coding sequence. The regulatory effect described here can be achieved by connecting the Poly (A) tail of the present application to the 3' end of an RNA molecule that does not contain a Poly (A) tail, or by replacing the original Poly (A) tail of the RNA with the Poly (A) tail of the present application.

[0199] As used herein, the percentage of "identity", such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to the degree of similarity between amino acid sequences or between nucleotide sequences determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, the proportion of the number of positions with the same base or amino acid residue to the total number of positions determined after two sequences have the same residue at as many positions as possible by introducing spaces, etc. The percentage of "identity" can be determined using software programs known in the art. Preferably, the comparison is performed using default parameters. A preferred comparison 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.

[0200] As used herein, the "complementarity" of nucleic acids refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, 10 out of 10 are about 50%, 60%, 70%, 80%, 90% and 100% complementary, respectively). "Complete complementarity" refers to the formation of hydrogen bonds between all consecutive residues of a nucleic acid sequence and the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% in a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. For a single base or a single nucleotide, according to the Watson-Crick base pairing principle, when A is paired with T or U, C is paired with G or I, it is called complementary or matching, and vice versa; and other base pairings are called non-complementary. In this application, the "complementary polynucleotide sequence" of a polynucleotide sequence refers to a polynucleotide sequence that is completely complementary to the polynucleotide sequence.

[0201] As used herein, a "conservative substitution variant" of a protein, polypeptide or amino acid sequence refers to one in which one or more amino acid residues undergo amino acid substitution without changing the overall conformation and function of the protein or enzyme, including but not limited to replacing the amino acids in the amino acid sequence of the parent protein in the manner described by the aforementioned "conservative substitution". Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes with more than 60% amino acid identity as determined by the BLAST or FASTA algorithm, preferably more than 75%, preferably more than 85%, and even more than 90% as the best, and have the same or substantially similar properties or functions compared to the native or parent protein or enzyme.

[0202] In the context of the present application, the terms "DNA" and "RNA" refer to single-stranded or double-stranded DNA or RNA molecules. Unless otherwise indicated, the terms "DNA" and "DNA molecule" refer to double-stranded DNA molecules consisting of A, C, G and / or T nucleotides, while the terms "RNA" and "RNA molecule" refer to single-stranded RNA molecules consisting of A, C, G and / or U nucleotides. In this article, the A, C, G, T and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine and uracil as their respective nitrogenous bases.

[0203] RNA molecules include coding RNA or non-coding RNA (ncRNA), such as Pre-mRNA, mature mRNA or long noncoding RNA (lncRNA).

[0204] As used herein, the "DNA and RNA hybrid molecule" is a molecule comprising a polynucleotide sequence consisting of deoxyribonucleotides and ribonucleotides. The DNA and RNA hybrid molecule can be obtained by:

[0205] Replace one or more deoxyribonucleotides in DNA with ribonucleotides;

[0206] Substituting one or more ribonucleotides in the RNA with deoxyribonucleotides; or

[0207] De novo synthesis using deoxyribonucleotides and ribonucleotides as raw materials by biological or chemical synthesis. It should be noted that the method of obtaining a hybrid molecule of DNA and RNA is not limited to the above method, and the hybrid molecule of DNA and RNA obtained by any method belongs to the category of "hybrid molecule of DNA and RNA" defined in this application.

[0208] As used herein, if two nucleic acid molecules are described as having "the same genetic information", it means that the two nucleic acid molecules are complementary, or contain exactly the same base sequence, or one of the nucleic acid molecules differs from the other nucleic acid molecule only in that one or more bases in its base sequence are replaced by another base having the same biological function. The same biological function means that the base and the other base can undergo traditional Watson-Crick base pairing with the same base, for example, thymine (T) and uracil (U) can both undergo Watson-Crick base pairing with adenine, hypoxanthine (I) and cytosine (C) can both undergo Watson-Crick base pairing with guanine (G), etc. Therefore, any two of DNA, RNA, and hybrid molecules of DNA and RNA can have the same genetic information. Among them, the term "base sequence" refers to the order of arrangement of bases in a polynucleotide molecule. Those skilled in the art will appreciate that, unless otherwise indicated, the base sequences or polynucleotide sequences described in this application may be used to describe DNA sequences, with "T" representing thymine, but when the base sequences or polynucleotide sequences are used to describe RNA (e.g., mRNA), "T" will be replaced by "U" (uracil). Therefore, any DNA disclosed by a specific sequence number (SEQ ID NO) herein also discloses an RNA (e.g., mRNA or Poly (A) tail) sequence that is complementary or corresponding to the DNA, wherein each "T" in the DNA sequence is replaced by "U".

[0209] As used herein, "a hybrid molecule of DNA and RNA" refers to a nucleic acid molecule that contains both deoxyribonucleotides and ribonucleotides. The "hybrid molecule of DNA and RNA" may be a DNA molecule in which one or more deoxyribonucleotides are replaced by ribonucleotides, or one or more ribonucleotides in an RNA molecule are replaced by deoxyribonucleotides.

[0210] Poly(A) tail and its uses

[0211] Provided herein is an engineered Poly (A) tail comprising one, two, three or more miRNA binding sites. Preferably, the engineered Poly (A) tail of the present invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNA binding sites. The one, two or more miRNA binding sites can be combined with the same or different miRNAs. In some embodiments, the Poly (A) tail provided herein is obtained by inserting one, two, three or more miRNA binding sites into any Poly (A) tail that does not contain miRNA binding sites. In some embodiments, the Poly (A) tail provided herein is obtained by inserting one or more of the miRNA binding sites into any Poly (A) tail that does not contain miRNA binding sites. In some embodiments, the Poly (A) tail provided herein is obtained by replacing one or more non-A bases in any Poly (A) tail that does not contain miRNA binding sites or one or more elements that are structurally equivalent to element d with one or more miRNA binding sites. In some embodiments, the Poly (A) tail provided in the present application is obtained by replacing one or more non-A bases in any Poly (A) tail that does not contain a miRNA binding site or one or more elements that are structurally equivalent to element d with a miRNA binding site. The "element that is structurally equivalent to element d" is a poly (A) tail that does not contain a miRNA binding site, consisting of any two or more consecutive nucleotides, the nucleotides at the 5' and 3' ends are not A, and does not contain a polynucleotide fragment of more than three consecutive A, such as element d in PCT application PCT / CN2023 / 079037. The "Poly (A) tail without a miRNA binding site" can be selected from any Poly (A) tail described in PCT / CN2023 / 079037, WO2022028559A1, WO2020 / 074642, US10717982B2, and any Poly (A) tail containing only A.

[0212] As used herein, the term "Poly A tail" or "Poly A sequence" refers to an uninterrupted or uninterrupted sequence of adenylic acid residues that is typically located at the 3'-end of an RNA molecule. In RNA, in the presence of a 3'-UTR, the Poly-A sequence is connected to the 3' end of the 3'-UTR. An uninterrupted poly-A tail is characterized by continuous adenylic acid residues. The Poly-A tail can be of any length. In some embodiments, the Poly-A tail comprises, or consists of at least 20, at least 30, at least 40, at least 80 or at least 100 and at most 500, at most 400, at most 300, at most 200 or at most 150 adenylic acid (A), particularly about 120 A. Typically, the vast majority of nucleotides in the PolyA tail are adenosine, where the vast majority refers to at least 75%, at least 80%, at least 85%, at least 90% of the nucleotides, etc., but the remaining nucleotides are allowed to be nucleotides other than A (non-A nucleotides), such as U (uridylic acid), G (guanylic acid) or C (cytidylic acid).

[0213] In some embodiments, the in vitro preparation process of the RNA is a prokaryotic fermentation process, that is, the coding nucleic acid of the RNA molecule containing the Poly (A) tail is introduced into a prokaryotic cell, and the prokaryotic cell is amplified to achieve the purpose of amplifying the coding nucleic acid, and then the amplified coding nucleic acid is transcribed into the RNA. In some embodiments, the in vitro preparation process of the RNA is to connect the RNA fragment containing the protein coding sequence with the Poly (A) tail by homologous recombination, enzyme ligation, or other non-homologous recombination, and the Poly (A) tail is prepared by a prokaryotic fermentation process, and the coding nucleic acid containing the Poly (A) tail is introduced into a prokaryotic cell during the prokaryotic fermentation process, and the prokaryotic cell is amplified to achieve the purpose of amplifying the coding nucleic acid, and then the amplified coding nucleic acid is transcribed into RNA containing the Poly (A) tail. In some embodiments, the aforementioned coding nucleic acid is linear. In some embodiments, the aforementioned coding nucleic acid is circular. In some embodiments, the aforementioned coding nucleic acid is a plasmid. In some embodiments, the aforementioned coding nucleic acid is single-stranded or double-stranded. In some embodiments, the aforementioned coding nucleic acid is chemically modified before being introduced into a prokaryotic cell. In some embodiments, the aforementioned encoding nucleic acid before being introduced into the prokaryotic cell is chemically synthesized. In some embodiments, the encoding nucleic acid is inserted into the nucleoid / nucleoid genomic DNA of the prokaryotic cell. In some embodiments, the encoding nucleic acid is free in the cytoplasm of the prokaryotic cell or outside the nucleoid / nucleoid. In some embodiments, the prokaryotic cell is Escherichia coli.

[0214] Based on this, the present application provides a series of engineered Poly (A) tails, which include miRNA binding site sequences. In some embodiments, the bases of the engineered Poly (A) tail except the miRNA binding site are all A. In some embodiments, the engineered Poly (A) tail further has a high degree of conservation during the in vitro preparation of RNA. In some embodiments, the engineered Poly (A) tail can improve the stability of mRNA. In some embodiments, the engineered Poly (A) tail is formed by inserting the miRNA binding site between any two nucleotides of the conserved Poly (A) tail. In some embodiments, the engineered Poly (A) tail is formed by the miRNA binding site being directly connected (adjacent) to the 5' terminal nucleotide of the conserved Poly (A) tail on the 5' side of the conserved Poly (A) tail. In some embodiments, the engineered Poly (A) tail is formed by the miRNA binding site being directly connected (adjacent) to the 3' terminal nucleotide of the conserved Poly (A) tail on the 3' side of the conserved Poly (A) tail. In some embodiments, the engineered Poly(A) tail is formed by replacing any one or more elements of the conserved Poly(A) tail with one or more miRNA binding sites.

[0215] As used herein, the term "conservative Poly (A) tail" is a type of Poly (A) tail containing at least one non-A base, which is highly conserved during the in vitro preparation of RNA and / or can improve the stability of mRNA compared to a Poly (A) tail in which all bases are A. Conservative Poly (A) can be a natural Poly (A) tail or an artificially synthesized Poly (A) tail, including but not limited to various Poly (A) tails described in PCT application PCT / CN2023 / 079037, Chinese patent application CN112805386A, and U.S. Patent US10717982B2. The above-mentioned application or patent text is hereby incorporated herein in its entirety.

[0216] As used herein, the two elements connected by "-" herein are directly connected, and the "direct connection" means that there is no nucleotide between the two elements, so the "direct connection" can be connected by any connection mode allowed between nucleotides. In some embodiments, the "direct connection" refers to being connected by a chemical bond. In some embodiments, the "direct connection" refers to being connected by a phosphate bond.

[0217] In addition, the present application also provides the use of the above-mentioned Poly (A) tail for low expression of the target gene in specific organs, tissues and / or cells, wherein the specific organs, tissues and / or cells highly express the miRNA.

[0218] Engineered DNA molecules

[0219] The present application also provides an engineered DNA molecule that can be replicated in a cell, which comprises the coding sequence of the aforementioned engineered Poly (A) tail or its complementary sequence. In some embodiments, the engineered DNA molecule can be replicated in a cell. In some embodiments, the engineered DNA molecule can express the aforementioned engineered Poly (A) tail. In some embodiments, the engineered DNA molecule can express the aforementioned engineered Poly (A) tail and can be replicated in a cell. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the prokaryotic cell is Escherichia coli. Those skilled in the art will appreciate that, in addition to the coding sequence of the engineered Poly (A) tail, the engineered DNA molecule should also include structural elements that enable the DNA molecule to replicate in a cell, or to replicate efficiently. The structural elements that enable the engineered DNA molecule to replicate in a cell or to replicate efficiently are known in the art, including, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further comprises a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of DNA elements, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing a plasmid that comprises the marker gene, and can be selected from, for example, antibiotic resistance genes. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0220] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid consisting of a double-stranded DNA molecule. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" can also encompass linear DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, cutting a circular plasmid with a restriction endonuclease, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid, and linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify in cells independently of the genomic genetic information stored in the nucleoid or nucleoid of prokaryotes, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid according to the present invention is a medium copy or high copy plasmid, more preferably a high copy plasmid. Examples of such high copy plasmids are such vectors: they are based on pUC, pTZ plasmids or any other plasmids (e.g., pMB1, pCoIE1) etc. that contain an ORI that supports high copies of plasmids.

[0221] In some embodiments, the DNA molecule is a DNA molecule or a fragment thereof constituting a prokaryotic nucleoid or nucleoid, that is, the coding sequence or its complementary sequence comprising the aforementioned engineered Poly (A) tail can be replicated along with the prokaryotic genome.

[0222] 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 is free DNA when introduced into a eukaryotic cell. In some embodiments, the DNA is a viral vector.

[0223] In some embodiments, the DNA molecule is further connected to a target gene fragment on the 5' side of the engineered Poly (A) tail coding sequence, and the target gene fragment and the engineered Poly (A) tail coding sequence jointly encode RNA. In some embodiments, the target gene fragment and the engineered Poly (A) tail coding sequence jointly encode mRNA. The target gene fragment comprises a coding sequence of a protein, a polypeptide or a fragment thereof. The coding sequence of the protein, polypeptide or a fragment thereof can be ultimately translated into one or more proteins, or one or more polypeptides, such as short peptides, oligopeptides, polypeptides, fusion proteins, proteins and fragments thereof, such as parts of known proteins, such as functional parts. The functional part can be, for example, a biologically active part of a protein or an antigenic part that can effectively produce antibodies, such as an antigenic epitope. The two ends of the coding sequence of the protein, polypeptide or fragment thereof respectively comprise a start codon (5' end) and a stop codon (3' end), which are the first three nucleotides and the last three nucleotides that can be translated of the mRNA molecule, respectively.

[0224] In some embodiments, the mRNA of the present invention further comprises 5'UTR, 3'UTR, etc. In some embodiments, the mRNA of the present invention comprises at least the sequence of 5'UTR, the target gene sequence, the sequence of 3'UTR, and the Poly(A) tail sequence of the present invention in order from 5' to 3'.

[0225] The 5'UTR usually 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. The RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the start of translation. Its activity can be optimized by changing the length and sequence of a given RBS or translation revelation site and the distance from the start codon. Alternatively or optionally, the 5'UTR includes an internal ribosome entry site or IRES. The 3'UTR may contain one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of mRNA molecules, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0226] The coding sequence of the protein, polypeptide or fragment thereof comprises codons that can be translated into an amino acid sequence. All the codons contained in the coding sequence may be naturally occurring codons encoding amino acids, or may be partially or entirely composed of artificially synthesized codons. In some embodiments, some or all of the codons are codon optimized. In some embodiments, some or all of the codons encode non-natural amino acids.

[0227] In some embodiments, the DNA molecule further comprises a structural element necessary for initiating or regulating transcription of the RNA on the 5' end side of the target gene fragment, and the structural element is known in the art. In some embodiments, the structural element comprises at least a promoter. Promoters and their sequences are known in the art, including weak promoters, medium strength promoters, strong promoters, mini promoters or core promoters, etc. 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 engineered Poly (A) tail in prokaryotes. In some embodiments, the promoter can initiate transcription of the target gene fragment and / or engineered Poly (A) tail in eukaryotic cells. The "promoter" comprises at least one transcription recognition site and a subsequent transcription factor binding site. The recognition and binding sites can interact with an amino acid sequence that mediates or regulates transcription. Compared with the recognition site, the binding site is closer to the aforementioned target gene fragment. The binding site can be, for example, a Pribnow box in prokaryotes or a TATA box in eukaryotes. For example, in some embodiments, when using the Pribnow box, the transcription recognition site can be located at about 35bp upstream of the transcription start site, and the transcription factor binding site can be located at about 10bp upstream of the transcription start site. In some embodiments, the promoter comprises at least one other regulatory element, such as an upstream element rich in AT at about 40 and / or 60 nucleotides before the transcription start site, and / or an additional regulatory element of the enhancing promoter activity between the recognition site and the binding site. In some embodiments, the promoter is a strong promoter, i.e., the promoter comprises a sequence that promotes the transcription of the aforementioned RNA coding sequence. Strong promoters are known to those skilled in the art, such as OXB18, OXB19 and OXB20 promoters derived from the RecA promoter of Escherichia coli, or can be identified or synthesized by conventional laboratory procedures. In some embodiments, the promoter is a T7 promoter. In some embodiments, the promoter also comprises other regulatory elements before the promoter, such as an enhancer that can promote the transcription of the aforementioned RNA coding sequence in a DNA plasmid.

[0228] In addition, the present application also provides the use of the above-mentioned engineered DNA molecule in the stable amplification of the coding sequence of the engineered Poly (A) tail or the coding sequence of RNA with the engineered Poly (A) tail.

[0229] Engineered RNA

[0230] The present application provides an engineered RNA or engineered RNA molecule, which comprises the aforementioned engineered Poly(A) tail and a target gene fragment on the 5' end of the Poly(A) tail encoding sequence. In some embodiments, the RNA is mRNA.

[0231] In some embodiments, the mRNA molecule of the present application further comprises a 5'UTR and / or a 3'UTR.

[0232] In some embodiments, the mRNA molecule of the present application further comprises a 5' cap. In a preferred embodiment, the 5' cap is m7G(5')ppp(5')(2'-OMeA)pG.

[0233] In some embodiments, the mRNA molecule of the present application further comprises a chemical modification, such as modifying all or part of the uridine acid in the polynucleotide sequence to N1-methyl pseudouridine.

[0234] As used herein, "mRNA" (messenger RNA) is any RNA that encodes at least one protein, polypeptide, or fragment thereof, naturally occurring, non-naturally occurring, or modified, and the mRNA has the ability to be translated to produce the encoded protein, polypeptide, or fragment thereof in vitro, in vivo, in situ, or ex vivo. Therefore, the mRNA can be a mature mRNA or a pre-mature mRNA, and the elements or structures that it must contain or selectively contain are known in the art. In some embodiments, the mRNA contains coding sequences for a plurality of necessary functional components to express, regulate, or enhance the expression level of the protein, polypeptide, or fragment thereof. The functional components include, but are not limited to, a 5' cap, a 5' UTR, a 3' UTR, and the like. Both the 5' UTR and the 3' UTR are typically transcribed from genomic DNA and are elements that pre-mature mRNAs have.

[0235] As a mature mRNA, the term "5' cap" is usually located at the 5' end of the mRNA, including methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate to form a 5',5'-triphosphate connection with its adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are called O type, I type and II type respectively. O type refers to the ribose of the terminal nucleotide is not methylated, I type refers to the ribose of one terminal nucleotide is methylated, and II type refers to the ribose of both terminal nucleotides is methylated. In some embodiments, the 5' cap can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to produce a 5'-guanosine cap structure: 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, MA), or m7G(5')ppp(5')(2'-OMeA)pG (CleanCapAG) according to the manufacturer's protocol. For example, in some embodiments, 5' capping of the modified RNA can be accomplished post-transcriptionally using a vaccinia virus capping enzyme to produce an O-type cap structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Type I cap structures can be generated using both vaccinia virus capping enzyme and 2'-O methyl-transferase to generate m7G(5')ppp(5')(2'-OMeA)pG. Type II cap structures can be generated from Type I cap structures followed by 2'-O-methylation of the 5'-third to last nucleotide using 2'-O methyl-transferase. Type III cap structures can be generated from Type II cap structures followed by 2'-O-methylation of the 5'-fourth to last nucleotide using 2'-O methyl-transferase.

[0236] In some embodiments, the mRNA further comprises a stabilizing element. Stabilizing elements may include, for example, a histone stem-loop. In some embodiments, the mRNA comprises a coding region, at least one histone stem-loop and optionally a poly(A) sequence or a polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the mRNA comprises a combination of a poly(A) sequence or a polyadenylation signal and at least one histone stem-loop, although both have alternative mechanisms in nature, their synergistic effect can increase protein expression to a level that exceeds that observed for any single element. The synergistic effect of the combination of poly(A) and at least one histone stem-loop is independent of the order of the elements or the length of the poly(A) sequence. In some embodiments, the histone stem-loop is generally derived from a histone gene and comprises two adjacent partial or complete reverse complementary sequences separated by a spacer (composed of a short sequence) and the intramolecular base pairing forms a loop. Unpaired loop regions are generally unable to base pair with any of the stem-loop elements. The stability of the stem-loop structure generally depends on the length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may occur. In some embodiments, the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.

[0237] In some embodiments, one or more AU-rich sequences of the mRNA can be removed. These sequences are sometimes referred to as AURES, which are destabilizing sequences found in the 3'UTR. AURES can be removed from the mRNA. Alternatively, AURES can be retained in the mRNA.

[0238] cell

[0239] The application also provides cells comprising the aforementioned engineered DNA molecules, which can be stored and / or amplified in the cells. In some embodiments, the cells are prokaryotic cells, and the DNA molecules can be replicated in the prokaryotic cells. In some embodiments, the cells are prokaryotic cells, and the DNA molecules can be replicated and / or transcribed in the prokaryotic cells. In some embodiments, the DNA molecules are eukaryotic cells, and the DNA molecules can be replicated in the cells. In some embodiments, the DNA molecules can be transcribed and / or replicated in the DNA cells.

[0240] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterium, an actinomycete, a cyanobacterium, a mycoplasma, a rickettsia, and a chlamydia. In some embodiments, the cell is selected from the group consisting of: Bacillus subtilis, Lactobacillus, Acetobacter, Corynebacterium, Brevibacterium, Arthrobacter, Pseudomonas, and Micrococcus. In some embodiments, the cell is a recA - In some embodiments, the cell is Escherichia coli. In some embodiments, the cell is Escherichia coli, and the Escherichia coli is selected from: K-12 and its derivative strains, and B strain and its derivative strains. In some embodiments, the Escherichia coli is selected from: MG1655, DH5 or DH5α, DH10B, BL21, DB3.1, HB101, JM109, JM110, MC1061, MG1655, Pir1, Stbl2, Stbl3, Top10, XL1Blue, XL10 Gold, BLR, HMS174, Tuner, Rostetta2, Lemo21, T7Express, Origami2, etc. In some embodiments, the cell is selected from Streptomyces, Micromonospora and Nocardia. In some embodiments, the cell is a fungus. In some embodiments, the cell is selected from yeast or mold.

[0241] Lipid Nanoparticles

[0242] The present application also provides lipid nanoparticles comprising the engineered Poly (A) tail, engineered RNA molecule, and / or engineered DNA molecule of the present application.

[0243] In some embodiments, lipid is mixed with the engineered RNA to form lipid nanoparticles. In some embodiments, the engineered Poly (A) tail, engineered RNA molecule, and / or engineered DNA molecule of the present application are formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticle is first formed into an empty lipid nanoparticle, and is combined or wrapped with the engineered Poly (A) tail, engineered RNA molecule, and / or engineered DNA molecule before being used (e.g., within a few minutes to an hour).

[0244] The lipid nanoparticles generally comprise ionizable lipids, non-cationic lipids, sterols and PEG lipid components and target nucleic acids, such as the engineered Poly (A) tail, engineered RNA molecules, and / or engineered DNA molecules of the present application.

[0245] The lipid nanoparticles of the present invention comprise ionizable lipids, phospholipids, structural lipids and PEG lipids; preferably, the molar ratio of the ionizable lipids, phospholipids, structural lipids and PEG lipids is: (20-60): (5-25): (25-55): (0.5-15), more preferably (40-55): (5-15): (30-50): (1-3);

[0246] The phospholipids are preferably selected from one or more of the following compounds:

[0247] Dilauroyl phosphatidylcholine (DLPC),

[0248] Dimyristoylphosphatidylcholine (DMPC),

[0249] Dioleoylphosphatidylcholine (DOPC),

[0250] Dipalmitoylphosphatidylcholine (DPPC),

[0251] Distearoylphosphatidylcholine (DSPC),

[0252] Dioleoylphosphatidylcholine (DUPC),

[0253] Palmitoyloleoylphosphatidylcholine (POPC),

[0254] 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC),

[0255] 1-oleoyl-2-cholesteryl dimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC),

[0256] l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0257] 1,2-Divinyl-sn-glycero-3-phosphocholine,

[0258] 1,2-Diaryl acyl-sn-glycero-3-phosphocholine,

[0259] 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE),

[0260] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

[0261] 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine,

[0262] 1,2-Divinyl-sn-glycero-3-phosphoethanolamine,

[0263] 1,2-Diaryl-sn-glycero-3-phosphoethanolamine,

[0264] 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

[0265] 1,2-dioleoyl-sn-glycero-3-phospho-(1-glycerol) sodium salt (DOPG) or sphingomyelin,

[0266] For example, the phospholipid is DOPE or DSPC;

[0267] The structural lipid is preferably selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol; for example, the structural lipid is cholesterol; and / or

[0268] The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol. For example, the PEG lipid is DMG-PEG2000.

[0269] The lipid nanoparticles of the present disclosure can be produced using components, compositions and methods as generally known in the art, see, e.g., PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 01428 ... S2016 / 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, all of which are incorporated herein by reference in their entirety.

[0270] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art to which the present application belongs. Therefore, those of ordinary skill in the art should recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments. Example

[0271] Example 1: Construction of luciferase mRNA containing polyA variants of the present invention

[0272] 1) Construction of a vector containing the luciferase protein coding region

[0273] The vector uses the E. coli cloning vector pUC57 as a vector backbone, and sequentially arranges a T7 promoter sequence (SEQ ID NO: 2), a 5'UTR, a Kozak sequence (GCCACC), a luciferase protein coding sequence, a 3'UTR and a poly(dA:dT) numbered RG008 between the Xba I restriction site and the Sap I restriction site of the multiple cloning site as a control for the poly(A) variant designed in the present invention.

[0274] 2) Replace the polyadenylic acid string poly(dA:dT) in the universal vector

[0275] All the sequences required for constructing different poly(A) variants (as shown in the sequence table at the end of the article) were synthesized, and double digestion was performed with two restriction endonucleases to remove the variant poly(dA:dT) numbered RG008 from the universal vector constructed in 1), and then the different poly(A) variants of the present invention were ligated with the vector from which poly(dA:dT) was removed using T4 DNA ligase 1, thereby completing the replacement of poly(dA:dT) in the universal vector.

[0276] Schematic diagram as Figure 1A and Figure 1B shown.

[0277] Example 2: Preparation of mRNA-LNP of the present invention

[0278] To the luciferase mRNA stock solution, add acetic acid solution to a final acetic acid concentration of 20mmol / L and a final mRNA concentration of 200μg / ml, stir and mix, and use as the mRNA working solution. The mRNA working solution and the mixed lipid solution (prepared according to Table 1) are mixed at a flow ratio of 2:1 to 4:1 through a T mixing device to prepare LNP. Then, the LNP is diluted 2 to 5 times with a 2mmol / L acetic acid solution, and then replaced with a 2mmol / L acetic acid solution by not less than 3 times, and the feed liquid is concentrated to the target concentration. Sucrose solution is added to adjust the osmotic pressure, and the pH is adjusted to 7.0 to 8.0 with a Tris solution to obtain the mRNA-lipid nanoparticles (LNP) of the present invention.

[0279] Table 1: Lipid-mixed solution formula

[0280] Fat mixture mol% Ionizable fat 40-55 cholesterol 30-50 Phospholipids 5-15 DMG-PEG2000 1-3

[0281] Example 3 Expression test of luciferase mRNA containing five poly(A)s of miR-142 binding sites in mice

[0282] Using luciferase as the protein coding region, 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; Poly(A)-0A insert miR-142) were investigated in the liver of mice with low miR-142 expression and the spleen of mice with high miR-142 expression, in order to detect whether the insertion of miR-142 binding sites can downregulate the expression level of mRNA in organs with high miR-142 expression.

[0283] C57BL / 6 mice were randomly divided into groups according to body weight, with 6 mice in each group, and adaptively fed for 2-3 days. The mice were administered drugs by tail vein injection, with each mouse being injected with 0.05 mg / kg of mRNA-LNPs (1 μg) and an injection volume of 100 μL. The blank control was injected with an equal volume of PBS. 6 hours after administration, Luciferin (200 μL) was injected intraperitoneally at a dose of 150 mg / kg. After 10 minutes, 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 in total, and the measured values ​​of each mouse were taken to make a bar graph. The results are shown in Figure 2A-2B shown.

[0284] Conclusion: The miR-142 binding site was inserted into RG008 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) to form different Poly (A) variants. When mRNA with such variants was encapsulated by LNPs and injected into mice through the tail vein, the mRNA was taken up and degraded in the spleen where macrophages (highly expressing miR-142) were enriched, and the expression was inhibited. In terms of spleen expression level, the reduction level when the miR-142 binding site was inserted into 3'UTR, 0A, 14A, 19A, and 30A was similar, which was significantly better than that when it was inserted into 60A.

[0285] In terms of liver expression levels, when the miR-142 binding site was inserted into positions 14A, 19A, 30A, and 60A, it had little effect on the liver expression level. When the insertion position was 14A and 30A, it was even better than the control group without miR-142 binding sites. However, when inserted into position 0A, the liver expression level decreased significantly.

[0286] Taking into account the liver expression level and the spleen expression level, the present invention screened out the insertion position 14A-30A as the optimal miR-142 binding site insertion position, which can significantly downregulate the spleen expression level without affecting the liver expression level, and is suitable for the application scenario of mRNA liver expression protein to treat diseases.

[0287] Example 4 Expression test of luciferase mRNA containing five poly(A)s of miR-122 binding sites in mice

[0288] 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; Poly(A)-0AinsertmiR-122) were investigated in the spleen of mice with low miR-122 expression and the liver of mice with high miR-122 expression.

[0289] C57BL / 6 mice were randomly divided into groups according to body weight, with 6 mice in each group, and adaptively fed for 2-3 days. The mice were dosed by tail vein injection, with each mouse being injected with 0.05 mg / kg of mRNA-LNPs (1 μg) in a volume of 100 μL. The blank control was injected with an equal volume of PBS. 6 hours after administration, Luciferin (200 μL) was injected intraperitoneally at a dose of 150 mg / kg. After 10 minutes, 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 in total, and the measured values ​​of each mouse were made into a bar graph, where the absolute values ​​of fluorescence imaging of the liver and spleen were as follows: Figure 3A-3B shown.

[0290] Conclusion: The miR-122 binding site is inserted after 0A, 14A, 19A, 30A, and 60A of RG008 to form different Poly (A) variants. When mRNA with such variants is encapsulated by LNPs and injected into mice through the tail vein, the mRNA will be taken up and significantly degraded in the liver, and the expression is inhibited. The reduction level of liver expression of miR-122 binding site inserted at 0A, 14A, and 19A of RG008 is significantly better than that inserted at 30A and 60A. The spleen expression level is reduced in all groups. Based on the liver expression level and spleen expression level, the present invention screens out the insertion positions 0A, 14A, and 19A as the better insertion positions of miR-122 binding sites, which can maintain the spleen expression level on the basis of significantly downregulating the liver expression level, and is suitable for the application scenario of mRNA spleen expression protein treatment of diseases.

[0291] Example 5 Stability experiment of plasmids containing different poly(A) variants in Escherichia coli

[0292] The effects of two groups 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; Poly(A)-0A insert miR-122) on the stability in Escherichia coli were investigated.

[0293] After the vector plasmid constructed according to the method in Example 1 was confirmed to be correct by sequencing, it was transformed into Escherichia coli DH5α, and the transformed plate was grown at 30°C. The next day, the plate was sent to a sequencing service company to randomly select 50 clones to complete the sequencing work. After the sequencing was completed, the situation of different Poly (A) variants was analyzed and calculated according to the sequencing results.

[0294] The results are as follows Figure 4 As shown in the figure, the proportion of base deletions when two different miRNA binding sites are inserted into the poly(A) variant 14A-30A position is lower than that when inserted into the 0A or 60A position and into the 3'UTR. The results show that the plasmid stability is better when the miRNA binding site is inserted into the RG008 poly(A) variant 14A-30A position.

[0295] Example 6 Expression test of mRNA containing poly(A) variants of miR-142 binding sites at different insertion positions in cells

[0296] The 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 monocytic macrophage leukemia RAW 264.7 cells with high expression of miR-142 were investigated.

[0297] RAW 246.7 cells were cultured in 24-well cell culture plates at a cell density of 1x10 5 cells / well, and each well was transfected with 500ng of in vitro synthesized mRNA using Lipofectamine 3000 (purchased from Thermo Fisher Scientific). 24h after transfection, the cell culture medium was removed, and 200μL of cell lysate was added to each well. After 10min, the cell lysate was transferred to a 1.5mL EP tube and centrifuged at 12000rpm and 4℃ for 5min. Then, 20μL of the lysate supernatant was transferred to a black opaque 96-well plate, and 200μL of fluorescein substrate was added using a fluorescence detector and the value was read. The results are shown in Figure 5 shown.

[0298] Conclusion: The miR-142 binding sites inserted after 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), and 14A (SEQ ID NO: 9) of RG008 constitute different Poly (A) variants. When mRNA carrying such variants is delivered to miR-142 overexpressing cell lines, such as Raw 264.7 cells, the mRNA will be degraded by miR-142 and its expression will be inhibited.

[0299] Example 7 Study on the mutual influence of different microRNA binding sites in the poly (A) tail after tandem connection

[0300] The expression levels of two Poly(A) variants (Poly(A)-0A insert miR-142+miR-122; Poly(A)-0A insertmiR-122+miR-142) in mouse mononuclear macrophage leukemia RAW 264.7 cells with high expression of miR-142 were investigated to examine whether the insertion of tandem binding sites of miR-122 and miR-142 affects the expression level of mRNA downregulated by the miR-142 binding site.

[0301] RAW 246.7 cells were cultured in 24-well cell culture plates at a cell density of 1x10 5 cells / well, and each well was transfected with 500ng of in vitro synthesized mRNA using Lipofectamine 3000 (purchased from Thermo Fisher Scientific). 24h after transfection, the cell culture medium was removed, and 200μL of cell lysate was added to each well. After 10min, the cell lysate was transferred to a 1.5mL EP tube and centrifuged at 12000rpm and 4℃ for 5min. Then, 20μL of the lysate supernatant was transferred to a black opaque 96-well plate, and 200μL of fluorescein substrate was added and read using a fluorescence detector. The results are shown in Figure 6 shown.

[0302] Conclusion: The miR-142+miR-122 or miR-122+miR-142 binding sites were inserted after 0A of RG008 to form different Poly(A) variants. When mRNA with such variants was delivered to miR-142-overexpressing cell lines, such as Raw 264.7 cells, via LNPs, the mRNA would be degraded by miR-142 and its expression would be inhibited. The different sequences of tandem connection of miR-142 and miR-122 binding sites did not affect the expression inhibition effect.

[0303] Example 8 Study on the precise expression of mRNA containing multiple microRNA binding site Poly (A) variants in cells

[0304] The six poly(A) variants (poly(A)-0A insert miR-142+miR-126+miR-148a; poly(A)-0A insert miR-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; poly(A)-0A insert miR-148a+miR-126+miR-142) were investigated in mouse monocytic macrophage leukemia RAW cells with high expression of miR-142. The expression levels in 264.7 cells, mouse liver sinusoidal endothelial cells LSEC with high expression of miR-126, mouse hepatic stellate cells HSC with high expression of miR-148a, and liver parenchymal cells AML,

[0305] RAW246.7, LSEC, HSC, and AML12 cells were cultured in 24-well cell culture plates at a cell density of 1x10 5 cells / well, and each well was transfected with 500ng of in vitro synthesized mRNA using Lipofectamine 3000 (purchased from Thermo Fisher Scientific). 24h after transfection, the cell culture medium was removed, and 200μL of cell lysate was added to each well. After 10min, the cell lysate was transferred to a 1.5mL EP tube, centrifuged at 12000rpm, 4℃ for 5min, and then 20μL of the lysate supernatant was transferred to a black opaque 96-well plate, and 200μL of fluorescein substrate was added and read using a fluorescence detector. Calculation of the reduction ratio relative to liver parenchymal expression: The data of the without miRNA ts group was defined as 1, and the relative expression levels of different groups in different cell line experiments relative to those without miRNAts were calculated to bridge the direct expression data of different cell lines. On this basis, the reduction ratio of AML12 expression in target cells (RAW 264.7 cells, liver sinusoidal endothelial cells LSEC, hepatic stellate cells HSC) relative to liver parenchymal cells was calculated for each design. The calculation method was: (1-relative expression level of target cells / relative expression level of liver parenchymal cells)*100%.

[0306] Conclusion: When miR-148a, miR-142, and miR-126 are tandemly expressed, when the mRNA is transfected into RAW 264.7 cells, the Poly(A) variant contains a miR-142 binding site, and the expression of the mRNA in this cell line is reduced. When the mRNA is transfected into LSEC cells, the Poly(A) variant contains a miR-126 binding site, and the expression of the mRNA in this cell line is reduced. When the mRNA is transfected into HSC cells, the Poly(A) variant contains a miR-148a binding site, and the expression of the mRNA in this cell line is reduced. In addition, the combination of miR-126+miR-148a+miR-142 and miR-148a+miR-142+miR-126 performed better than the other sequences in the three cells. See Figures 7A-7D , Figures 8A-8C .

[0307] The liver is mainly composed of hepatocytes, hepatic macrophages, hepatic sinusoidal endothelial cells, and hepatic stellate cells. This example screened out a combination of microRNA binding sites specifically expressed by hepatocytes with excellent performance, providing a reliable tool for targeted treatment of hepatocytes.

[0308] The results of the present invention show that inserting miRNA binding sites into Poly(A) variants can cause mRNA to show different expression differences in different cell lines and different organs of mice. Therefore, by inserting one or more miRNA binding sites into the same sequence, highly specific expression regulation can be achieved.

[0309] The sequences used in the above examples of the present application are shown in the following sequence table. It should be understood that the following sequences are only exemplary sequences of the present application embodiment, rather than any limitation to the present application scheme. The nucleic acid sequences in the following sequence table may represent DNA sequences or RNA sequences, and when they represent RNA sequences, "T" therein represents uridine. Moreover, in the present application, a single T also refers to uracil or uridine in the context of RNA.

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

Claims

1. An engineered Poly(A) tail comprising a miRNA binding site.

2. The engineered Poly(A) tail according to claim 1, wherein the Poly(A) tail comprises or is of formula (I): nA-miRNA binding site-mA formula (I); in, The miRNA binding site is formed by one or more miRNA binding sites connected by one or more nucleotides or directly connected; nA represents n consecutive adenylate nucleotides (A) adjacent to the 5' end of the miRNA binding site, mA represents the m consecutive adenylate nucleotides (A) adjacent to the 3' end of the miRNA binding site, m and n are natural numbers, and 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 3' and 5' ends of the miRNA binding site do not contain any A except the A contained in the miRNA binding site itself.

3. An engineered Poly(A) tail according to claim 2, wherein n=0 or n≥1.

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

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

6. An engineered Poly(A) tail according to any one of claims 1 to 5, wherein the length of the Poly(A) tail is 80 to 240 nt, such as 100 to 200 nt, 101 to 150 nt, 120 to 150 nt, 130 to 140 nt, 123 nt to 135 or 125 to 139 nt.

7. The engineered Poly(A) tail according to any one of claims 2 to 6, wherein the Poly(A) tail: The 3' side of formula (I) further comprises a tail segment directly connected to the 3' end of the structure of formula (I), wherein the nucleotide at the 5' end of the tail segment is not A; and / or The 5' side of formula (I) further comprises a head fragment directly connected to the 5' end of the structure of formula (I), and the nucleotide at the 3' end of the head fragment is not A.

8. The engineered Poly(A) tail of claim 7, wherein: The tail segment or the head segment is composed of one or more elements c and / or one or more elements d and one or more A, The element c is a non-A nucleotide; The element d is composed of any two or more consecutive nucleotides, the nucleotides at the 5' and 3' ends of the element d are not A, and the element d does not contain more than three consecutive A; The length range of the element d is 2nt≤d≤20nt; The element c and the element d are not adjacent.

9. The engineered Poly (A) tail according to claim 8, wherein the number of the element c it comprises is 2 to 10, 3 to 8, or 4 to 6 or 2-5, preferably 2; and / or element c is G.

10. The engineered Poly(A) tail according to any one of claims 8-9, wherein the element d is selected from any one or more of the sequences shown in SEQ ID NO: 60 to 64, preferably, the element d is shown in SEQ ID NO:

60.

11. The engineered Poly (A) tail according to any one of claims 8 to 10, comprising 0 to 5 elements d, preferably 1 to 3, and more preferably 1.

12. The engineered Poly (A) tail according to any one of claims 1-11, does not comprise a head fragment, and the structure of the tail fragment is: element d-19A-element c-19A-element c-17A or element c-19A-element c-17A.

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

14. The engineered Poly (A) tail according to any one of claims 1 to 13, which has a structure of nA-miRNA binding site-mA-SEQ ID NO: 60-19A-G-19A-G-17A.

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

16. The engineered Poly (A) tail according to any one of claims 1 to 15, wherein The miRNAs include miRNA-142, miR-148a and miR-126, and in the miRNA binding site of the Poly(A), the arrangement order of each miRNA binding site from 5' to 3' is: 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 miR-142 binding site, miR-126 binding site, and miR-148a binding site; The miR-126 binding site, miR-148a binding site and miR-142 binding site are adjacent to each other.

17. The engineered Poly (A) tail according to any one of claims 1-16, wherein n=0, m=60, and mA and the tail fragment constitute a polynucleotide sequence as shown in SEQ ID NO:

1.

18. The engineered Poly(A) tail according to any one of claims 1 to 16, wherein the Poly(A) tail comprises any one polynucleotide sequence selected from the following: SEQ ID NO: 6-16, SEQ ID NO: 19-31, SEQ ID NO: 33-52, SEQ ID NO: 54-59 and SEQ ID NO: 65-71.

19. An engineered RNA molecule, which is an mRNA or a non-coding RNA, comprising 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 at least a 5'UTR sequence, a target gene sequence, a 3'UTR sequence and the Poly (A) tail according to any one of claims 1 to 18 in sequence from the 5' to the 3' end, wherein the nA in formula (I) of the Poly (A) tail is directly connected to the 3'UTR; preferably, the RNA molecule has a 5' cap and / or all or part of the uridylic acid is modified to N1-methyl pseudouridine.

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; preferably, the DNA molecule is 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, an engineered DNA molecule according to claim 20, wherein the cell is a eukaryotic or prokaryotic cell; preferably the prokaryotic cell is Escherichia coli.

22. The engineered Poly (A) tail according to any one of claims 1-18 is used for 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, and preferably the regulation is to lowly express the target gene in a specific organ, tissue and / or cell; more preferably, the specific organ, tissue and / or cell is a liver or spleen organ, tissue and / or cell.

23. Use of the engineered Poly (A) tail according to any one of claims 1-18 for specifically expressing a target gene in a specific organ, tissue and / or cell, preferably, the organ or tissue is preferably a liver or spleen organ or tissue; more preferably, the cell is a hepatocyte.

24. Use of the engineered Poly(A) tail according to any one of claims 1-18 for making the replication of the DNA coding sequence encoding the Poly(A) tail in a host cell more conservative, preferably, the host cell is a prokaryotic cell, more preferably Escherichia coli.

25. A lipid nanoparticle comprising an engineered Poly(A) tail according to any one of claims 1-18, an engineered RNA molecule according to claim 19, or an engineered DNA molecule according to claim 20.

26. The lipid nanoparticle according to claim 25, wherein the lipid nanoparticle comprises ionizable lipids, phospholipids, structural lipids and PEG lipids; preferably, the molar ratio of the ionizable lipids, phospholipids, structural lipids and PEG lipids is: (20-60): (5-25): (25-55): (0.5-15), more preferably (40-55): (5-15): (30-50): (1-3); The phospholipids are preferably selected from one or more of the following compounds: Dilauroyl phosphatidylcholine (DLPC), Dimyristoylphosphatidylcholine (DMPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DUPC), Palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl dimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Divinyl-sn-glycero-3-phosphocholine, 1,2-Diaryl acyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl alcohol-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-phospho-(1-glycerol) sodium salt (DOPG) or sphingomyelin, For example, the phospholipid is DOPE or DSPC; The structural lipid is preferably selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol; for example, the structural lipid is cholesterol; and / or The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol. For example, the PEG lipid is DMG-PEG2000.

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