WO2024251232A1 - 表达可调控的工程化rna分子 - Google Patents

表达可调控的工程化rna分子 Download PDF

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Publication number
WO2024251232A1
WO2024251232A1 PCT/CN2024/097944 CN2024097944W WO2024251232A1 WO 2024251232 A1 WO2024251232 A1 WO 2024251232A1 CN 2024097944 W CN2024097944 W CN 2024097944W WO 2024251232 A1 WO2024251232 A1 WO 2024251232A1
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Prior art keywords
mir
binding site
poly
tail
engineered
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PCT/CN2024/097944
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English (en)
French (fr)
Inventor
张卫国
董翊洁
陈瑞
祁锐
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RinuaGene International HK Ltd
RinuaGene Biotechnology Co Ltd
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RinuaGene International HK Ltd
RinuaGene Biotechnology Co Ltd
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Application filed by RinuaGene International HK Ltd, RinuaGene Biotechnology Co Ltd filed Critical RinuaGene International HK Ltd
Priority to EP24818762.7A priority Critical patent/EP4726038A1/en
Priority to KR1020267000677A priority patent/KR20260022409A/ko
Priority to AU2024285035A priority patent/AU2024285035A1/en
Publication of WO2024251232A1 publication Critical patent/WO2024251232A1/zh
Priority to US19/408,745 priority patent/US20260092293A1/en
Priority to MX2025014784A priority patent/MX2025014784A/es
Anticipated expiration legal-status Critical
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Definitions

  • 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.
  • 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.
  • 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.
  • poly (A) sequence after the 3'UTR which can prevent exonuclease degradation.
  • 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 mRNA stability regulation and translation initiation.
  • PABP poly binding protein binding protein
  • 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.
  • 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.
  • miRNA also known as microRNA
  • miRNA 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.
  • 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.
  • IVTT in vitro transcription
  • the poly(dA:dT) repeat sequence in the plasmid is unstable during replication in Escherichia coli, and deletion mutations often occur, resulting in shortening of poly(dA:dT).
  • 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.
  • 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.
  • the present application provides:
  • 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.
  • the two or more miRNA binding site sequences are adjacent to each other or separated by one or more bases.
  • the miRNA binding site includes one or more miRNA binding sites.
  • nA represents n consecutive adenylate nucleotides (A) adjacent to the 5' end of the miRNA binding site
  • mA represents m consecutive adenylate nucleotides (A) adjacent to the 3' end of the miRNA binding site
  • n 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.
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • the element c is a non-A nucleotide; for example, T, U, C or G;
  • 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;
  • the length of the element d is in the range of 2nt ⁇ d ⁇ 30nt; preferably 6nt ⁇ d ⁇ 20nt, more preferably 6nt ⁇ d ⁇ 12nt;
  • the element c and the element d are not adjacent.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the 1/2 portion of the Poly(A) tail close to the 3' end contains one or more non-A nucleotides.
  • the Poly(A) tail comprises one or more non-A nucleotides in the 1/3 near the 3' end.
  • the Poly(A) tail comprises one or more non-A nucleotides in the 1/4 near the 3' end.
  • 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.
  • the miRNA binding site is inserted between any two As in element a or element b.
  • 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;
  • 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;
  • the element c consists of a non-A nucleotide
  • 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.
  • 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,
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the engineered Poly (A) tail does not contain a head fragment.
  • the engineered Poly (A) tail consists of a tail fragment and formula (I).
  • the engineered Poly (A) tail consists of a head fragment, a tail fragment and formula (I).
  • nA-miRNA binding site-mA-element c-element b-element c-element b, wherein 110 ⁇ m+n ⁇ 70, preferably, m+n 79;
  • the structure is selected as:
  • 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.
  • nA-miRNA binding site-mA-G-19A-G-17A, m+n 79;
  • 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.
  • nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, m+n 60 and 30 ⁇ n ⁇ 14; preferably, 19 ⁇ n ⁇ 14.
  • nA-miRNA binding site-mA-SEQ ID NO: 60-19A-G-19A-G-17A, m+n 60;
  • 60A-SEQ ID NO: 60-nA-miRNA binding site-mA-G-19A-G-17A, m+n 19;
  • nA-miRNA binding site-mA-G-19A-G-17A, m+n 79;
  • 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.
  • the miRNA is selected from one or more of miR-142, miR-122, miR-126, and miR-148a.
  • the miRNA is selected from one or more of miR-142-3p, miR-122-5p, miR-126-3p and miR-148a-3p.
  • 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.
  • 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.
  • the miRNA comprises or consists of: miR-142, miR-122, miR-126, and miR-148a.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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 miR-148a binding site, miR-126 binding site and miR-142 binding site
  • 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.
  • 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 miR-148a binding site, miR-126 binding site and miR-142 binding site
  • 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 separated from each other by one or more nucleotides.
  • 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:
  • miR-126 binding site miR-148a binding site, and miR-142 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 miR-148a binding site, miR-126 binding site and miR-142 binding site
  • miRNA binding sites are adjacent to each other, and the other two adjacent miRNA binding sites are separated by one or more nucleotides.
  • the structure is selected from any of the following:
  • nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, wherein m+n 60, n ⁇ 20 or n ⁇ 14 or n ⁇ 10;
  • nA-miRNA binding site-mA-element d-19A-G-19A-G-17A, wherein m+n 60, 14 ⁇ n ⁇ 30; preferably 14 ⁇ n ⁇ 19;
  • 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 NO: 60 to 64.
  • the structure is selected from any of the following:
  • 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.
  • 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.
  • 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.
  • nA in formula (I) in the Poly(A) tail is directly connected to the 3'UTR.
  • the DNA molecule is a plasmid or a viral vector.
  • 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.
  • 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.
  • the organ or tissue is preferably liver or spleen organ tissue; more preferably, the cell is a hepatocyte.
  • the host cell is a prokaryotic cell, preferably Escherichia coli.
  • 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.
  • 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.
  • 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 the present application, or be an equivalent solution to the solution of the present application.
  • Figures 1A-1B show schematic diagrams of the composition of five types of Poly(A) containing miR-142/miR-122 binding sites independently designed by the present invention
  • 2A-2B show the absolute numerical results of fluorescence imaging of spleen/liver of mice dissected after expressing luciferase mRNA with 5 poly(A)s containing miR-142 binding sites independently designed by the present invention in mice.
  • 3A-3B show the absolute numerical results of fluorescence imaging of liver/spleen obtained by dissection after luciferase mRNA carrying five poly(A)s containing miR-122 binding sites independently designed by the present invention were expressed in mice.
  • FIG. 4 shows 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 E. coli DH5 ⁇ .
  • Figure 5 shows the absolute numerical results of enzyme activity detection of luciferase mRNA containing 8 poly(A)s containing miR-142 binding sites independently designed by the present invention in the mammalian cell line Raw 264.7.
  • Figure 6 shows the absolute numerical results of 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.
  • Figures 7A-7D show the absolute numerical results of enzyme activity detection of luciferase mRNA containing the independently designed poly (A) variant of the present invention in the mammalian cell line Raw 264.7/LSEC/HSC/AML.
  • Figures 8A-8C show the ratio of the reduced expression of luciferase mRNA carrying the independently designed poly(A) variant of the present invention in Raw 264.7/LSEC/HSC relative to the reduced expression in hepatocyte AML.
  • 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.
  • 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.
  • a large number of RNAs containing poly (A) tails with determined sequences are obtained based on the DNA.
  • 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.
  • element c 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”.
  • “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:
  • 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.
  • 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".
  • 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.
  • miRNA i.e., microRNA or microRNA
  • miRNA binding site is a miRNA binding site that downregulates mRNA expression after binding to miRNA.
  • 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.
  • RNA ribonucleotide
  • DNA deoxyribonucleotide
  • 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.
  • a DNA molecule containing genetic information that can be transcribed into an RNA molecule is referred to as the "coding nucleic acid” of the RNA molecule; an RNA molecule containing genetic information that can be translated into an amino acid sequence is referred to as the "coding nucleic acid" of the amino acid sequence.
  • 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.
  • “5' end” and “5' end” can be used interchangeably, and “3' end” and “3' end” can be used interchangeably.
  • “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.
  • nucleotides 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.
  • sequence segment is closer to the "5' end" of the polynucleotide sequence relative to the other sequence segment.
  • 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.
  • 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.
  • a nucleic acid molecule e.g., a Poly (A) tail
  • 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.
  • 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.
  • 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.
  • the two or more elements 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the "complementary polynucleotide sequence" of a polynucleotide sequence refers to a polynucleotide sequence that is completely complementary to the polynucleotide sequence.
  • 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.
  • Constant 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.
  • DNA and RNA refer to single-stranded or double-stranded DNA or RNA molecules.
  • DNA and RNA molecule refer to double-stranded DNA molecules consisting of A, C, G and/or T nucleotides
  • RNA and RNA molecule refer to single-stranded RNA molecules consisting of A, C, G and/or U nucleotides.
  • A, C, G, T and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine and uracil as their respective nitrogenous bases.
  • RNA molecules include coding RNA (coding RNA) or non-coding RNA (ncRNA), such as Pre-mRNA, mature mRNA or long noncoding RNA (lncRNA).
  • coding RNA coding RNA
  • ncRNA non-coding RNA
  • Pre-mRNA Pre-mRNA
  • mature mRNA mature mRNA
  • long noncoding RNA lncRNA
  • 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:
  • 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.
  • nucleic acid molecules are described as having "the same genetic information"
  • 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.
  • any two of DNA, RNA, and hybrid molecules of DNA and RNA can have the same genetic information.
  • the term "base sequence” refers to the order of arrangement of bases in a polynucleotide molecule.
  • the base sequence or polynucleotide sequence described in this application may use "T” to represent thymine when used to describe a DNA sequence, but when the base sequence or polynucleotide sequence is used to describe RNA (such as mRNA), "T” will be replaced by "U” (uracil).
  • any DNA disclosed by a specific sequence number (SEQ ID NO) in this article also discloses an RNA (such as 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”.
  • 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.
  • an engineered Poly (A) tail comprising one, two, three or more miRNA binding sites.
  • 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.
  • 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.
  • 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.
  • 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.
  • Poly A tail refers to an uninterrupted or uninterrupted sequence of adenylic acid residues that is typically located at the 3'-end of an RNA molecule.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the aforementioned coding nucleic acid is linear.
  • 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.
  • the present application provides a series of engineered Poly (A) tails, which include miRNA binding site sequences.
  • the bases of the engineered Poly (A) tail except the miRNA binding site are all A.
  • the engineered Poly (A) tail further has a high degree of conservation during the in vitro preparation of RNA.
  • the engineered Poly (A) tail can improve the stability of mRNA.
  • the engineered Poly (A) tail is formed by inserting the miRNA binding site between any two nucleotides of the conserved Poly (A) tail.
  • 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.
  • 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.
  • 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.
  • the "direct connection” refers to being connected by a chemical bond.
  • the "direct connection” refers to being connected by a phosphate bond.
  • 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.
  • 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.
  • the engineered DNA molecule can be replicated in a cell.
  • the engineered DNA molecule can express the aforementioned engineered Poly (A) tail.
  • the engineered DNA molecule can express the aforementioned engineered Poly (A) tail and can be replicated in a cell.
  • the cell is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell is Escherichia coli.
  • 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).
  • 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).
  • MCS multiple cloning site
  • 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.
  • the DNA molecule is a DNA plasmid.
  • DNA plasmid refers to a plasmid consisting of a double-stranded DNA molecule.
  • the "plasmid” is a circular DNA molecule.
  • the "plasmid” can also encompass linear DNA molecules.
  • 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.
  • the DNA plasmid according to the present invention is a medium copy or high copy plasmid, more preferably a high copy plasmid.
  • 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.
  • 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.
  • the DNA molecule is genomic DNA, such as viral genomic DNA or eukaryotic genomic DNA.
  • the DNA is mitochondrial DNA.
  • the DNA is free DNA when introduced into a eukaryotic cell.
  • the DNA is a viral vector.
  • 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.
  • 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 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 include 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.
  • the mRNA of the present invention further comprises 5'UTR, 3'UTR, etc. In some embodiments, the mRNA of the present invention at least comprises 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'.
  • 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.
  • RBS ribosome binding site
  • 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.
  • 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.
  • 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.
  • 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.
  • the DNA molecule further comprises a structural element necessary for initiating or regulating transcription of the RNA on the 5' end of the target gene fragment, and the structural element is known in the art.
  • 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.
  • 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.
  • the transcription recognition site when using the Pribnow box, 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.
  • 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.
  • 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.
  • the promoter is a T7 promoter.
  • 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.
  • 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.
  • 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.
  • the RNA is mRNA.
  • the mRNA molecule of the present application further comprises a 5'UTR and/or a 3'UTR.
  • the mRNA molecule of the present application further comprises a 5' cap.
  • the 5' cap is m7G(5')ppp(5')(2'-OMeA)pG.
  • the mRNA molecule of the present application further comprises a chemical modification, such as modifying all or part of the uridine nucleotides in the polynucleotide sequence to N1-methyl pseudouridine.
  • mRNA messenger RNA
  • 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.
  • 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.
  • 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
  • II type refers to the ribose of both terminal nucleotides is methylated.
  • the 5' cap can be accomplished simultaneously during an in vitro transcription reaction to produce a 5'-guanosine cap structure using the following chemical RNA cap analogs according to the manufacturer's protocol: 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).
  • 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.
  • the mRNA further comprises a stabilizing element.
  • Stabilizing elements may include, for example, a histone stem-loop.
  • 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.
  • 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.
  • 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.
  • wobble base pairing non-Watson-Crick base pairing
  • the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.
  • 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.
  • the application also provides cells comprising the aforementioned engineered DNA molecules, which can be stored and/or amplified in the cells.
  • the cells are prokaryotic cells, and the DNA molecules can be replicated in the prokaryotic cells.
  • the cells are prokaryotic cells, and the DNA molecules can be replicated and/or transcribed in the prokaryotic cells.
  • the DNA molecules are eukaryotic cells, and the DNA molecules can be replicated in the cells.
  • the DNA molecules can be transcribed and/or replicated in the DNA cells.
  • the cell is a prokaryotic cell.
  • the cell is a bacterium, actinomycetes, cyanobacteria, mycoplasma, rickettsia and chlamydia.
  • the cell is selected from: Bacillus subtilis, lactobacillus, acetic acid bacteria, corynebacterium, brevis, arthrobacter, pseudomonas and micrococcus.
  • the cell is a recA - bacterium.
  • the cell is Escherichia coli.
  • the cell is Escherichia coli, and the Escherichia coli is selected from: K-12 and its derivative strains, and B strains and their derivative strains.
  • the E. coli is selected from: MG1655, DH5 or DH5 ⁇ , DH10B, BL21, DB3.1, HB101, JM109, JM110, MC1061, MG1655, Pir1, Stbl2, Stbl3, Top10, XL1Blue, XL10 Gold, BLR, HMS174, Tuner, Rostetta2, Lemo21, T7Express, Origami2, etc.
  • the cell is selected from Streptomyces, Micromonospora and Nocardia.
  • the cell is a fungus.
  • the cell is selected from yeast or mold.
  • 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.
  • lipid is mixed with the engineered RNA to form lipid nanoparticles.
  • the engineered Poly (A) tail, engineered RNA molecule, and/or engineered DNA molecule of the present application are formulated in lipid nanoparticles.
  • 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).
  • 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.
  • target nucleic acids such as the engineered Poly (A) tail, engineered RNA molecules, and/or engineered DNA molecules of the present application.
  • 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);
  • the phospholipids are preferably selected from one or more of the following compounds:
  • DLPC Dilauroyl phosphatidylcholine
  • DMPC Dimyristoylphosphatidylcholine
  • DOPC Dioleoylphosphatidylcholine
  • DPPC Dipalmitoylphosphatidylcholine
  • DSPC Distearoylphosphatidylcholine
  • DUPC Dioleoylphosphatidylcholine
  • DOPE 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine
  • 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.
  • the PEG lipid is DMG-PEG2000.
  • 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 ...
  • Example 1 Construction of luciferase mRNA containing polyA variants of the present invention
  • the vector uses the Escherichia coli cloning vector pUC57 as the vector backbone, and arranges the T7 promoter sequence (SEQ ID NO: 2), 5'UTR, Kozak sequence (GCCACC), luciferase protein coding sequence, 3'UTR and poly (dA: dT) numbered RG008 in sequence between the Xba I restriction site and the SapI restriction site of its multiple cloning site as a control for the poly (A) variant designed in the present invention.
  • T7 promoter sequence SEQ ID NO: 2
  • 5'UTR 5'UTR
  • Kozak sequence GCCACC
  • luciferase protein coding sequence 3'UTR
  • poly (dA: dT) numbered RG008 poly sequence between the Xba I restriction site and the SapI restriction site of its multiple cloning site as a control for the poly (A) variant designed in the present invention.
  • FIG. 1A Schematic diagrams are shown in FIG. 1A and FIG. 1B .
  • acetic acid solution 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 200 ⁇ g/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.
  • 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) in the liver of mice with low miR-142 expression and the spleen of mice with high miR-142 expression were investigated to detect whether the insertion of miR-142 binding sites could downregulate the expression level of mRNA in organs with high miR-142 expression.
  • mice were randomly divided into groups according to body weight, with 6 mice in each group, and adaptively raised for 2-3 days. Mice were administered by tail vein injection, each injected with 0.05 mg/kg mRNA-LNPs (1 ⁇ g), the injection volume was 100 ⁇ L, and the blank control was injected with an equal volume of PBS. 6 hours after administration, Luciferin (200 ⁇ L) was injected intraperitoneally at a dose of 150 mg/kg, and the mice were anesthetized 10 minutes later, and the small animal imager was used for imaging. The liver and spleen of the mice were dissected and imaged again. The experiment was performed once, and the measured values of each mouse were taken to make a bar graph, and the results are shown in Figures 2A-2B.
  • the miR-142 binding site is inserted into the position 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) of RG008 to form different Poly (A) variants.
  • mRNA with such variants is encapsulated by LNPs and injected into mice through the tail vein, the mRNA will be taken up and degraded in the spleen where macrophages (highly expressing miR-142) are enriched, and the expression is inhibited.
  • the reduction level when the miR-142 binding site is inserted into the 3'UTR, 0A, 14A, 19A, and 30A positions is comparable, which is significantly better than the insertion position of 60A.
  • 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.
  • 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.
  • mice C57BL/6 mice were randomly divided into groups according to body weight, with 6 mice in each group, and adaptively raised for 2-3 days.
  • the mice were administered by tail vein injection, with each mouse injected with 0.05 mg/kg of mRNA-LNPs (1 ⁇ g) and an injection volume of 100 ⁇ L.
  • the blank control was injected with an equal volume of PBS.
  • Luciferin 200 ⁇ L was injected intraperitoneally at a dose of 150 mg/kg.
  • the mice were anesthetized and imaged using a small animal imager.
  • the liver and spleen of the mice were dissected and imaged again.
  • the experiment was performed once, and the measured values of each mouse were taken to make a bar graph, where the absolute values of fluorescence imaging of the liver and spleen are shown in Figures 3A-3B, respectively.
  • the miR-122 binding site is inserted after 0A, 14A, 19A, 30A, and 60A of RG008 to form different Poly (A) variants.
  • 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.
  • 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.
  • 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.
  • the vector plasmid constructed according to the method in Example 1 was confirmed to be correct by sequencing, it was transformed into Escherichia coli DH5 ⁇ , 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.
  • Example 6 Expression test of mRNA containing poly(A) variants of miR-142 binding sites at different insertion positions in cells
  • RAW 246.7 cells were cultured in a 24-well cell culture plate with a cell density of 1x10 5 cells/well. Each well was transfected with 500ng of in vitro synthesized mRNA using Lipofectamine 3000 (purchased from Thermo Fisher Scientific). 24 hours after transfection, the cell culture medium was removed, 200 ⁇ L of cell lysate was added to each well, and after 10 minutes, the cell lysate was transferred to a 1.5mL EP tube and centrifuged at 12000rpm and 4°C for 5 minutes. Subsequently, 20 ⁇ L of the lysate supernatant was transferred to a black, light-proof 96-well plate, and 200 ⁇ L of fluorescein substrate was added and read using a fluorescence detector. The results are shown in Figure 5.
  • the miR-142 binding sites were 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 to form different Poly (A) variants.
  • mRNA carrying such variants was delivered to miR-142 overexpressing cell lines, such as Raw 264.7 cells, the mRNA would be degraded by miR-142 and its expression would be inhibited.
  • RAW 246.7 cells were cultured in a 24-well cell culture plate with a cell density of 1x10 5 cells/well. Each well was transfected with 500ng of in vitro synthesized mRNA using Lipofectamine 3000 (purchased from Thermo Fisher Scientific). 24 hours after transfection, the cell culture medium was removed, 200 ⁇ L of cell lysate was added to each well, and after 10 minutes, the cell lysate was transferred to a 1.5mL EP tube and centrifuged at 12000rpm and 4°C for 5 minutes. Subsequently, 20 ⁇ L of the lysate supernatant was transferred to a black, light-proof 96-well plate, and 200 ⁇ L of fluorescein substrate was added and read using a fluorescence detector. The results are shown in Figure 6.
  • RAW246.7, LSEC, HSC, AML12 cells were cultured in 24-well cell culture plates with a cell density of 1x10 5 cells/well. Each well was transfected with 500ng of in vitro synthesized mRNA using Lipofectamine 3000 (purchased from Thermo Fisher Scientific). 24 hours after transfection, the cell culture medium was removed, 200 ⁇ L of cell lysate was added to each well, and after 10 minutes, the cell lysate was transferred to a 1.5mL EP tube and centrifuged at 12000rpm and 4°C for 5 minutes.
  • the Poly(A) variant contains a miR-142 binding site, and the expression of mRNA in this cell line is reduced.
  • the Poly(A) variant contains a miR-126 binding site, and the expression of mRNA in this cell line is reduced.
  • the Poly(A) variant contains a miR-148a binding site, and the expression of mRNA in this cell line is reduced.
  • miR-126+miR-148a+miR-142 and miR-148a+miR-142+miR-126 performed better than other sequences in the three cells. See Figures 7A-7D, Figures 8A-8C.
  • 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.
  • 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.
  • 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.

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Abstract

—种工程化RNA分子、编码所述RNA分子的DNA分子及其用途。所述工程化RNA分子包含含有MRNA结合位点的Poly（A）尾序列。所述Poly（A）尾可用于使目的基因实现器官、组织和/或细胞的精准表达。

Description

表达可调控的工程化RNA分子
相关申请
本申请要求于2023年6月9日提交的中国临时申请PCT/CN2023/099512、于2023年9月26日提交的中国临时申请PCT/CN2023/121779、和于2024年6月3日提交的中国临时申请202410711025.6的优先权。在先申请的全部内容通过引用整体并入本文。
技术领域
本申请涉及生物技术领域，具体涉及一种包含Poly(A)尾的工程化RNA分子。所述Poly(A)尾使得所述工程化RNA分子在特定器官、组织和/或细胞中精准表达。
 背景技术
脱氧核糖核酸(DNA)和信使核糖核酸(mRNA)是生物遗传物质的主要组成物质，其主要作用是携带和传递遗传信息。在真核细胞内，完整的mRNA主要包括5’的帽子结构、5’非翻译区(5’untranslated region,5’UTR)、编码区(ORF)、3’非翻译区(3’untranslated region,3’UTR)以及3’多聚腺苷酸尾部结构poly(A)。5’的帽子结构主要调控mRNA的稳定性以及mRNA翻译的启动，帽子结构通过将5’端封闭起来，以避免mRNA被核酸外切酶水解，并且其可以通过被帽子结合蛋白(cap binding protein，eIF-4E)识别并结合，调控mRNA与核糖体结合，进而启动翻译过程。5’UTR是帽子与起始密码子之间的一段较短的序列，过短或过长都不利于mRNA的翻译起始。一般认为3’UTR主要参与转录后调控，包括调节mRNA的体内半衰期，比如，很多miRNA可以与靶基因mRNA的3’UTR结合，通过降解或者结合抑制的形式降低靶基因的表达。在3’UTR之后还有一段多聚A(poly(A))的序列，可以防止外切酶降解，此外，poly(A)尾巴序列还可以与多聚腺苷酸结合蛋白(PABP)结合，并进一步招募eIF4G、eIF4B、Paip-1等多种蛋白形成复合体，参与mRNA稳定性调节及翻译起始过程。真核生物细胞内天然mRNA的poly(A)尾巴是通过多聚腺苷酸聚合酶在转录后添加的，在包括从真菌、植物到动物的进化过程中该聚合酶具有对腺苷酸的底物特异性。至今未发现明确的证据表明在天然的poly(A)尾结构中能形成具有特异调控功能的复杂非腺苷酸motif。
mRNA疗法的应用领域广泛，根据mRNA疗法不同应用主要分为以下方向：传染病疫苗方向、肿瘤免疫治疗方向、单抗药物及其他蛋白类药物替代方向、基因编辑方向。mRNA疫苗在新冠流行期间的成功也证实了mRNA平台的巨大价值。mRNA疗法通常通过基于脂质的载体系统(包括脂质体和脂质纳米颗粒)来递送mRNA。这些脂质载体通常封装mRNA并且可以改善mRNA的细胞内递送和有效性。脂质纳米颗粒(LNP)制剂代表了核酸递送领域的一场革命。LNP通常包括一种或多种阳离子脂质和/或氨基(可离子化)脂质、磷脂、结构脂质(如胆固醇)和/或含有聚乙二醇的脂质(PEG脂质)。阳离子和/或可离子化的脂质包括，例如，含有胺的脂质，可以很容易地质子化。
尽管LNP介导的核酸递送相关研究已取得长足进步,但在目前的递送体系中几乎都有很强的嗜肝性。由于LNP递送脂质的构效关系不清晰，难以理性设计器官、组织高度特异的、尤其是同一个器官和组织的不同细胞类型特异的递送载体，导致mRNA-LNP会广泛积聚到多个器官、组织和/或细胞类型中。如何将mRNA递送到特定器官、组织和/或细胞中以实现所递送的mRNA在特定器官、组织和/或细胞中特异性表达以及减少其他器官、组织和/或细胞的副作用也是此类疗法的主要挑战。
miRNA(又称microRNA)是在植物、动物和一些病毒中发现的小型单链、19-25个核苷酸长度的非编码RNA分子,其功能主要是mRNA降解或沉默和基因表达的转录后调节。它们通过Watson-Crick碱基配对与目标mRNA分子3'非翻译区(3'UTR)内的特定序列互补进而导致这些mRNA分子被沉默。miRNA的表达在器官、组织、细胞方面具有高度特异性，miRNA在组织特异性表达的例子有肝脏(miRNA-122)、脾脏(miRNA-142)等。miRNA结合序列通常位于mRNA的3’UTR。本领域已经记载了将miRNA结合位点整合到3'UTR中以改善脱靶表达。
在mRNA领域，体外制备mRNA药物的第一步，是以含有设计好的产品序列的线性化质粒为模板通过体外转录(IVT)合成，Poly(A)尾则通常以共转录的方式添加在3’UTR下游。为实现共转录添加Poly(A)，需要在模板质粒中包含相对应的poly(dA:dT)序列。然而，质粒中poly(dA:dT)重复序列在大肠杆菌中复制过程中不稳定，经常发生缺失突变导致poly(dA:dT)变短。这种现象不利于通过发酵规模化生产体外转录模板质粒的制备工艺，Poly(A)截短对mRNA的体内稳定性和生物学活性产生显著影响。因此，mRNA的质粒复制稳定性是mRNA药物/疫苗的重要考察因素。
mRNA领域目前仍亟需开发可递送至特定器官、组织和/或细胞且质量、活性可控，易于工业化生产的mRNA疫苗或药物,以及调节所递送的mRNA在特定器官、组织和/或细胞中的表达的工具及方法。
申请概述
天然mRNA的polyA中不存在miRNA结合位点，本申请发明人创造性地通过将miRNA结合位点整合入Poly(A)尾，在增强mRNA在特定器官、组织和/或细胞中表达的特异性的同时，兼顾了mRNA的质粒复制稳定性，以使生产出的mRNA质量可控、药效稳定。
具体地，本申请提供了：
1、一种工程化Poly(A)尾，所述Poly(A)尾包含miRNA结合位点。所述工程化Poly(A)尾包含的miRNA结合位点可以是一个、两个或更多个。
在一些实施方案中，所述两个或更多个miRNA结合位点序列彼此毗邻或由一个或多个碱基相分隔。
2、根据方案1的工程化Poly(A)尾，其中所述Poly(A)尾包含如下的式(I)：
nA-miRNA结合处-mA    式(I)；
其中，miRNA结合处包含一个或多个miRNA结合位点，
nA表示与所述miRNA结合处5'末端毗邻的连续n个腺苷酸(A)，
mA表示与所述miRNA结合处3'末端毗邻的连续m个腺苷酸(A)，
m和n为自然数，且m+n≤150、m+n≤120、m+n≤100、m+n≤80、m+n≤60、m+n≤30、m+n≤19、或m+n≤14。在一些实施方案中，所述m与n之和为：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、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、或150。
在一些实施方案中，miRNA结合处中一个或多个miRNA结合位点直接相连或通过间隔序列相连。所述间隔序列由一个或多个核苷酸组成。在一些实施方案中，所述间隔序列由一个或多个非A核苷酸组成。
在一些实施方案中，式(I)中的miRNA结合处由1、2、3、4、5或6个miRNA结合位点组成。在一些实施方案中，式(I)中的miRNA结合处由一个miRNA结合位点组成。在一些实施方案中，式(I)中的miRNA结合处包含两个或更多个相毗邻或通过一个或多个核苷酸相连接的miRNA结合位点。在一些实施方案中，式(I)中的miRNA结合处包含三个或更多个相毗邻或通过一个或多个核苷酸相连接的miRNA结合位点。在一些实施方案中，所述Poly(A)尾中仅有一个式(I)。在一些实施方案中，所述Poly(A)尾为式(I)。
3、根据方案2的工程化Poly(A)尾，其中n＝0或n≥1。
4、根据方案2的工程化Poly(A)尾，其中m＝0。
5、根据方案2的工程化Poly(A)尾，其中n≤80、n≤60、n≤30、n≤19、n≤14或n≤10。在一些实施方案中，14≤n≤30、14≤n≤19或19≤n≤30。在一些实施方案中，所述n为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或80。
6、根据方案1至5中任一项的工程化Poly(A)尾，其中所述Poly(A)尾的长度为80至240nt，例如100至200nt，101至150nt、120至150nt、130至140nt、123nt至135或125至139nt。在一些实施方案中，所述Poly(A)尾的长度为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、143nt、144nt、145nt、146nt、147nt、148nt、149nt、150nt、151nt、152nt、153nt、154nt、155nt、156nt、157nt、158nt、159nt、160nt、161nt、162nt、163nt、164nt、165nt、166nt、167nt、168nt、169nt、170nt、171nt、172nt、173nt、174nt、175nt、176nt、177nt、178nt、179nt、180nt、181nt、182nt、183nt、184nt、185nt、186nt、187nt、188nt、189nt、190nt、191nt、192nt、193nt、194nt、195nt、196nt、197nt、198nt、199nt、200nt、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、或239nt。
7、根据方案1至6中任一项所述的工程化Poly(A)尾，其中所述Poly(A)尾：
在式(I)的3'一侧进一步包含与式(I)结构的3'末端直接相连的尾部片段，优选所述尾部片段5’末端的核苷酸不为A；和/或
在式(I)的5'一侧进一步包含与式(I)结构的5'末端直接相连的头部片段，优选所述头部片段3’末端的核苷酸不为A。
在一些实施方案中，所述尾部片段或头部片段由一个或多个非A核苷酸以及多个A组成，例如包括1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个非A核苷酸。
在一些实施方案中，所述尾部片段或头部片段的核苷酸数量为p,其中p≤80、p≤60、p≤30、p≤19、或p≤14。在一些实施方案中，所述p为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。
在一些实施方案中，所述尾部片段或头部片段由一个或多个元件c和/或一个或多个元件d与一个或多个A组成，
所述元件c为一个非A的核苷酸；例如T、U、C或G；
所述元件d由任意两个或更多个连续的核苷酸组成，元件d的5’及3’末端的核苷酸不为A核苷酸；
所述元件d的长度范围为2nt≤d≤30nt；优选6nt≤d≤20nt，更优选6nt≤d≤12nt；
其中所述元件c和元件d不相邻。
在一些实施方案中，所述元件d的长度为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或30nt。
在一些实施方案中，所述元件d由任意两个或更多个连续的核苷酸组成，元件d不包含三个以上连续的A，所述核苷酸选自A、U、C、G核苷酸，其中元件d的5’及3’末端的核苷酸不为A核苷酸，优选所述元件d的长度范围为2nt≤d≤30nt，优选6nt≤d≤20nt，更优选6nt≤d≤12nt。
在一些实施方案中，其中所述元件c的个数为0个、1个、2至10个、3个至8个、或4至6个或2-5个，例如7个。在一些实施方案中，元件c的个数为0、1、2、3、4、5、6、7、8、9、10个。
在一些实施方案中，其中所述元件d的个数为0-5，优选1-3个，例如2或4个。在一些实施方案中，元件d的个数为0、1、2、3、4、5个。
在一些实施方案中，其中当元件c和元件d同时存在时，所述元件c和元件d的个数总和为2-15个，优选3-5个，进一步优选3个。在一些实施方案中，所述元件c和元件d的个数总和为4、5、6、7、8、9、10、11、12、13或14个。
在一些实施方案中，其中在所述Poly(A)尾靠近3’末端的1/2部分，包含一个或多个非A核苷酸。
在一些实施方案中，在所述Poly(A)尾靠近3’末端的1/3处包含一个或多个非A核苷酸。
在一些实施方案中，在所述Poly(A)尾靠近3’末端的1/4处包含一个或多个非A核苷酸。
根据前述任一种方案中所述的工程化Poly(A)尾，其在所述miRNA结合处的5'一侧包含0至60、0至30、0至20、0至14、或0至10个核苷酸，例如1、2、3、4、5、6、7、8、9、10、11、12或13个核苷酸。
8.根据方案1至7中任一项的工程化Poly(A)尾,所述工程化Poly(A)尾由所述miRNA结合处插入如下所示的Poly(A)尾结构中形成：
元件a-元件c-元件b-元件c-元件b-元件c-元件b-元件c-元件b；
元件b-元件c-元件b-元件c-元件a-元件d-元件b-元件c-元件b-元件c-元件b；
元件b-元件c-元件b-元件c-元件b-元件d-元件a-元件c；
元件a-元件d-元件b-元件c-元件b-元件c-元件b；或
元件b-元件c-元件b-元件c-元件b-元件d-元件a；
元件a-元件d-元件b；
元件a-元件d-元件a；
元件b-元件d-元件a；
元件b-元件d-元件b；
元件a-元件d-A
元件a-元件c-A
可选地：
1)由所述miRNA结合处替换任1个、2个、3个、4个、5个或多个元件形成；
2)由所述miRNA结合处插入到任意两个元件之间形成；或
3)由所述miRNA结合处插入到元件a或元件b中的任一两个A之间形成。
在所述Poly(A)尾结构中，所述元件a由多个连续的腺嘌呤(A)核苷酸组成，所述元件a的长度范围为100nt≥a＞30nt，优选80nt≥a≥60nt；
所述元件b由多个连续的A核苷酸组成，所述元件b的长度范围所述元件b的长度范围为10nt≤b≤30nt，优选14nt≤b≤20nt；
所述元件c由一个非A的核苷酸组成；
所述元件d由任意两个或更多个连续的核苷酸组成，元件d不包含三个以上连续的A，所述核苷酸选自A、T、C、G核苷酸，其中元件d的5’及3’末端的核苷酸不为A核苷酸，优选所述元件d的长度范围为2nt≤d≤30nt，优选6nt≤d≤20nt，更优选6nt≤d≤12nt。
在一些实施方案中，所述元件a为31nt、32nt、33nt、34nt、35nt、36nt、37nt、38nt、39nt、40nt、41nt、42nt、43nt、44nt、45nt、46nt、47nt、48nt、49nt、50nt、51nt、52nt、53nt、54nt、55nt、56nt、57nt、58nt、59nt、60nt、61nt、62nt、63nt、64nt、65nt、66nt、67nt、68nt、69nt、70nt、71nt、72nt、73nt、74nt、75nt、76nt、77nt、78nt、79nt、80nt、81nt、82nt、83nt、84nt、85nt、86nt、87nt、88nt、89nt、90nt、91nt、92nt、93nt、94nt、95nt、96nt、97nt、98nt、99nt、100nt。
在一些实施方案中，所述元件b为10nt、11nt、12nt、13nt、14nt、15nt、16nt、17nt、18nt、19nt、20nt、21nt、22nt、23nt、24nt、25nt、26nt、27nt、28nt、29nt或30nt。
在一些实施方案中，所述元件d为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、29n或30nt。
9、根据方案1至7中任一项的工程化Poly(A)尾,所述工程化Poly(A)尾由所述miRNA结合处插入到WO2020074642A1、WO2022028559A1、US_10717982中记载的Poly(A)尾结构中形成。
10、根据方案1-9中任一项所述的工程化Poly(A)尾，其中所述元件c为G。
11、根据方案1-10中任一项所述的工程化Poly(A)尾，其中所述元件d包含回文序列。
12、根据方案1-11中任一项所述的工程化Poly(A)尾，其中所述元件d长度为6nt。
13、根据方案1-12中任一项所述的工程化Poly(A)尾，其中所述元件d选自以下序列中的任一种或多种：GATATC(SEQ ID NO：60)、GTATAC(SEQ ID NO：61)、GAATCT(SEQ ID NO：62)、GCATATGACT(SEQ ID NO：63)及GATATCGTATAC(SEQ ID NO：64)。
14、根据方案1-12中任一项所述的工程化Poly(A)尾，其中所述元件d如GATATC(SEQ ID NO：60)所示。
15、根据方案1-14中任一项所述的工程化Poly(A)尾，其中所述Poly(A)尾在miRNA结合处5'一侧包含的核苷酸个数与所述Poly(A)尾在miRNA结合处3'一侧包含的核苷酸个数的比值小于1/1、1/2、1/3、1/4、1/5、1/6、1/7、1/8、1/9、或1/10。
16、根据方案6-15中任一项所述的工程化Poly(A)尾，所述尾部片段的结构为：元件d-元件b-元件c-元件b-元件c-元件b或元件c-元件b-元件c-元件b,优选的所述尾部片段的结构为：元件d-19A-元件c-19A-元件c-17A或元件c-19A-元件c-17A，更优选的所述尾部片段的结构为：GATATC-19A-G-19A-G-17A。在一些实施方案中，所述的工程化Poly(A)尾不包含头部片段。在一些实施方案中，所述工程化Poly(A)尾由尾部片段和式(I)组成。在一些实施方案中，所述工程化Poly(A)尾由头部片段、尾部片段和式(I)组成。
在根据前述1-15中任一项所述的工程化Poly(A)尾，其结构选自以下的任一种：
1)nA-miRNA结合处-mA，其中150≥m+n≥50，优选的，m+n＝120或m+n＝60；
2)nA-miRNA结合处-mA-元件d-元件b-元件c-元件b-元件c-元件b，其中80≥m+n≥60，优选的，m+n＝60；
3)元件a-元件d-nA-miRNA结合处-mA-元件c-元件b-元件c-元件b，其中30≥m+n≥10,优选的，m+n＝19；
4)nA-miRNA结合处-mA-元件c-元件b-元件c-元件b，其中110≥m+n≥70，优选的，m+n＝79；
5)nA-miRNA结合处-mA-元件c-元件b-元件c-元件b-元件d-元件a或元件b-元件c-nA-miRNA结合处-mA-元件c-元件b-元件d-元件a,其中19≥m+n≥14,优选的，m+n＝19。
在根据前述1-15中任一项所述的工程化Poly(A)尾中，其结构选为：
nA-miRNA结合处-mA-元件d-元件b-元件c-元件b-元件c-元件b，其中80≥m+n≥60，优选的，m+n＝60；进一步优选地，30≥n≥14；更优选地，19≥n≥14。
在根据前述1-15中任一项所述的工程化Poly(A)尾中，其结构选自以下的任一种：
1)nA-miRNA结合处-mA，其中m+n＝120或m+n＝100或m+n＝60；
2)nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，m+n＝60；
3)60A-元件d-nA-miRNA结合处-mA-G-19A-G-17A，m+n＝19；
4)nA-miRNA结合处-mA-G-19A-G-17A，m+n＝79；
5)nA-miRNA结合处-mA-G-19A-G-19A-元件d-60A或19A-G-nA-miRNA结合处-mA-G-19A-元件d-60A,其中m+n＝19。
在根据前述1-15中任一项所述的工程化Poly(A)尾中，其结构为：
nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，m+n＝60且30≥n≥14；优选地，19≥n≥14。
在根据前述1-15中任一项所述的工程化Poly(A)尾中，其结构选自以下的任一种：
1)nA-miRNA结合处-mA，其中m+n＝120或m+n＝100；
2)nA-miRNA结合处-mA-SEQ ID NO：60-19A-G-19A-G-17A，m+n＝60；
3)60A-SEQ ID NO：60-nA-miRNA结合处-mA-G-19A-G-17A，m+n＝19；
4)nA-miRNA结合处-mA-G-19A-G-17A，m+n＝79；
5)nA-miRNA结合处-mA-G-19A-G-19A-SEQ ID NO：60 -60A或19A-G-nA-miRNA结合处-mA-G-19A-元件d-60A,其中m+n＝19。
在根据前述1-15中任一项所述的工程化Poly(A)尾中，其结构为：
nA-miRNA结合处-mA-SEQ ID NO：60-19A-G-19A-G-17A，m+n＝60且30≥n≥14，优选地，19≥n≥14。
17、根据方案1-16中任一项所述的工程化Poly(A)尾，其中，所述miRNA结合处所结合的miRNA选自以下的一种或多种：
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和miR-204。
在一些实施方案中，所述miRNA选自miR-142,miR-122,miR-126,和miR-148a中的一种或多种。
在一些实施方案中，所述miRNA选自miR-142-3p,miR-122-5p,miR-126-3p和miR-148a-3p中的一种或多种。
在一些实施方案中，所述miRNA包括或由其组成：miR142和miR-122，miR142和miR-126，miR-142和miR-148a，miR-122和miR-126,miR-122和miR-148a,或miR-126和miR-148a。
在一些实施方案中，所述miRNA包括或由其组成：miR-142、miR-122和miR-126，miR-142、miR-122和miR-148a，miR-142、miR-126和miR-148a，或miR-122、miR-126和miR-148a。
在一些实施方案中，所述miRNA包括或由其组成：miR-142、miR-122、miR-126和miR-148a。
在一些实施方案中，所述miRNA包括miR142和miR-122或由miR142和miR-122组成，且所述Poly(A)的miRNA结合处中，miR142结合位点位于miR-122结合位点的5'一侧或所述miR142结合位点位于miR-122结合位点的3'一侧，所述miR142结合位点与miR-122结合位点彼此毗邻。
在一些实施方案中，所述miRNA包括miR142和miR-122，且所述Poly(A)的结合处中，miR142结合位点位于miR-122结合位点的5'一侧或所述miR142位点位于miR-122位点的3'一侧，所述miR142结合位点与miR-122结合位点由一个或多个核苷酸相分隔。
在一些实施方案中，所述miRNA包括miR142和miR-126或由miR142和miR-126组成，且所述Poly(A)的结合处中，miR142结合位点位于miR-126结合位点的5'一侧或所述miR142位点位于miR-126位点的3'一侧，所述miR142结合位点与miR-126结合位点彼此毗邻。
在一些实施方案中，所述miRNA包括miR142和miR-126，且所述Poly(A)的结合处中，miR142结合位点位于miR-126结合位点的5'一侧或所述miR142位点位于miR-126位点的3'一侧，所述miR142结合位点与miR-126结合位点由一个或多个核苷酸相分隔。
在一些实施方案中，所述miRNA包括miR-142和miR-148a或由miR-142和miR-148a组成，且在所述Poly(A)的结合处中，miR-142结合位点位于miR-148a结合位点的5'一侧或miR-142结合位点位于miR-148a结合位点的3'一侧，所述miR-142结合位点与miR-148a结合位点彼此毗邻。
在一些实施方案中，所述miRNA包括miR-142和miR-148a，且在所述Poly(A)的结合处中，miR-142结合位点位于miR-148a结合位点的5'一侧或miR-142结合位点位于miR-148a结合位点的3'一侧，所述miR-142结合位点与miR-148a结合位点由一个或多个核苷酸相分隔。
在一些实施方案中，所述miRNA包括miR-122和miR-126或由miR-122和miR-126组成，且在所述Poly(A)的结合处中，miR-122结合位点位于miR-miR-126结合位点的5'一侧或miR-122结合位点位于miR-126结合位点的3'一侧，所述miR-122结合位点与miR-126结合位点彼此毗邻。
在一些实施方案中，所述miRNA包括miR-122和miR-126，且在所述Poly(A)的结合处中，miR-122结合位点位于miR-126结合位点的5'一侧或miR-122结合位点位于miR-126结合位点的3'一侧，所述miR-122结合位点与miR-126结合位点由一个或多个核苷酸相分隔。
在一些实施方案中，所述miRNA包括miR-122和miR-148a或由miR-122和miR-148a组成，且在所述Poly(A)的结合处中，miR-122结合位点位于miR-148a结合位点的5'一侧或miR-122结合位点位于miR-148a结合位点的3'一侧，所述miR-122结合位点与miR-148a结合位点彼此毗邻。
在一些实施方案中，所述miRNA包括miR-122和miR-148a，且在所述Poly(A)的结合处中，miR-122结合位点位于miR-148a结合位点的5'一侧或miR-122结合位点位于miR-148a结合位点的3'一侧，所述miR-122结合位点与miR-148a结合位点由一个或多个核苷酸相分隔。
在一些实施方案中，所述miRNA包括miR-126和miR-148a或由miR-126和miR-148a组成，且在所述Poly(A)的结合处中，miR-126结合位点位于miR-148a结合位点的5'一侧或miR-126结合位点位于miR-148a结合位点的3'一侧，所述miR-126结合位点与miR-148a结合位点彼此毗邻。
在一些实施方案中，所述miRNA包括miR-126和miR-148a，且在所述Poly(A)的结合处中，miR-126结合位点位于miR-148a结合位点的5'一侧或miR-126结合位点位于miR-148a结合位点的3'一侧，所述miR-126结合位点与miR-148a结合位点由一个或多个核苷酸相分隔。
在一些实施方案中，所述miRNA包括miRNA-142、miR-148a和miR-126，并且在所述Poly(A)的miRNA结合处中，各miRNA结合位点从5'至3'的排列顺序为：
miR-126结合位点、miR-148a结合位点及miR-142结合位点；
miR-148a结合位点、miR-142结合位点及miR-126结合位点；
miR-142结合位点、miR-148a结合位点和miR-126结合位点；
miR-126结合位点、miR-142结合位点及miR-148a结合位点；
miR-148a结合位点、miR-126结合位点及miR-142结合位点；或
miR-142结合位点、miR-126结合位点和miR-148a结合位点；
其中miR-126结合位点、miR-148a结合位点和miR-142结合位点彼此毗邻。
在一些实施方案中，所述miRNA包括miRNA-142、miR-148a和miR-126，并且在所述Poly(A)的miRNA结合处中，各miRNA结合位点从5'至3'的排列顺序为：
miR-126结合位点、miR-148a结合位点及miR-142结合位点；
miR-148a结合位点、miR-142结合位点及miR-126结合位点；
miR-142结合位点、miR-148a结合位点和miR-126结合位点；
miR-126结合位点、miR-142结合位点及miR-148a结合位点；
miR-148a结合位点、miR-126结合位点及miR-142结合位点；或
miR-142结合位点、miR-126结合位点和miR-148a结合位点；
其中miR-126结合位点、miR-148a结合位点和miR-142结合位点彼此之间由一个或多个核苷酸相分隔。
在一些实施方案中，所述miRNA包括miRNA-142、miR-148a和miR-126，并且在所述Poly(A)的miRNA结合处中，各miRNA结合位点从5'至3'的排列顺序为：
miR-126结合位点、miR-148a结合位点及miR-142结合位点；
miR-148a结合位点、miR-142结合位点及miR-126结合位点；
miR-142结合位点、miR-148a结合位点和miR-126结合位点；
miR-126结合位点、miR-142结合位点及miR-148a结合位点；
miR-148a结合位点、miR-126结合位点及miR-142结合位点；或
miR-142结合位点、miR-126结合位点和miR-148a结合位点；
其中miR-126结合位点、miR-148a结合位点和miR-142结合位点中有两个相邻的miRNA结合位点彼此毗连，另外两个相邻的miRNA结合位点由一个或多个核苷酸相分隔。
在根据前述任一项所述的工程化Poly(A)尾中，其结构选自以下的任一种：
1)nA-miRNA结合处-mA，其中n＝0，m＝120或m＝100或m＝60；
2)nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，其中m+n＝60,n≤20或n≤14或n≤10；
3)nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，其中n＝0，m＝60；
4)nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，其中m+n＝60,14≤n≤30；优选14≤n≤19；
5)nA-miRNA结合处-mA-G-19A-G-17A，其中n＝60，m＝19；
其中优选地所述元件d为回文序列，更优选的所述元件d的多核苷酸序列如SEQ ID NO:60至64中任一项所示。
在根据前述任一项所述的工程化Poly(A)尾中，其结构选自以下的任一种：
1)nA-miRNA结合处-mA，其中n＝0，m＝120或m＝100或m＝60，所述miRNA结合处为miR-122、miR-126或miR-142的结合位点；
2)nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，其中n＝14，m＝46，所述miRNA结合处为miR-122、miR-126或miR-142的结合位点；
3)nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，其中n＝19，m＝41，所述miRNA结合处为miR-122、miR-126或miR-142的结合位点；
4)nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，其中n＝30，m＝30，所述miRNA结合处为miR-122、miR-126或miR-142的结合位点；
5)nA-miRNA结合处-mA-G-19A-G-17A，其中n＝60，m＝19，所述miRNA结合处为miR-122、miR-126或miR142的结合位点；
6)nA-miRNA结合处-mA-元件d-19A-G-19A-G-17A，其中n＝0，m＝60，miRNA结合处为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、或miR148a-miR126-miR142的结合位点；或
7)nA-miR-122结合处-mA-元件d-19A-G-19A-G-17A，其中n＝1、2、3、4、5、6、7、8、9或10，且n+m＝60，
8)nA-miR-142结合处-mA-元件d-19A-G-19A-G-17A，其中n＝1、2、3、4、5、6、7、8、9或10，且n+m＝60，
9)nA-miR-126结合处-mA-元件d-19A-G-19A-G-17A，其中n＝1、2、3、4、5、6、7、8、9或10，且n+m＝60，
10)nA-miR-148a结合处-mA-元件d-19A-G-19A-G-17A，其中n＝1、2、3、4、5、6、7、8、9或10，且n+m＝60，
其中优选地所述元件d为回文序列，更优选的所述元件d的多核苷酸序列如SEQ ID NO:60至64中任一项所示，优选SEQ ID NO:60。
在一些实施方案中，所述工程化Poly(A)尾的miRNA结合处由选自以下一个、两个、三个、四个或更多个miRNA结合位点序列组成：SEQ ID NO：4、SEQ ID NO：17、SEQ ID NO：32或SEQ ID NO：53。在一些实施方案中，所述工程化Poly(A)尾的miRNA结合处由选自以下一个、两个、三个、四个或更多个miRNA结合位点序列：SEQ ID NO：4、SEQ ID NO：17、SEQ ID NO：32或SEQ ID NO：53以及所述miRNA结合位点之间的0个、1个或多个核苷酸组成。
18、根据方案1-17中任一项所述的工程化Poly(A)尾，其中所述Poly(A)尾的序列包含选自以下的多核苷酸序列或为下述多核苷酸序列中的任一种：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。
19、工程化RNA分子，其为mRNA或非编码RNA，其包含根据方案1-18中任一项所述Poly(A)尾以及位于所述Poly(A)尾5’一侧的目的基因序列。在一些实施方案中，所述Poly(A)尾中的式(I)中的nA与3’UTR直接相连。
[根据细则91更正 07.06.2024]
20、工程化DNA分子，其编码包含根据方案1-18中任一项所述的工程化Poly(A)尾，或根据方案19所述的工程化RNA序列。
21、根据方案20所述的工程化DNA分子，所述DNA分子为质粒或病毒载体。
22、DNA与RNA的杂合分子，其携带与根据方案1-18中任一项所述的工程化Poly(A)尾，根据方案19所述的工程化RNA分子或根据方案20或21所述的工程化DNA分子相同的遗传信息。
[根据细则91更正 07.06.2024]
23、工程化细胞，其包含根据方案1-18中任一项所述的工程化Poly(A)尾，根据方案19所述的工程化RNA分子，根据方案20或21所述的工程化DNA分子，或根据方案22所述的杂合分子，其中所述细胞为的真核或原核细胞。
24、根据方案23所述的工程化细胞，其中所述原核细胞为大肠杆菌。
25、将根据方案1-18中任一项所述的工程化Poly(A)尾用于使目的基因在特定器官、组织和/或细胞中低表达的用途，所述特定器官、组织和/或细胞高表达所述miRNA或
将根据方案1-18中任一项所述的工程化Poly(A)尾用于使目的基因在特定器官、组织和/或细胞中特异性表达的用途，优选地，所述器官或组织优选肝或脾器官组织；更优选地，所述细胞为肝实质细胞。
26、将根据方案20或21所述的工程化DNA分子，或根据方案22所述的杂合分子用于使编码所述Poly(A)尾的DNA编码序列在宿主细胞中的复制更加保守的用途。
27、根据方案26的用途，所述宿主细胞为原核细胞，优选大肠杆菌。
28、脂质纳米颗粒，其包含根据方案1-18中任一项所述的工程化Poly(A)尾，根据方案19所述的工程化RNA分子，根据20或21所述的工程化DNA分子，或根据方案22所述的杂合分子。
29、病毒颗粒，其包含根据方案1-18中任一项所述的工程化Poly(A)尾，根据方案19所述的工程化RNA分子，根据20或21所述的工程化DNA分子，或根据方案22所述的杂合分子。
本领域技术人员应当知晓，在所述miRNA结合处5’末端核苷酸的5’一侧还可以进一步包含0个、1个或更多个非A核苷酸，该技术方案，也应属于本申请涵盖的范畴，或作为本申请方案的等同方案。
应当理解，本文描述的本申请的方面和实施方案包括“包含”，“组成”和“基本上由……组成”的方面和实施方案。以上详细描述了本申请的优选实施方案，但是，本申请并不限于此。在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本申请所公开的内容，均属于本申请的保护范围。
 附图说明
图1A-1B示出了本发明自主设计的含有miR-142/miR-122结合位点的5种Poly(A)的组成示意图；
图2A-2B示出了带有本发明自主设计的含有miR-142结合位点的5种poly(A)的荧光素酶mRNA在小鼠中表达后，解剖得到小鼠脾脏/肝脏进行的荧光成像的绝对数值结果。
图3A-3B示出了带有本发明自主设计的含有miR-122结合位点的5种poly(A)的荧光素酶mRNA在小鼠中表达后，解剖得到小鼠肝脏/脾脏进行的荧光成像的绝对数值结果。
图4示出了带有本发明自主设计的含有miRNA结合位点miR-142/miR-122的不同poly(A)的荧光素酶DNA质粒在大肠杆菌DH5α中的碱基缺失统计。
图5示出了带有本发明自主设计的含有miR-142结合位点的8种poly(A)的荧光素酶mRNA在哺乳动物细胞系Raw 264.7中的酶活性检测的绝对数值结果。
图6示出了带有miR-142结合位点和miR-122结合位点poly(A)的荧光素酶mRNA在哺乳动物细胞系Raw 264.7中的酶活性检测的绝对数值结果。
图7A-7D示出了带有本发明自主设计poly(A)变体的荧光素酶mRNA在哺乳动物细胞系Raw 264.7/LSEC/HSC/AML中的酶活性检测的绝对数值结果。
图8A-8C示出了带有本发明自主设计poly(A)变体的荧光素酶mRNA在Raw 264.7/LSEC/HSC的表达相对于肝实质细胞AML表达降低的比例。
发明详述
本申请首先提供了一种实现特定器官、组织和/或细胞特异性表达的工具，即通过将miRNA结合位点整合入Poly(A)尾，特异性地关闭不希望mRNA表达的器官、组织和/或细胞中的mRNA表达，以减少mRNA全身表达的脱靶毒副作用。
本申请还提供了在体外稳定扩增Poly(A)尾转录模板DNA的方法，以减少所述DNA在细胞中大量复制时，其中Poly(A)尾转录模板序列的突变频率。从而，基于所述DNA获得大量包含序列确定的Poly(A)尾的RNA。在此基础上，使得经过工程化设计而具有某种特定功能的Poly(A)尾的RNA，例如mRNA，得以通过体外发酵，实现规模化生产。
术语
在本申请中，Poly(A)尾中的“元件c”、“元件d”、nA、mA、尾部片段、头部片段、miRNA结合位点及miRNA结合处均可以术语“元件”指代。其中，“miRNA结合处”、“nA”、“mA”、“元件c”及“元件d”互不重合也互不包含，确定“miRNA结合处”、“nA”、“mA”、“元件c”及“元件d”的方式可以是：
首先通过序列比对的方式，在Poly(A)尾中确定miRNA结合位点，然后以所有miRNA结合位点中最靠近所述Poly(A)尾5’末端的miRNA结合位点的5'一侧最邻近的A为nA的3’末端，从而确定所述连续n个A为nA；并以所有miRNA结合位点中最靠近所述Poly(A)尾3’末端的miRNA结合位点的3'一侧最邻近的A为mA的5’末端，从而确定所述连续m个A为mA；nA和mA之间的部分即为miRNA结合处。所述Poly(A)尾如果在nA的5’一侧具有多核苷酸或多核苷酸序列，则所述多核苷酸或多核苷酸序列为头部片段；所述Poly(A)尾如果在mA的3’一侧具有多核苷酸或多核苷酸序列，则所述多核苷酸或多核苷酸序列为尾部片段。确定“元件d”和“元件c”的方式是首先从目标序列中确定元件d，所述元件d为目标序列中一段包含非A碱基的片段，由任意两个或更多个连续的核苷酸组成，元件d的5’及3’末端的核苷酸不为A核苷酸，元件d不包含三个以上连续的A，且元件d的5’及3’末端均与至少2个A毗邻，在元件d确定后，再从所述目标序列中元件d以外的部分中确定所有非A碱基分别为“元件c”。在一些实施方案中，所述Poly(A)尾包含多个miRNA结合处，且每个结合处中的各miRNA结合位点之间直接相连或通过非A碱基相连。在一些实施方案中，所述Poly(A)尾包含多个miRNA结合处，且每个结合处中的各miRNA结合位点之间直接相连或通过核酸序列相连，所述核酸序列中不包含三个以上连续的A。
如本文所用，“miRNA”，即微RNA或microRNA，是一类短非编码RNA，长度通常小于22bp。其通过与mRNA上的靶序列(即miRNA结合位点)结合，发挥转录后调控作用。如本文所用，优选的，“miRNA结合位点”即与miRNA结合后，下调mRNA表达的miRNA结合位点。
在本文中，“编码”是指i)DNA序列中包含可被转录成RNA分子的遗传信息，和/或ii)RNA分子中包含可被翻译成氨基酸序列的遗传信息。因此，如本文所用，“编码序列”可用以指代mRNA前体或成熟mRNA中可以被翻译为蛋白质的核糖核苷酸(RNA)序列或其片段，亦可指代作为模板用以转录所述mRNA前体或成熟mRNA的脱氧核糖核苷酸(DNA)序列的互补序列或其片段。此外，本申请的“编码序列”还可以进一步包含编码蛋白、功能性核酸、或其片段，例如miRNA、shRNA、dsRNA、向导RNA、Poly(A)尾、5’UTR、3’UTR等的多核苷酸序列。其中，包含可被转录成RNA分子的遗传信息的DNA分子称为所述RNA分子的“编码核酸”；包含可被翻译成氨基酸序列的遗传信息的RNA分子称为所述氨基酸序列的“编码核酸”。
在本申请中，所有多核苷酸序列中的核苷酸由5’末端向3’末端编号，即5’末端的核苷酸为第一个核苷酸，3’末端的核苷酸为最后一个核苷酸。如无特别指明，“5’端”与“5’末端”可互换使用，“3’端”与“3’末端”可互换使用。特别的，“5’末端”与“3’末端”可分别用于描述一条核酸序列或一条核酸序列的某一个区段的第一个及最后一个核苷酸所在的位置。此外，“5’”与“3’”尤其用于描述同一条核酸序列中，核苷酸之间，核苷酸序列区段之间，或核苷酸与核苷酸序列区段之间的相对位置关系；例如“5’一侧”用以描述同一条多核苷酸序列中两段相互之间无重叠部分的序列的相对位置关系，当描述一段序列位于另一段序列的5’一侧，则是指所述一段序列相对于另一段序列更加靠近所述多核苷酸序列的“5’末端”。同理，当描述一段序列位于另一段序列的3’一侧，则是指一段序列相对于所述另一段序列更加靠近所述多核苷酸序列的“3’末端”，且所述一段序列与所述另一段序列彼此不包含重叠部分。此外，如本文所用，“5’部分”是指以所述多核苷酸序列“中心位置”为界，靠近所述多核苷酸序列5’末端的一半。“3’部分”则指以所述多核苷酸序列中心位置为界，靠近所述多核苷酸序列3’末端的一半。本申请所述“中心位置”至所述5’末端和至所述3’末端的核苷酸数量相等。
如本文所用，“毗邻”是指核酸分子(例如Poly(A)尾)的两个元件之间无任何核苷酸或碱基插入(如非特别指明，本申请中术语“核苷酸”与术语“碱基”可互换使用)，即：所述两个元件的一个元件的多核苷酸序列的3'末端的3’一侧的第一个核苷酸是另一个元件的5'末端核苷酸。例如“与所述miRNA结合位点5'末端毗邻的连续n个A”是指所述连续的n个A位于所述miRNA结合位点5'一侧，且所述miRNA结合位点5'末端核苷酸与所述n个A之间无其他核苷酸或碱基插入。例如“与所述miRNA结合位点3'末端毗邻”是指所述连续的n个A位于所述miRNA结合位点3'一侧，且所述miRNA结合位点3'末端核苷酸与所述n个A之间无其他核苷酸或碱基插入。此外，“毗邻”还可用于描述多个元件之间的位置关系，以表示所述多个元件中的每一个元件与所述多个元件中的任一个其他元件相毗邻，且所述多个元件的任意两个元件之间不包含所述多个元件以外的其他元件或核苷酸。
在本申请中，当描述两个或多个元件的位置关系为“不相邻”，则是指两个或多个元件两两之间不毗邻。换句话讲，即所述两个或多个元件两两之间至少包含所述两个元件的核苷酸以外的一个或多个其他核苷酸或碱基。
如本文所用，将术语“保守”用于描述核酸分子的复制时，是指所述复制过程中出现突变的概率低。在此语境中，“保守”是个相对的概念。例如当描述“Poly(A)尾的DNA编码序列用于使编码RNA的DNA分子在宿主细胞中的复制更加保守”时，是指编码RNA的亲代DNA分子复制为子代DNA分子后，如果所述RNA分子包含该Poly(A)尾，则相对于不包含所述Poly(A)尾(例如包含某种其他Poly(A)尾的RNA分子)的RNA分子的编码DNA，所述子代DNA分子与所述亲代DNA分子具有100％序列同一性的概率更高；或由所述亲代DNA分子复制获得的多条所述子代DNA分子与所述亲代DNA分子之间的平均序列同一性更高。
在本申请中，当描述“调控”RNA分子表达时，所述“调控”是指：在相同的时长内使所述RNA分子表达的蛋白或功能RNA的总量提高或降低；或使所述RNA可以在更长或更短的时间范围内表达蛋白或功能RNA，所述提高、降低、或更长或更短的时间范围是相较于另一表达相同蛋白或功能RNA的RNA分子而言的。当描述“调控”蛋白表达时，则指调控包含所述蛋白编码序列的RNA分子表达。这里描述的调控作用可以通过将本申请的Poly(A)尾连接在不包含Poly(A)尾的RNA分子的3’末端实现，也可以通过将RNA原有的Poly(A)尾替换为本申请的Poly(A)尾实现。
如本文所用，“同一性”的百分比，例如85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、98.5％、99％、99.5％同一性，是指氨基酸序列之间或核苷酸序列之间，通过序列比对确定的相似程度，是85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、98.5％、99％、99.5％。例如，通过引入空位等方式可以使两条序列在尽可能多的位置上具有相同残基后，确定的具有相同碱基或氨基酸残基的位置数量占位置总数的比例。“同一性”的百分比可以用本领域已知的软件程序来确定。优选的是使用默认参数进行比对。一个优选的比对程序是BLAST。优选的程序是BLASTN和BLASTP。这些程序的细节可以在以下互联网地址找到：ncbi.nlm.nih.gov/cgi-bin/BLAST。
如本文所用，核酸的“互补”是指一条核酸通过传统的Watson-Crick碱基配对与另一条核酸形成氢键的能力。百分比互补性表示核酸分子中可与另一核酸分子形成氢键(即，Watson-Crick碱基配对)的残基的百分比(例如，10个中的约5、6、7、8、9、10个分别为约50％，60％，70％，80％，90％和100％互补)。“完全互补”是指核酸序列的所有连续残基与第二核酸序列中相同数量的连续残基形成氢键。如本文所用，“基本上互补”是指在约40、50、60、70、80、100、150、200、250或更多个核苷酸的区域内，至少约70％，75％，80％，85％，90％，95％，96％，97％，98％，99％或100％中的任何一个的互补程度，或指在严格条件下杂交的两条核酸。对于单个碱基或单个核苷酸，按照Watson-Crick碱基配对原则，A与T或U、C与G或I配对时，被称为互补或匹配，反之亦然；而除此以外的碱基配对都称为不互补。本申请中某多核苷酸序列的“互补多核苷酸序列”则是指与该某多核苷酸序列完全互补的多核苷酸序列。
如本文所用，某个蛋白、多肽或氨基酸序列的“保守取代变体”是指其中一个或多个氨基酸残基经过氨基酸取代而不改变蛋白质或酶的整体构象和功能，这包括但不限于以前述“保守取代”描述的方式取代亲本蛋白质中氨基酸序列中的氨基酸。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
在本申请的上下文中，术语“DNA”和“RNA”是指单链或双链DNA或RNA分子。除非另有说明，否则术语“DNA”和“DNA分子”是指由A、C、G和/或T核苷酸构成的双链DNA分子，而术语“RNA”和“RNA分子”是指由A、C、G和/或U核苷酸构成的单链RNA分子。在本文中，所述A、C、G、T和U核苷酸是指包含腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶作为各自的含氮碱基的核苷酸。
RNA分子包括编码RNA(coding RNA)或非编码RNA(non-coding RNA,ncRNA)，例如Pre-mRNA、成熟mRNA或长链非编码RNA(long noncoding RNA,lncRNA)。
如本文所用，所述“DNA与RNA的杂合分子”是一个包含由脱氧核糖核苷酸和核糖核苷酸组成的多核苷酸序列的分子。所述DNA与RNA的杂合分子可以通过以下方式获得：
将DNA中的一个或多个脱氧核糖核苷酸替换为核糖核苷酸；
将RNA中的一个或多个核糖核苷酸替换为脱氧核糖核苷酸；或
通过生物或化学合成的方式，以脱氧核糖核苷酸以及核糖核苷酸为原料从头合成。需注意，获得DNA与RNA的杂合分子的方式不限于以上方式，由任何方式获取的DNA与RNA的杂合分子均属于本申请定义的“DNA与RNA的杂合分子”的范畴。
如本文所用，如果描述两个核酸分子具有“相同的遗传信息”，则是指所述两个核酸分子互补，或包含完全相同的碱基序列，或其中一个核酸分子相对于另一个核酸分子，其区别仅在于其碱基序列中一个或多个碱基由与其具有相同生物功能的另一个碱基替代。所述相同生物功能是指所述碱基与所述另一个碱基可与同一种碱基发生传统的Watson-Crick碱基配对，例如胸腺嘧啶(T)与尿嘧啶(U)均可与腺嘌呤发生Watson-Crick碱基配对，次黄嘌呤(I)与胞嘧啶(C)均可与鸟嘌呤(G)发生Watson-Crick碱基配对等。因此，DNA、RNA以及DNA与RNA的杂合分子中的任意两种均可具有相同的遗传信息。其中，术语“碱基序列”指多核苷酸分子中碱基的排列顺序。本领域技术人员应当知晓，除非另有说明，否则本申请中所述的碱基序列或多核苷酸序列可在用于描述DNA序列时，以“T”代指胸腺嘧啶，但在所述碱基序列或多核苷酸序列用于描述RNA(例如mRNA)时，“T”将由“U”(尿嘧啶)取代。因此，由本文中的特定序列号(SEQ ID NO)公开的任何DNA也公开与所述DNA互补或对应的RNA(例如mRNA或Poly(A)尾)序列，其中所述DNA序列的每个“T”被“U”取代。
如本文所用“DNA与RNA的杂合分子”是指既包含脱氧核糖核苷酸又包含核糖核苷酸的核酸分子。所述“DNA与RNA的杂合分子”可以是一条DNA分子中的一个或多个脱氧核糖核苷酸被核糖核苷酸替代，也可以是一条RNA分子中的一个或多个核糖核苷酸被脱氧核糖核苷酸替代。
Poly(A)尾及其用途
本文提供了一种工程化Poly(A)尾，其包含一个、两个、三个或更多个miRNA结合位点。优选的，本发明工程化Poly(A)尾包含1、2、3、4、5、6、7、8、9、10或更多个miRNA结合位点。所述一个、两个或更多个miRNA结合位点可以与相同或不同的miRNA相结合。在一些实施方案中，本申请提供的Poly(A)尾通过将一个、两个、三个或更多个miRNA结合位点插入任何不含miRNA结合位点的Poly(A)尾获得。在一些实施方案中，本申请提供的Poly(A)尾通过将一个或多个所述miRNA结合处插入任何不含miRNA结合位点的Poly(A)尾获得。在一些实施方案中，本申请提供的Poly(A)尾为通过将任何不含miRNA结合位点的Poly(A)尾中的一个或多个非A碱基或一个或多个在结构上等同于元件d的元件替换为一个或多个miRNA结合位点获得。在一些实施方案中，本申请提供的Poly(A)尾通过将任何不含miRNA结合位点的Poly(A)尾中的一个或多个非A碱基或一个或多个在结构上等同于元件d的元件替换为miRNA结合处获得。所述“在结构上等同于元件d的元件”即不包含miRNA结合位点的Poly(A)尾中，由任意两个或更多个连续的核苷酸组成，5’及3’末端的核苷酸不为A，且不包含连续三个以上A的多核苷酸片段，例如PCT申请PCT/CN2023/079037中的元件d。所述“不含miRNA结合位点的Poly(A)尾”可选自描述于PCT/CN2023/079037、WO2022028559A1、WO2020/074642、US10717982B2中的任一种Poly(A)尾，及任何仅含有A的Poly(A)尾。
如本文所用，术语"PolyA尾"或"PolyA序列"是指通常位于RNA分子3'-末端的不间断或不中断的腺苷酸残基序列。在RNA中，在有3'-UTR存在的情况下，Poly-A序列与3'-UTR的3’端相连。不间断的poly-A尾的特点是有连续的腺苷酸残基。Poly-A尾可以是任何长度的。在一些实施方案中，Poly-A尾包含，或由至少20、至少30、至少40、至少80或至少100和至多500、至多400、至多300、至多200或至多150个腺苷酸(A)组成，特别是约120个A。通常，PolyA尾中的绝大多数核苷酸都是腺苷，所述绝大多数是指至少75％，至少80％，至少85％，至少90％的核苷酸等，但允许剩余的核苷酸是A以外的核苷酸(非A核苷酸)，例如U(尿苷酸)、G(鸟苷酸)或C(胞苷酸)。
在一些实施方案中，所述RNA的体外制备过程是原核发酵过程，即将包含所述Poly(A)尾的RNA分子的编码核酸导入原核细胞中，通过扩增所述原核细胞，达到扩增所述编码核酸的目的，随后，将扩增后的所述编码核酸转录为所述RNA。在一些实施方案中，所述RNA的体外制备过程是将包含蛋白质编码序列的RNA片段与Poly(A)尾通过同源重组、酶切连接、或其他非同源重组等方式连接，且所述Poly(A)尾通过原核发酵过程制备，所述原核发酵过程时将包含所述Poly(A)尾的编码核酸导入原核细胞中，通过扩增所述原核细胞，达到扩增所述编码核酸的目的，随后，将扩增后的所述编码核酸转录为包含所述Poly(A)尾的RNA。在一些实施方案中，前述编码核酸是线性的。在一些实施方案中，前述编码核酸是环状的。在一些实施方案中，前述编码核酸是质粒。在一些实施方案中，前述编码核酸是单链或双链。在一些实施方案中，前述编码核酸在导入原核细胞前经过了化学修饰。在一些实施方案中，导入原核细胞前的前述编码核酸通过化学合成。在一些实施方案中，所述编码核酸插入所述原核细胞的类核/拟核基因组DNA中。在一些实施方案中，所述编码核酸游离存在于所述原核细胞的细胞质中或类核/拟核以外。在一些实施方案中，所述原核细胞是大肠杆菌。
基于此，本申请提供了一系列的工程化Poly(A)尾，所述工程化Poly(A)尾包含miRNA结合位点序列。在一些实施方案中，所述工程化Poly(A)尾除miRNA结合位点以外的碱基均为A。在一些实施方案中，所述工程化Poly(A)尾还进一步在RNA的体外制备过程中具有较高的保守性。在一些实施方案中，所述工程化Poly(A)尾可提升mRNA稳定性。在一些实施方案中，所述工程化Poly(A)尾是由所述miRNA结合位点插入保守Poly(A)尾的任一两个核苷酸之间形成的。在一些实施方案中，所述工程化Poly(A)尾是由所述miRNA结合位点在保守Poly(A)尾的5'一侧与保守Poly(A)尾的5’末端核苷酸直接相连(毗邻)形成的。在一些实施方案中，所述工程化Poly(A)尾是由所述miRNA结合位点在保守Poly(A)尾的3'一侧与保守Poly(A)尾的3’末端核苷酸直接相连(毗邻)形成的。在一些实施方案中，所述工程化Poly(A)尾是由一个或多个miRNA结合位点替换保守Poly(A)尾的任一个或多个元件形成的。
如本文所用，术语“保守Poly(A)尾”是一类包含至少一个非A碱基的Poly(A)尾，其在RNA的体外制备过程中具有较高的保守和/或相较于所有碱基均为A的Poly(A)尾可使mRNA的稳定性得到提升。保守Poly(A)可以是天然Poly(A)尾或人工合成的Poly(A)尾，包括但不限于PCT申请PCT/CN2023/079037、中国专利申请CN112805386A、美国专利US10717982B2中所描述的各种Poly(A)尾。上述申请或专利文本在此全部并入本文。
如本文所用，本文中“-”连接的两个元件之间直接相连，所述“直接相连”是指所述两个元件之间不包含任何核苷酸，因此所述“直接相连”可以通过任何核苷酸间允许的连接方式相连。在一些实施方案中，所述“直接相连”是指通过化学键相连。在一些实施方案中，所述“直接相连”是指通过磷酸酯键相连。
此外，本申请也提供了将上述Poly(A)尾用于使目的基因在特定器官、组织和/或细胞中低表达的用途，所述特定器官、组织和/或细胞高表达所述miRNA。
工程化DNA分子
本申请还提供了一种可在细胞中复制的工程化DNA分子，其包含前述工程化Poly(A)尾的编码序列或其互补序列。在一些实施方案中，所述工程化DNA分子可在细胞中复制。在一些实施方案中，所述工程化DNA分子可表达前述工程化Poly(A)尾。在一些实施方案中，所述工程化DNA分子可表达前述工程化Poly(A)尾且可在细胞中复制。在一些实施方案中，所述是细胞是原核细胞或真核细胞。在一些实施方案中，所述原核细胞是大肠杆菌。本领域技术人员应当知晓，除所述工程化Poly(A)尾的编码序列以外，所述工程化DNA分子还应包含可使所述DNA分子在细胞中复制，或高效复制所必须的结构元件。使所述工程化DNA分子在细胞中复制或高效复制所述必须的结构元件是本领域已知的，包括例如复制起点(ORI)。在一些实施方案中，所述工程化DNA分子还进一步包含标记基因或其片段和/或报道基因或其片段、和允许插入DNA元件的独特的限制性内切酶位点，优选多克隆位点(MCS)形式的限制性内切酶位点。所述标记基因有利于鉴定含有包含所述标记基因的质粒的细胞，可选自，例如抗生素抗性基因。所述MCS中的每一个限制性内切酶位点均可被不同的限制性内切酶特异性识别。
在一些实施方案中，所述DNA分子是DNA质粒。如本文所用，术语“DNA质粒”是指由双链DNA分子组成的质粒。在一些实施方案中，所述“质粒”是环状DNA分子。在一些实施方案中，所述“质粒”还可以涵盖线性DNA分子。具体的，术语“质粒”还涵盖通过例如用限制性内切酶切割环状质粒，进而使该环状质粒分子转变成线性分子而使该环状质粒线性化所得到的分子，以及可在原核生物中复制的线性分子。质粒可以复制，即在细胞中独立于原核细胞拟核或类核存储的基因组遗传信息而扩增，并且可以用于克隆，即用于在细菌细胞中扩增遗传信息。优选地，根据本发明的DNA质粒是中拷贝或高拷贝质粒，更优选地是高拷贝质粒。此类高拷贝质粒的实例是这样的载体：其基于pUC、pTZ质粒或包含支持质粒高拷贝的ORI的任意其它质粒(例如pMB1、pCoIE1)等。
在一些实施方案中，所述DNA分子是构成原核生物拟核或类核的DNA分子或其片段，即所述包含前述工程化Poly(A)尾的编码序列或其互补序列可随原核生物基因组进行复制。
在一些实施方案中所述DNA分子是基因组DNA，例如病毒基因组DNA或真核生物基因组DNA。在一些实施方案中，所述DNA为线粒体DNA。在一些实施方案中，所述DNA当导入真核细胞中后，为游离DNA。在一些实施方案中，所述DNA为病毒载体。
在一些实施方案中，所述DNA分子在所述工程化Poly(A)尾编码序列的5’一侧进一步连接目的基因片段，所述目的基因片段与所述工程化Poly(A)尾编码序列共同编码RNA。在一些实施方案中，所述目的基因片段与所述工程化Poly(A)尾编码序列共同编码mRNA。所述目的基因片段包含蛋白、多肽或其片段的编码序列。所述蛋白、多肽或其片段的编码序列可被最终翻译为一个或多个蛋白、或一条或多条多肽，例如短肽、寡肽、多肽、融合蛋白、蛋白质及其片段，如已知蛋白质的部分，例如功能性部分。所述功能性部分可以是例如，蛋白质的生物活性部分或可以有效地产生抗体的抗原部分，例如抗原表位。所述蛋白、多肽或其片段的编码序列的两端分别包含起始密码子(5’端)和终止密码子(3’端)，其分别是所述mRNA分子的可被翻译的前三个核苷酸和后三个核苷酸。
在一些实施方案中，本发明mRNA还包括5’UTR、3’UTR等。在一些实施方案中，本发明mRNA从5’至3’至少依次包含5'UTR的序列，目的基因序列、3'UTR的序列、本发明的Poly(A)尾序列。
5'UTR通常包含至少一个核糖体结合位点(RBS)，如原核生物中的Shine-Dalgarno序列，或至少一个翻译起始位点，如真核生物中的Kozak序列。RBS通过在翻译起始时募集核糖体来促进mRNA分子的有效且准确的翻译。可以通过改变给定的RBS或翻译启示位点的长度和序列以及距起始密码子的距离来优化其活性。可选地或任选地，5'UTR包括内部核糖体进入位点或IRES。3'UTR可包含一个或多个调控序列，如增强mRNA分子稳定性的氨基酸序列的结合位点、调控RNA分子(如miRNA分子)的结合位点、和/或参与mRNA分子的胞内运输的信号序列。
所述蛋白、多肽或其片段的编码序列包含可以翻译成氨基酸序列的密码子。所述编码序列包含的全部密码子中，可以全部是天然存在的编码氨基酸的密码子，也可以有部分或全部由人工合成的密码子组成。在一些实施方案中，所述部分或全部密码子经过了密码子优化。在一些实施方案中，所述部分或全部密码子编码非天然氨基酸。
在一些实施方案中，所述DNA分子在述目的基因片段的5’端一侧还进一步包含可启动或调控所述RNA转录所必须的结构元件，所述结构元件是本领域已知的。在一些实施方案中，所述结构元件至少包含启动子。启动子及其序列是本领域已知的，包括弱启动子、中等强度启动子、强启动子、mini启动子或核心启动子等。在一些特定的实施方案中，所述启动子为强启动子。在一些实施方案中，所述启动子可在原核细胞中启动所述目的基因片段和/或工程化Poly(A)尾转录。在一些实施方案中，所述启动子可以在真核细胞中启动所述目的基因片段和/或工程化Poly(A)尾转录。所述“启动子”包含至少一个转录识别位点及其后的转录因子结合位点。所述识别和结合位点可以与介导或调节转录的氨基酸序列相互作用。与识别位点相比，结合位点更靠近前述目的基因片段。结合位点可以是，例如原核生物中的Pribnow框或真核生物中的TATA框。例如，在一些实施方案中，当使用Pribnow框时，所述转录识别位点可以位于转录起始位点上游约35bp处，而转录因子结合位点可以位于转录起始位点上游约10bp处。在一些实施方案中，所述启动子包含至少一个另外的调控元件，如位于转录起始位点之前约40和/或60个核苷酸处富含AT的上游元件，和/或位于识别位点和结合位点之间的增强启动子活性的另外的调控元件。在一些实施方式中，所述启动子是强启动子，即所述启动子包含促进前述RNA编码序列转录的序列。强启动子是本领域技术人员已知的，例如来自大肠杆菌的RecA启动子衍生的OXB18、OXB19和OXB20启动子，或者可以通过常规实验室程序鉴定或合成。在一些实施方案中，所述启动子为T7启动子。在一些实施方案中，所述启动子前还包含另外的调控元件，如包含在DNA质粒中可促进前述RNA编码序列转录的增强子。
此外，本申请还提供了上述工程化DNA分子在稳定扩增工程化Poly(A)尾编码序列或带有工程化Poly(A)尾的RNA的编码序列中的用途。
工程化RNA
本申请提供了一种工程化RNA或工程化RNA分子，其包含前述工程化Poly(A)尾，以及Poly(A)尾编码序列5’端一侧的目的基因片段。在一些实施方案中，所述RNA为mRNA。
在一些实施方式中，本申请的mRNA分子还包含5’UTR和/或3’UTR。
在一些实施方式中，本申请的mRNA分子还包含5’帽子。在优选的实施方式中，所述5’帽子m7G(5’)ppp(5’)(2’-OMeA)pG。
在一些实施方式中，本申请的mRNA分子还包含化学修饰，例如将所述多核苷酸序列中的全部或部分尿苷酸修饰为N1-甲基假尿苷。
如本文所用“mRNA”(信使RNA)是编码至少一种蛋白质、多肽或其片段的，天然存在、非天然存在或经修饰的任何RNA，所述mRNA具备经翻译以在体外、体内、原位或离体产生所编码的蛋白质、多肽或其片段的能力。因此，所述mRNA可以是成熟的mRNA或成熟前的mRNA，其必须包含或选择性包含的元件或结构是本领域已知的。在一些实施方案中，所述mRNA包含多个必要的功能组件的编码序列，以表达、调节、或增强所述蛋白、多肽或其片段的表达水平。所述功能组件包括但不限于5’帽子、5’UTR、3’UTR等。5'UTR和3'UTR两者通常都从基因组DNA转录，并且是成熟前的mRNA就具有的元件。
作为成熟mRNA，通常术语“5’帽子”位于mRNA的5’最末端，包含甲基化鸟苷酸，所述甲基化鸟苷酸经焦磷酸连接于mRNA的5'末端，与其相邻的核苷酸形成5',5'-三磷酸连接。5’帽子结构通常有三种类型(m7G5'ppp5'Np、m7G5'ppp5'NmpNp、m7G5'ppp5'NmpNmpNp)，分别称为O型、I型和II型。O型指末端核苷酸的核糖未甲基化，I型指末端一个核苷酸的核糖甲基化，II型指末端两个核苷酸的核糖均甲基化。在一些实施方案中，所述5’帽子可根据制造商的方案，使用以下化学RNA帽类似物在体外转录反应期间同时完成对多核苷酸的5'加帽以产生5'-鸟苷帽结构：3′-O-Me-m7G(5')ppp(5')G[ARCA帽]、G(5')ppp(5')A、G(5')ppp(5')G、m7G(5')ppp(5')A、m7G(5')ppp(5')G(NewEnglandBioLabs、Ipswich、MA)、或m7G(5’)ppp(5’)(2’-OMeA)pG(CleanCapAG)。例如在一些实施方案中，可使用牛痘病毒加帽酶在转录后完成对经修饰RNA的5'加帽，以产生O型帽子结构：m7G(5')ppp(5')G(New England BioLabs,Ipswich,MA)。可使用牛痘病毒加帽酶和2'-O甲基-转移酶两者来产生I型帽子结构，以产生m7G(5’)ppp(5’)(2’-OMeA)pG。可从I型帽子结构接着使用2'-O甲基-转移酶对5'-倒数第三核苷酸进行2'-O-甲基化来产生II型帽子结构。可从II型帽子结构接着使用2'-O甲基-转移酶对5'-倒数第四核苷酸进行2'-O-甲基化来产生III型帽子结构。
在一些实施方案中，所述mRNA还包含稳定化元件。稳定化元件可包括例如组蛋白茎环。在一些实施方案中，所述mRNA包含编码区、至少一个组蛋白茎环和任选地poly(A)序列或多聚腺苷酸化信号。所述poly(A)序列或多聚腺苷酸化信号通常应增强所编码蛋白质的表达水平。在一些实施方案中，所述mRNA包含poly(A)序列或多聚腺苷酸化信号与至少一个组蛋白茎环的组合，尽管两者在自然界中具有替代机制，但其协同作用可使蛋白质表达增加至超过任一单个元件所观察到的水平。poly(A)与至少一个组蛋白茎环的组合的协同效应不依赖于元件的次序或poly(A)序列的长度。在一些实施方案中，所述组蛋白茎环通常源自组蛋白基因，并且包括由间隔区(由短序列组成)分隔的两个相邻部分或完全反向互补序列的分子内碱基配对形成环。未配对的环区通常无法与茎环元件中的任一者进行碱基配对。茎环结构的稳定性通常取决于长度、错配或膨出部的数目以及配对区的碱基组成。在一些实施方案中，可产生摆动碱基配对(非Watson-Crick碱基配对)。在一些实施方案中，所述至少一个组蛋白茎环序列包含15至45个核苷酸长度。
在一些实施方案中，可将所述mRNA的一个或多个富含AU的序列去除。这些序列有时称为AURES，其是在3'UTR中发现的去稳定化序列。可将AURES从mRNA中去除。或者，可使AURES保留在mRNA中。
细胞
本申请还提供了包含前述工程化DNA分子的细胞，所述DNA分子可在所述细胞中储存和/或扩增。在一些实施方案中，所述细胞为原核细胞，所述DNA分子可在所述原核细胞中复制。在一些实施方案中，所述细胞为原核细胞，所述DNA分子可在所述原核细胞中复制和/或转录。在一些实施方案中，所述DNA分子为真核细胞，所述DNA分子可在所述细胞中复制。在一些实施方案中，所述DNA分子可在所述DNA细胞中转录和/或复制。
在一些实施方案中，所述细胞为原核细胞。在一些实施方案中，所述细胞为细菌、放线菌、蓝细菌、支原体、立克次氏体和衣原体。在一些实施方案中，所述细胞选自：枯草芽孢杆菌、乳酸杆菌、醋酸杆菌、棒状杆菌、短杆菌、节杆菌、假单胞菌、及小球菌。在一些实施方案中，所述细胞为recA-的细菌。在一些实施方案中，所述细胞为大肠杆菌。在一些实施方案中，所述细胞为大肠杆菌，所述大肠杆菌选自：K-12及其衍生菌株，以及B菌株及其衍生菌株。在一些实施方案中，所述大肠杆菌选自：MG1655、DH5或DH5α、DH10B、BL21、DB3.1、HB101、JM109、JM110、MC1061、MG1655、Pir1、Stbl2、Stbl3、Top10、XL1Blue、XL10 Gold、BLR、HMS174、Tuner、Rostetta2、Lemo21、T7Express、Origami2等。在一些实施方案中，所述细胞选自链霉菌属、小单孢菌属和诺卡氏菌属。在一些实施方案中，所述细胞为真菌。在一些实施方案中，所述细胞选自酵母菌或霉菌。
脂质纳米颗粒
本申请还提供了包含本申请的工程化Poly(A)尾、工程化RNA分子、和/或工程化DNA分子的脂质纳米颗粒。
在一些实施方案中，脂质与所述工程化RNA混合形成脂质纳米颗粒。在一些实施方案中，将本申请的工程化Poly(A)尾、工程化RNA分子、和/或工程化DNA分子配制在脂质纳米颗粒中。在一些实施方案中，所述脂质纳米颗粒首先形成为空的脂质纳米颗粒，并且在即将施用之前(例如在几分钟至一小时内)与所述工程化Poly(A)尾、工程化RNA分子、和/或工程化DNA分子组合或包裹。
所述脂质纳米颗粒通常包含可电离的脂质、非阳离子脂质、固醇和PEG脂质组分以及目标核酸，例如本申请的工程化Poly(A)尾、工程化RNA分子、和/或工程化DNA分子。
本发明所述脂质纳米颗粒包含可电离脂、磷脂、结构脂质和PEG脂质；优选地，所述可电离脂、磷脂、结构脂质和PEG脂质的摩尔比为：(20～60)：(5～25)：(25～55)：(0.5-15)，更优选地(40～55)：(5～15)：(30～50)：(1～3)；
其中所述磷脂优选选自下述化合物中的一种或两种以上：
二月桂酰基卵磷脂(DLPC)、
二肉豆蔻酰磷脂酰胆碱(DMPC)、
二油酰基卵磷脂(DOPC)、
二棕榈酰磷脂酰胆碱(DPPC)、
二硬脂酰磷脂酰胆碱(DSPC)、
二油酰基磷脂酰胆碱(DUPC)、
棕榈酰油酰磷脂酰胆碱(POPC)、
1,2-二-O-十八烷基-sn-甘油-3-磷酸胆碱(18:0Diether PC)、
1-油酰基-2-胆甾醇二甲基琥珀酸-sn-甘油-3-磷酸胆碱(OChemsPC)、
l-十六烷基-sn-甘油-3-磷酸胆碱(C16 Lyso PC)、
1,2-二乙烯基-sn-甘油-3-磷酸胆碱、
1,2-二芳基酰基-sn-甘油-3-磷酸胆碱、
1,2-二油酰-SN-甘油-3-磷酰乙醇胺(DOPE)、
1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺、
1,2-二乙烯醇基-sn-甘油-3-磷酸乙醇胺、
1,2-二乙烯基-sn-甘油-3-磷酸乙醇胺、
1,2-二芳基-sn-甘油-3-磷酸乙醇胺、
1,2-二硫代六烯酸-sn-甘油-3-磷酸乙醇胺、
1,2-二油酰基-sn-甘油-3-磷酸-(1-甘油)钠盐(DOPG)或鞘磷脂，
例如，所述磷脂为DOPE或DSPC；
所述结构脂质优选选自胆固醇、粪甾醇、谷甾醇、麦角甾醇、豆甾醇中的一种或两种以上；例如，所述结构脂质是胆固醇；和/或
所述PEG脂质优选选自PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油或PEG修饰的二烷基甘油中的一种或两种以上，例如，所述PEG脂质为DMG-PEG2000。
可使用如本领域中通常已知的组分、组合物和方法来产生本公开的脂质纳米颗粒，参见例如PCT/US2016/052352、PCT/US2016/068300、PCT/US2017/037551、PCT/US2015/027400、PCT/US2016/047406、PCT/US2016000129、PCT/US2016/014280、PCT/US2016/014280、PCT/US2017/038426、PCT/US2014/027077、PCT/US2014/055394、PCT/US2016/52117、PCT/US2012/069610、PCT/US2017/027492、PCT/US2016/059575和PCT/US2016/069491，其全部通过引用整体并入本文。
应当理解，本申请包含本文所描述的各种方面、实施方案以及所述方面和/或实施方案的组合。以上描述以及随后的实施例旨在说明而不是限制本申请的范围。在本申请范围内的其他方面、改进和修改对于本申请所属领域的技术人员将是显而易见的。因此，本领域的普通技术人员应该认识到，本申请的范围还包括对所述方面和实施方案的所述改进和修改。
 实施例
实施例1：包含本发明polyA变体的荧光素酶mRNA的构建
1)构建包含荧光素酶蛋白编码区的载体
该载体以大肠杆菌克隆载体pUC57作为载体骨架，在其多克隆位点的Xba I酶切位点和SapI酶切位点之间按顺序排列T7启动子序列(SEQ ID NO：2)、5'UTR、Kozak序列(GCCACC)、荧光素酶蛋白编码序列、3'UTR以及编号为RG008的poly(dA:dT)，作为本发明中设计的poly(A)变体的对照。
[根据细则91更正 07.06.2024]
2)将通用载体中的多聚腺苷酸串poly(dA:dT)进行替换合成构建不同poly(A)变体所需的全部序列(如文末序列表所示)，用2个限制性内切酶进行双酶切以便从1)中构建的通用载体中切除编号为RG008的变体poly(dA:dT)，之后用T4 DNA连接酶1将本发明不同poly(A)变体与切除poly(dA:dT)的载体连接，完成了对通用载体中poly(dA:dT)的替换。
[根据细则91更正 07.06.2024]
示意图如图1A和图1B所示。
实施例2：本发明mRNA-LNP的制备
向荧光素酶mRNA原液中，加入醋酸溶液至醋酸终浓度为20mmol/L、mRNA终浓度为200μg/ml，搅拌混匀，作为mRNA工作液。将mRNA工作液与混脂溶液(根据表1配制)经T混流装置按流量比为2:1～4:1混合，制备成LNP。然后，用2mmol/L醋酸溶液将LNP稀释2～5倍，再用2mmol/L醋酸溶液置换不低于3倍，将料液浓缩至目标浓度。加入蔗糖溶液调节渗透压，并用Tris溶液调节pH至7.0～8.0，获得本发明的mRNA-脂质纳米颗粒(LNP)。
表1：混脂溶液配方
实施例3含有miR-142结合位点的5种poly(A)的荧光素酶mRNA在小鼠中的表达测试
以荧光素酶为蛋白编码区，考察5种Poly(A)变体(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)在miR-142低表达的小鼠肝脏以及miR-142高表达的小鼠脾脏中的表达水平，以检测miR-142结合位点的插入是否能够在miR-142高表达的器官中下调mRNA的表达水平。
C57BL/6小鼠按照体重随机分组，每组6只，适应性饲养2-3天。小鼠采取尾静脉注射方式给药，每只注射0.05mg/kg的mRNA-LNPs(1μg)，注射体积为100μL，空白对照注射等体积PBS。给药后6h，按照150mg/kg的计量在腹腔注射Luciferin(200μL)，10min后麻醉小鼠，采用小动物成像仪进行成像，解剖小鼠的肝脏和脾脏，再次成像，实验共做1次，取每只小鼠的测定值做柱状图，结果如图2A-2B所示。
结论：miR-142结合位点插入在RG008的0A(SEQ ID NO：16)、14A(SEQ ID NO:9)、19A(SEQ ID NO：8)、30A(SEQ ID NO：7)、60A(SEQ ID NO：6)之后的位置构成不同的Poly(A)变体，当带有这类变体的mRNA通过LNPs包裹，并通过尾静脉注射进小鼠体内时，在巨噬细胞(高表达miR-142)富集的脾脏中，mRNA会被摄取并降解，表达受到抑制。在脾脏表达水平方面，miR-142结合位点插入3’UTR、0A、14A、19A、30A位置处时下降水平相当，显著优于插入位置为60A处。
在肝脏表达水平方面，当miR-142结合位点插入至14A、19A、30A、60A位置处时，对肝脏的表达水平基本无影响，插入位置为14A、30A时甚至优于无miR-142结合位点的对照组，而插入至0A位置处肝脏表达水平下降明显。
综合肝脏表达水平和脾脏表达水平，本发明筛选出插入位置14A-30A处为较优的miR-142结合位点插入位置，其可在不影响肝脏表达水平的前提下显著下调脾脏表达水平，适用于mRNA肝部表达蛋白治疗疾病的应用场景。
实施例4含有miR-122结合位点的5种poly(A)的荧光素酶mRNA在小鼠中的表达测试
考察5种Poly(A)变体(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)-0Ainsert miR-122)在miR-122低表达的小鼠脾脏以及miR-122高表达的小鼠肝脏中的表达水平。
C57BL/6小鼠按照体重随机分组，每组6只，适应性饲养2-3天。小鼠采取尾静脉注射方式给药，每只注射0.05mg/kg的mRNA-LNPs(1μg)，注射体积为100μL，空白对照注射等体积PBS。给药后6h，按照150mg/kg的计量在腹腔注射Luciferin(200μL)，10min后麻醉小鼠，采用小动物成像仪进行成像。解剖小鼠的肝脏和脾脏，再次成像。实验共做1次，取每只小鼠的测定值做柱状图，其中肝脏及脾脏的荧光成像绝对值分别如图3A-3B所示。
结论：miR-122结合位点插入在RG008的0A、14A、19A、30A、60A之后的位置构成不同的Poly(A)变体，当带有这类变体的mRNA通过LNPs包裹，并通过尾静脉注射进小鼠体内时，在肝脏中，mRNA均会被摄取并明显降解，表达受到抑制。miR-122结合位点插入在RG008的0A、14A、19A的肝脏表达降低水平显著优于插入至30A、60A处。脾脏表达水平各组均有所降低。综合肝脏表达水平和脾脏表达水平，本发明筛选出插入位置0A、14A、19A处为较优的miR-122结合位点插入位置，其可在显著下调肝脏表达水平的基础上保持脾脏表达水平，适用于mRNA脾表达蛋白治疗疾病的应用场景。
实施例5含有不同poly(A)变体的质粒在大肠杆菌中复制的稳定性实验
考察两组共10种Poly(A)变体(Poly(A)-60A insert miR-142；Poly(A)-30Ainsert 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)-0Ainsert miR-122)在大肠杆菌中的稳定性的影响。
根据实施例1中的方法构建的载体质粒经测序确认无误后，转入大肠杆菌DH5α，转化后的平板在30℃生长，次日将平板寄送至测序服务公司随机挑选50个克隆，完成测序工作。测序完成后根据测序结果分析和计算不同Poly(A)变体的情况。
结果如图4所示，两种不同的miRNA结合位点插入到Poly(A)变体14A-30A的位置处发生碱基缺失的比例均低于插入至0A或60A位置处以及插入至3’UTR中。结果表明，miRNA结合位点插入到RG008 Poly(A)变体14A-30A位置处的质粒稳定性更好。
实施例6含有不同插入位置的miR-142结合位点的Poly(A)变体的mRNA在细胞中的表达测试
考察8种Poly(A)变体(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)在miR-142高表达的小鼠单核巨噬细胞白血病RAW 264.7细胞中的表达水平。
RAW 246.7细胞培养使用24孔细胞培养板，细胞的密度为1x105cells/孔，每孔采用Lipofectamine 3000(购自赛默飞)转染500ng体外合成的mRNA。转染24h后吸掉细胞培养基，每孔加入200μL细胞裂解液，10min后将细胞裂解液转移至1.5mL EP管中，12000rpm，4℃离心5min，随后将20μL裂解液上清转移至黑色不透光的96孔板中，采用荧光检测仪加入200μL荧光素底物并读值。结果如图5所示。
结论：miR-142结合位点插入在RG008的0A(SEQ ID NO：16)、1A(SEQ ID NO：15)、2A(SEQ ID NO：14)、3A(SEQ ID NO：13)、4A(SEQ ID NO：12)、5A(SEQ ID NO：11)、10A(SEQ ID NO：10)、14A(SEQ ID NO：9)之后的位置构成不同的Poly(A)变体，当带有这类变体的mRNA被递送至miR-142过表达的细胞系，如Raw 264.7细胞中时，mRNA均会被miR-142降解，表达受到抑制。
实施例7 Poly(A)尾中不同microRNA结合位点串联后相互影响研究
考察2种Poly(A)变体(Poly(A)-0A insert miR-142+miR-122；Poly(A)-0Ainsert miR-122+miR-142)在miR-142高表达的小鼠单核巨噬细胞白血病RAW 264.7细胞中的表达水平，以考察miR-122和miR-142串联结合位点的插入是否影响miR-142结合位点下调mRNA的表达水平。
RAW 246.7细胞培养使用24孔细胞培养板，细胞的密度为1x105cells/孔，每孔采用Lipofectamine 3000(购自赛默飞)转染500ng体外合成的mRNA。转染24h后吸掉细胞培养基，每孔加入200μL细胞裂解液，10min后将细胞裂解液转移至1.5mLEP管中，12000rpm，4℃离心5min，随后将20μL裂解液上清转移至黑色不透光的96孔板中，采用荧光检测仪加入200μL荧光素底物并读值。结果如图6所示。
结论：miR-142+miR-122或miR-122+miR-142结合位点插入在RG008的0A之后的位置构成不同的Poly(A)变体，当带有这类变体的mRNA通过LNPs递送至miR-142过表达的细胞系，如Raw 264.7细胞中时，mRNA会被miR-142降解，表达受到抑制，且miR-142和miR-122结合位点不同顺序串联后不影响表达抑制效果。
实施例8含有多个microRNA结合位点Poly(A)变体的mRNA的细胞精准表达研究
考察6种Poly(A)变体(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)在miR-142高表达的小鼠单核巨噬细胞白血病RAW 264.7细胞，miR-126高表达的小鼠肝窦内皮细胞LSEC，miR-148a高表达的小鼠肝星状细胞HSC以及肝实质细胞AML中的表达水平，
RAW246.7，LSEC，HSC，AML12细胞的培养使用24孔细胞培养板，细胞的密度为1x105cells/孔，每孔采用Lipofectamine 3000(购自赛默飞)转染500ng体外合成的mRNA。转染24h后吸掉细胞培养基，每孔加入200μL细胞裂解液，10min后将细胞裂解液转移至1.5mLEP管中，12000rpm，4℃离心5min，随后将20μL裂解液上清转移至黑色不透光的96孔板中，采用荧光检测仪加入200μL荧光素底物并读值。相对于肝实质表达的降低比例计算：将without miRNA ts组的数据定义为1，计算不同细胞系实验中不同组相对于without miRNA ts的相对表达量，以桥接不同细胞系直接的表达数据，以此为基础计算每个设计在目标细胞(RAW 264.7细胞、肝窦内皮细胞LSEC、肝星状细胞HSC)相对于肝实质细胞AML12表达降低的比例，计算方式为：(1-目标细胞相对表达量/肝实质细胞相对表达量)*100％。
结论：miR-148a，miR-142，miR-126串联时，当mRNA转染至RAW 264.7细胞时，Poly(A)变体中含有miR-142结合位点，mRNA在该细胞系中的表达就会降低。当mRNA转染至LSEC细胞时，Poly(A)变体中含有miR-126结合位点，mRNA在该细胞系中的表达就会降低。当mRNA转染至HSC细胞时，Poly(A)变体中含有miR-148a结合位点，mRNA在该细胞系中的表达就会降低。此外，miR-126+miR-148a+miR-142和miR-148a+miR-142+miR-126的组合在三个细胞中的表现优于其他顺序。参见图7A-7D，图8A-8C。
肝脏主要由肝实质细胞、肝巨噬细胞、肝窦内皮细胞、肝星状细胞组成，本实施例筛选出了表现优异的肝实质细胞特异性表达的microRNA结合位点组合，为肝实质细胞的靶向治疗提供了可靠的工具。
本发明结果表明，在Poly(A)变体中插入miRNA结合位点可以使mRNA在不同细胞系中以及小鼠的不同脏器中表现出不同的表达差异，因此，通过在同一个序列中插入一个或多个miRNA结合位点，可以实现高度特异的表达调控。
本申请以上实施例中使用的序列示于如下序列表中。应当理解，以下序列仅为本申请实施方案的示例性序列，而非对本申请方案的任何限制。以下序列表中的核酸序列可表示DNA序列或RNA序列，当其表示RNA序列时，其中的“T”代表尿苷。并且，本申请中单独的T在RNA的语境下，也指代尿嘧啶或尿苷。
序列表：

Claims (26)

  1. 一种工程化Poly(A)尾，所述Poly(A)尾包含miRNA结合位点。
  2. 根据权利要求1的工程化Poly(A)尾，其中所述Poly(A)尾包含或为如下的式(I)：nA-miRNA结合处-mA   式(I)；
    其中，miRNA结合处由一个或多个miRNA结合位点通过一个或多个核苷酸连接而成或直接连接而成；
    nA表示与所述miRNA结合处5'末端毗邻的连续n个腺苷酸(A)，
    mA表示与所述miRNA结合处3'末端毗邻的连续m个腺苷酸(A)，
    m和n为自然数，且m+n≤150、m+n≤120、m+n≤100、m+n≤80、m+n≤60、m+n≤30、m+n≤19、或m+n≤14；优选地，m+n＝60；
    miRNA结合处的3'及5'末端不包含除miRNA结合位点本身包含的A以外的其他A。
  3. 根据权利要求2的工程化Poly(A)尾，其中n＝0或n≥1。
  4. 根据权利要求2的工程化Poly(A)尾，其中n≤60、n≤30、n≤19、n≤14或n≤10。
  5. 根据权利要求2的工程化Poly(A)尾，其中14≤n≤30,优选19≤n≤30或14≤n≤19。
  6. 根据权利要求1至5中任一项的工程化Poly(A)尾，其中所述Poly(A)尾的长度为80至240nt，例如100至200nt，101至150nt、120至150nt、130至140nt、123nt至135或125至139nt。
  7. 根据权利要求2至6中任一项所述的工程化Poly(A)尾，其中所述Poly(A)尾：
    在式(I)的3'一侧进一步包含与式(I)结构的3'末端直接相连的尾部片段，所述尾部片段5’末端的核苷酸不为A；和/或
    在式(I)的5'一侧进一步包含与式(I)结构的5'末端直接相连的头部片段，所述头部片段3’末端的核苷酸不为A。
  8. 根据权利要求7所述的工程化Poly(A)尾，其中，所述尾部片段或头部片段由一个或多个元件c和/或一个或多个元件d与一个或多个A组成，
    所述元件c为一个非A的核苷酸；
    所述元件d由任意两个或更多个连续的核苷酸组成，元件d的5’及3’末端的核苷酸不为A，且元件d不包含三个以上连续的A；
    所述元件d的长度范围为2nt≤d≤20nt；
    其中所述元件c和元件d不相邻。
  9. 根据权利要求8所述的工程化Poly(A)尾，其包含的所述元件c的个数为2至10个、3个至8个、或4至6个或2-5个，优选2个；和/或元件c为G。
  10. 根据权利要求8-9中任一项所述的工程化Poly(A)尾，其中所述元件d选自如SEQ ID NO：60至64所示序列中的任一种或多种，优选地，所述元件d如SEQ ID NO：60所示。
  11. 根据权利要求8-10中任一项所述的工程化Poly(A)尾，其包含的所述元件d的个数为0-5，优选1-3个，进一步优选1个。
  12. 根据权利要求1-11中任一项所述的工程化Poly(A)尾，不包含头部片段，且所述尾部片段的结构为：元件d-19A-元件c-19A-元件c-17A或元件c-19A-元件c-17A。
  13. 根据权利要求1-12中任一项所述的工程化Poly(A)尾，其结构为nA-miRNA结合处-mA-元件d-19A-元件c-19A-元件c-17A。
  14. 根据权利要求1-13中任一项所述的工程化Poly(A)尾，其结构为nA-miRNA结合处-mA-SEQ ID NO：60-19A-G-19A-G-17A。
  15. 根据权利要求1-14中任一项所述的工程化Poly(A)尾，其中，所述miRNA选自以下的一种或多种：
    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,miR-101和miR-204；
    优选地，所述miRNA选自以下的一种或多种miR-142,miR-122,miR-126,miR-148a；
    优选地，所述miRNA选自以下的一种或多种：miR-142和miR-122的组合，miR-126和miR-148a的组合，miR-142，miR-148a和miR-126的组合。
  16. 根据权利要求1-15中任一项所述的工程化Poly(A)尾，其中，所述miRNA包括miRNA-142、miR-148a和miR-126，并且在所述Poly(A)的miRNA结合处中，各miRNA结合位点从5'至3'的排列顺序为：
    miR-126结合位点、miR-148a结合位点及miR-142结合位点；
    miR-148a结合位点、miR-142结合位点及miR-126结合位点；
    miR-142结合位点、miR-148a结合位点和miR-126结合位点；
    miR-126结合位点、miR-142结合位点及miR-148a结合位点；
    miR-148a结合位点、miR-126结合位点及miR-142结合位点；或
    miR-142结合位点、miR-126结合位点和miR-148a结合位点；
    其中miR-126结合位点、miR-148a结合位点和miR-142结合位点彼此毗邻。
  17. 根据权利要求1-16中任一项所述的工程化Poly(A)尾，其中，n＝0，m＝60，且mA与所述尾部片段组成如SEQ ID NO：1所示的多核苷酸序列。
  18. 根据权利要求1-16中任一项所述的工程化Poly(A)尾，其中所述Poly(A)尾包含选自以下的任一条多核苷酸序列：
    SEQ ID NO：6-16、SEQ ID NO：19-31、SEQ ID NO：33-52、SEQ ID NO：54-59及SEQ ID NO：65-71。
  19. 工程化RNA分子，其为mRNA或非编码RNA，其包含根据权利要求1-18中任一项所述Poly(A)尾以及位于所述Poly(A)尾5’一侧的目的基因序列,所述RNA序列从5’至3’端至少依次包含5'UTR序列，目的基因序列、3'UTR序列以及权利要求1-18中任一项所述Poly(A)尾，所述Poly(A)尾的式(I)中的nA与3’UTR直接相连；优选地所述RNA分子具有5’帽子和/或全部或部分尿苷酸被修饰为N1-甲基假尿苷。
  20. 工程化DNA分子，其编码包含根据权利要求1-18中任一项所述的工程化Poly(A)尾，或根据权利要求19所述的工程化RNA序列；优选地，所述DNA分子为质粒或病毒载体。
  21. [根据细则91更正 07.06.2024]
    工程化细胞，其包含根据权利要求1-18中任一项所述的工程化Poly(A)尾，根据权利要求19所述的工程化RNA分子，根据权利要求20所述的工程化DNA分子，其中所述细胞为真核或原核细胞；优选地所述原核细胞为大肠杆菌。
  22. 根据权利要求1-18中任一项所述的工程化Poly(A)尾用于调控目的基因在特定器官、组织和/或细胞中表达的用途，所述特定器官、组织和/或细胞高表达所述miRNA，优选的所述调控为使目的基因在特定器官、组织和/或细胞中低表达；更优选的，所述特定器官、组织和/或细胞为肝或脾器官、组织和/或细胞。
  23. 将根据权利要求1-18中任一项所述的工程化Poly(A)尾用于使目的基因在特定器官、组织和/或细胞中特异性表达的用途，优选地，所述器官或组织优选肝或脾器官或组织；更优选地，所述细胞为肝实质细胞。
  24. 根据权利要求1-18中任一项所述的工程化Poly(A)尾用于使编码所述Poly(A)尾的DNA编码序列在宿主细胞中的复制更加保守的用途，优选地，所述宿主细胞为原核细胞，更优选大肠杆菌。
  25. 脂质纳米颗粒，其包含根据权利要求1-18中任一项所述的工程化Poly(A)尾，根据权利要求19所述的工程化RNA分子，根据20所述的工程化DNA分子。
  26. 根据权利要求25所述的脂质纳米颗粒，其中所述脂质纳米颗粒包含可电离脂、磷脂、结构脂质和PEG脂质；优选地，所述可电离脂、磷脂、结构脂质和PEG脂质的摩尔比为：(20～60)：(5～25)：(25～55)：(0.5-15)，更优选地(40～55)：(5～15)：(30～50)：(1～3)；
    其中所述磷脂优选选自下述化合物中的一种或两种以上：
    二月桂酰基卵磷脂(DLPC)、
    二肉豆蔻酰磷脂酰胆碱(DMPC)、
    二油酰基卵磷脂(DOPC)、
    二棕榈酰磷脂酰胆碱(DPPC)、
    二硬脂酰磷脂酰胆碱(DSPC)、
    二油酰基磷脂酰胆碱(DUPC)、
    棕榈酰油酰磷脂酰胆碱(POPC)、
    1,2-二-O-十八烷基-sn-甘油-3-磷酸胆碱(18:0Diether PC)、
    1-油酰基-2-胆甾醇二甲基琥珀酸-sn-甘油-3-磷酸胆碱(OChemsPC)、
    l-十六烷基-sn-甘油-3-磷酸胆碱(C16 Lyso PC)、
    1,2-二乙烯基-sn-甘油-3-磷酸胆碱、
    1,2-二芳基酰基-sn-甘油-3-磷酸胆碱、
    1,2-二油酰-SN-甘油-3-磷酰乙醇胺(DOPE)、
    1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺、
    1,2-二乙烯醇基-sn-甘油-3-磷酸乙醇胺、
    1,2-二乙烯基-sn-甘油-3-磷酸乙醇胺、
    1,2-二芳基-sn-甘油-3-磷酸乙醇胺、
    1,2-二硫代六烯酸-sn-甘油-3-磷酸乙醇胺、
    1,2-二油酰基-sn-甘油-3-磷酸-(1-甘油)钠盐(DOPG)或鞘磷脂，
    例如，所述磷脂为DOPE或DSPC；
    所述结构脂质优选选自胆固醇、粪甾醇、谷甾醇、麦角甾醇、豆甾醇中的一种或两种以上；例如，所述结构脂质是胆固醇；和/或
    所述PEG脂质优选选自PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油或PEG修饰的二烷基甘油中的一种或两种以上，例如，所述PEG脂质为DMG-PEG2000。
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