WO2023116607A1 - 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 - Google Patents

一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 Download PDF

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WO2023116607A1
WO2023116607A1 PCT/CN2022/139942 CN2022139942W WO2023116607A1 WO 2023116607 A1 WO2023116607 A1 WO 2023116607A1 CN 2022139942 W CN2022139942 W CN 2022139942W WO 2023116607 A1 WO2023116607 A1 WO 2023116607A1
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nucleotide sequence
nucleotide
sirna
seq
nucleotides
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WO2023116607A9 (zh
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梁子才
张鸿雁
高山
丁学锋
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Suzhou Ribo Life Science Co Ltd
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Suzhou Ribo Life Science Co Ltd
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Priority to CN202280068763.2A priority Critical patent/CN118202049A/zh
Priority to CA3242059A priority patent/CA3242059A1/en
Priority to KR1020247021150A priority patent/KR20240117104A/ko
Priority to AU2022422864A priority patent/AU2022422864A1/en
Priority to JP2024533904A priority patent/JP2024546667A/ja
Priority to EP22909920.5A priority patent/EP4455284A1/en
Priority to US18/722,280 priority patent/US20250057870A1/en
Publication of WO2023116607A1 publication Critical patent/WO2023116607A1/zh
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    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/351—Conjugate

Definitions

  • the present disclosure relates to a nucleic acid capable of inhibiting mTORC1 activity and a pharmaceutical composition containing the nucleic acid and a siRNA conjugate.
  • the present disclosure also relates to preparation methods and uses of these nucleic acids, pharmaceutical compositions and siRNA conjugates.
  • Neurodegenerative diseases are progressive loss of neuronal structure and function, including neuronal death and glial homeostasis, leading to cognitive impairment such as dementia. Including Parkinson's disease (PD), Alzheimer's disease (AD), frontotemporal dementia (Frontotemporal dementia), Huntington's disease (Huntington disease, HD), amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis, ALS, commonly known as ALS) and spinal muscular atrophy (Spinal muscular atrophy, SMA) and so on.
  • AD and PD mainly occur in middle-aged and elderly people. With the aging of the population, the incidence of AD and PD is increasing, while HD, ALS and SMA may occur at all ages.
  • mTORC1 The mammalian target of rapamycin (mTOR) signaling pathway is a signaling pathway that regulates protein synthesis, cell growth, proliferation, etc.
  • mTORC1 is composed of RPTOR, Ras homolog enriched in brain (RHEB), DEP domain-containing mTOR-interacting protein (DEPTOR), mammalian yeast homolog A complex composed of lethal factor Sec13 protein 8 (mammalian lethal with SEC13 protein 8, mLST8) and 40kDa proline rich AKT substrate of 40kDa (PRAS40), which is a key negative regulator of autophagy regulation factor.
  • RHEB Ras homolog enriched in brain
  • DEPTOR DEP domain-containing mTOR-interacting protein
  • PRAS40 40kDa proline rich AKT substrate of 40kDa
  • mTORC1 is activated and mitophagy is inhibited in Alzheimer's Disease (AD) patients and animal models, which is the key cause of senile plaques, neurofibrillary tangles and cognitive decline in AD patients. It has been reported in the literature that inhibiting RPTOR can inhibit the activity of mTORC1, thereby promoting autophagy and mitophagy, thereby reducing misfolded proteins in the brain of patients with neurodegenerative diseases.
  • the inventors of the present disclosure unexpectedly found that the following siRNA and its modified sequence provided by the present disclosure can specifically inhibit the expression of RPTOR gene in cells, and the pharmaceutical composition and siRNA conjugate comprising the siRNA of the present disclosure can effectively inhibit the expression of the RPTOR gene
  • the disclosed siRNA is delivered to target tissues and/or cells, thereby showing high druggability in the treatment or prevention of neurodegenerative diseases, especially Alzheimer's disease.
  • the present disclosure provides an siRNA capable of inhibiting RPTOR gene expression
  • the siRNA contains a sense strand and an antisense strand
  • each nucleotide in the siRNA is independently a modified or unmodified nucleotide
  • the sense strand contains a nucleotide sequence I
  • the antisense strand contains a nucleotide sequence II
  • the nucleotide sequence I and the nucleotide sequence II are at least partially formed in reverse complementarity Double-stranded region, wherein, the nucleotide sequence I and the nucleotide sequence II are selected from a group of sequences shown in i)-iii) below:
  • nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and there are no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 2
  • nucleotide sequences shown are of equal length and differ by no more than 3 nucleotides:
  • Z a1 is A
  • Z a2 is U
  • the nucleotide sequence I includes a nucleotide Z a3 whose position corresponds to Z a1
  • the nucleotide sequence II includes a nucleoside whose position corresponds to Z a2 Acid Z a4 , said Z a4 being the first nucleotide at the 5' end of said antisense strand;
  • nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 123, and there are no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 124
  • nucleotide sequences shown are of equal length and differ by no more than 3 nucleotides:
  • Z b1 is A
  • Z b2 is U
  • the nucleotide sequence I includes a nucleotide Z b3 corresponding to Z b1
  • the nucleotide sequence II includes a nucleoside corresponding to Z b2 acid Z b4 , said Z b4 being the first nucleotide at the 5' end of said antisense strand;
  • nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 245, and there are no more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 246
  • nucleotide sequences shown are of equal length and differ by no more than 3 nucleotides:
  • Z c1 is G
  • Z c2 is C
  • the nucleotide sequence I contains a nucleotide Z c3 corresponding to Z c1
  • the nucleotide sequence II contains a nucleoside corresponding to Z c2 acid Z c4 , said Z c4 being the first nucleotide at the 5' end of the antisense strand.
  • the present disclosure provides a pharmaceutical composition comprising the siRNA of the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure provides an siRNA conjugate comprising the siRNA provided in the present disclosure and a conjugation group conjugated to the siRNA.
  • the present disclosure provides the use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure for the treatment and/or prevention of diseases related to the regulation of RPTOR function.
  • the present disclosure provides a method of treating a disease or symptom associated with modulation of RPTOR function, such as a disease or symptom associated with neurodegenerative or nonalcoholic steatohepatitis, particularly Alzheimer's disease, wherein The method includes administering an effective amount of the disclosed siRNA and/or pharmaceutical composition and/or siRNA conjugate to a subject in need thereof.
  • the present disclosure provides a method of inhibiting RPTOR gene expression in a cell, the method comprising contacting the cell with an effective amount of the siRNA and/or the pharmaceutical composition and/or the siRNA conjugate of the present disclosure.
  • the present disclosure also provides a kit containing the siRNA and/or the pharmaceutical composition and/or the siRNA conjugate of the present disclosure.
  • siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure have good stability, high RPTOR mRNA inhibitory activity, very low off-target effect, and/or can significantly treat and alleviate diseases related to RPTOR function regulation or symptom.
  • the siRNA, the pharmaceutical composition or the siRNA conjugate provided in the present disclosure exhibit excellent target gene expression inhibitory activity in in vitro cell experiments.
  • the siRNA of the present disclosure exhibited at least 40.5% RPTOR mRNA inhibition; at a concentration of 5 nM, the siRNA of the present disclosure exhibited at least 68.3% RPTOR mRNA inhibition rate; at a concentration of 50nM, the disclosed siRNA exhibits at least 71.1%, even up to 86.8% RPTOR mRNA inhibition rate, showing an excellent effect of inhibiting RPTOR gene expression.
  • the disclosed siRNAs with different lengths and modification schemes all have high inhibition rates.
  • the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure may have good stability and target mRNA inhibitory activity in vivo.
  • the siRNA conjugates of different modification schemes of the present disclosure showed 51.3% or 52.9% RPTOR mRNA expression inhibition rate in C57BL/6j mice, showing good RPTOR mRNA inhibitory activity.
  • siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure can inhibit the expression of RPTOR gene, effectively treat diseases related to mTORC1 activation and abnormal autophagy function, and have good application prospects.
  • Figure 1 is a bar graph showing the relative expression levels of RPTOR mRNA in HepG2 human liver cancer cells in vitro after transfection of siRNA of the present disclosure and a reference siRNA at a concentration of 50 nM.
  • Figure 2 is a bar graph showing the relative expression levels of RPTOR mRNA in HepG2 human liver cancer cells in vitro after transfection of different concentrations of siRNA of the present disclosure.
  • RPTOR mRNA refers to the sequence shown in Genbank accession number NM_020761.3.
  • target gene used in the present disclosure refers to the gene encoding the above-mentioned RPTOR mRNA
  • target mRNA refers to the above-mentioned RPTOR mRNA.
  • the uppercase letters C, G, U, and A represent the base composition of nucleotides;
  • the lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is methoxy Modified nucleotides;
  • the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s indicates that between the two nucleotides adjacent to the left and right of the letter s It is a phosphorothioate group connection;
  • P1 means that a nucleotide adjacent to the right side of the P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, and in some embodiments, P1 is Indicates specifically modified VP, Ps or P, wherein the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is vinyl phosphate (5'-(E)
  • fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine
  • non-fluorinated modified nucleotide refers to Nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribose group of a nucleotide with a non-fluorine group.
  • Nucleotide analog means a nucleic acid capable of replacing nucleotides, but is structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymus The group of pyrimidine deoxyribonucleotides. Such as isonucleotides, bridged nucleic acid (BNA) or acyclic nucleotides.
  • BNA bridged nucleic acid
  • methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • the expressions "complementary” or “reverse complementary” can be used interchangeably and have the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are each linked to the bases of the other strand. The bases on the base pair up in a complementary fashion.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA;
  • the purine base guanine (C) is always paired with the pyrimidine base cytosine Pyrimidines (G).
  • Each base pair consists of a purine and a pyrimidine.
  • mismatching means in the art that in a double-stranded nucleic acid, The bases at the corresponding positions are not paired in a complementary form.
  • substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementary” means that there is no more than one base mismatch between two nucleotide sequences; “complete reverse complement” means that there is no base mismatch between two nucleotide sequences.
  • nucleotide difference between a nucleotide sequence and another nucleotide sequence, which means that the base type of the nucleotide at the same position has changed between the former and the latter, For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is recognized as a difference between the two nucleotide sequences. There is a nucleotide difference at this position. In some embodiments, when the nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference occurs at that position.
  • Abasic nucleotides refer to monomeric compounds formed after nucleic acid bases in nucleotides are replaced by other groups or hydrogen atoms, such other groups include but are not limited to substituted or unsubstituted aryl or heteroaryl groups.
  • nucleoside monomer refers to the The type and sequence of nucleotides in siRNA or siRNA conjugates, modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites also known as Nucleoside phosphoramidites) used in solid-phase synthesis of phosphoramidites ).
  • Phosphoramidite solid phase synthesis is a method used in RNA synthesis well known to those skilled in the art.
  • the nucleoside monomers used in this disclosure are all commercially available.
  • conjugate means that two or more chemical moieties each having a specific function are covalently linked to each other; accordingly, a “conjugate” is Refers to the compound formed by covalent linkage between the various chemical moieties.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugates should be understood as a general term of multiple siRNA conjugates or siRNA conjugates represented by a certain chemical formula according to the context.
  • a "conjugate molecule” should be understood as a specific compound that can be conjugated to siRNA through a reaction, ultimately forming the siRNA conjugate of the present disclosure.
  • alkyl refers to straight and branched chains having the specified number of carbon atoms, typically 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms.
  • alkyl residue having a specific number of carbons all branched and straight chain forms having that number of carbons are intended to be encompassed; thus, for example, "butyl” is meant to include n-butyl, sec-butyl , isobutyl and tert-butyl; “propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl and refers to a divalent atomic group identical to alkyl but having two points of attachment.
  • alkenyl means an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double Obtained by removing a molecule of hydrogen.
  • the group can be in the cis or trans configuration of the double bond.
  • Typical alkenyl groups include, but are not limited to: vinyl; propenyl such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl base), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1- but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl and the like.
  • alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residues as alkenyl but with two points of attachment.
  • alkoxy refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentyloxy, etc.
  • An alkoxy group typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.
  • hydroxy protecting groups can be used in the present disclosure.
  • a protecting group renders a chemical functional group insensitive to specific reaction conditions and can be added to and removed from a molecule at that functional group without substantially damaging the rest of the molecule.
  • Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, cited in Each of the above-mentioned documents is incorporated herein in its entirety.
  • protecting groups are stable under basic conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups useful herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanth-9-yl (Mox).
  • non-exclusive examples of hydroxyl protecting groups useful herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy trityl) and TMTr (4,4',4"-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or a marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and any species poultry.
  • treatment refers to means of obtaining a beneficial or desired result, including but not limited to therapeutic benefit.
  • Treatment means eradicating or ameliorating the underlying disorder being treated.
  • therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, whereby improvement is observed in the subject, although the subject may still be afflicted by the underlying disorder.
  • Prevention refers to the means of obtaining a beneficial or desired result, including but not limited to prophylactic benefit.
  • siRNA, pharmaceutical composition, or siRNA conjugate may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physical symptoms of a disease, even if possible A diagnosis of the disease has not yet been made.
  • the present disclosure provides an siRNA capable of inhibiting RPTOR gene expression.
  • the siRNA of the present disclosure contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, and will not be repeated here.
  • the siRNA of the present disclosure contains a sense strand and an antisense strand, the lengths of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 Nucleotides.
  • the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/ 25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand and the antisense strand of the siRNA is 19/21, 21/21, 21/23 or 23/25.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partly reverse complementary to form a double-stranded region.
  • the siRNA disclosed herein may be the following first, second or third siRNA, and each siRNA will be described below.
  • the siRNA of the disclosure is a first siRNA.
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length, and have no more than 3 nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2
  • the nucleotide sequences shown are equal in length and differ by no more than 3 nucleotides:
  • Z a1 is A
  • Z a2 is U
  • the nucleotide sequence I includes a nucleotide Z a3 whose position corresponds to Z a1
  • the nucleotide sequence II includes a nucleoside whose position corresponds to Z a2 acid Z a4 , which is the first nucleotide at the 5' end of the antisense strand.
  • positional correspondence means being at the same position in the nucleotide sequence, counting from the same end of the nucleotide sequence.
  • position of the first nucleotide at the 3' end of nucleotide sequence I is the nucleotide corresponding to the first nucleotide at the 3' end of SEQ ID NO:1.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at Z a4 position, and Z a4 is selected from A, C or g.
  • Z a3 is a nucleotide complementary to Z a4 .
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two core There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementarity refers to no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity It means that there is no base mismatch between two nucleotide sequences.
  • the sense strand and the antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length; and the Described nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and described nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4:
  • Z a3 is selected from A, U, G or C, and Z a4 is a nucleotide complementary to Z a3 .
  • Z a3 is A and Z a4 is U.
  • the sense strand further comprises nucleotide sequence III
  • the antisense strand further comprises nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nuclei Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to 5 of the nucleotide sequence I ' end, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence
  • the second nucleotide sequence refers to the expression in the target mRNA that is identified by SEQ ID A nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by NO: 1 and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the core The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides, according to the 5' end In the direction to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, Both nucleotide sequences III and IV have a length of 3 nucleotides.
  • the base composition of the nucleotide sequence III is UCA
  • the base composition of the nucleotide sequence IV is UGA
  • the length ratio of the sense strand and the antisense strand is 22/22
  • the lengths of the nucleotide sequences III and IV are both 4 nucleotides, according to the direction from the 5' end to the 3' end, the core
  • the base composition of nucleotide sequence III is GUCA
  • the base composition of nucleotide sequence IV is UGAC; at this time, the length ratio of the sense strand and the antisense strand is 23/23.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • the siRNA of the disclosure is a second siRNA.
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 123 are equal in length, and there are no more than 3 nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 124 The nucleotide sequences shown are of equal length and differ by no more than 3 nucleotides:
  • Z b1 is A
  • Z b2 is U
  • the nucleotide sequence I includes a nucleotide Z b3 corresponding to Z b1
  • the nucleotide sequence II includes a nucleoside corresponding to Z b2 acid Z b4 , said Z b4 being the first nucleotide at the 5' end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 124 includes a difference at the Z b4 position, and Z b4 is selected from A, C or g.
  • Zb3 is a nucleotide complementary to Zb4 .
  • the siRNAs with the above-mentioned nucleotide differences have higher target mRNA inhibitory ability, and these siRNAs containing nucleotide differences are also within the protection scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two core There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementarity refers to no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity It means that there is no base mismatch between two nucleotide sequences.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 125
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 126:
  • Z b3 is selected from A, U, G or C, and Z b4 is a nucleotide complementary to Z b3 .
  • Zb3 is A and Zb4 is U.
  • the sense strand further comprises nucleotide sequence III
  • the antisense strand further comprises nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each 1-4 nuclei Nucleotide
  • the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary or completely reverse complementary
  • the nucleotide sequence III is connected to 5 of the nucleotide sequence I ' end
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence
  • the second nucleotide sequence refers to the expression in the target mRNA that is identified by SEQ ID A nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by NO: 123 and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the core The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides, according to the 5' end In the direction to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, Both nucleotide sequences III and IV have a length of 3 nucleotides.
  • the base composition of the nucleotide sequence III is UCA
  • the base composition of the nucleotide sequence IV is UGA
  • the length ratio of the sense strand and the antisense strand is 22/22
  • the lengths of the nucleotide sequences III and IV are both 4 nucleotides, according to the direction from the 5' end to the 3' end, the core
  • the base composition of the nucleotide sequence III is AUCA
  • the base composition of the nucleotide sequence IV is UGAU; at this time, the length ratio of the sense strand and the antisense strand is 23/23.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • the siRNA of the disclosure is a third siRNA.
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 245 are equal in length, and have no more than 3 nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 246 The nucleotide sequences shown are of equal length and differ by no more than 3 nucleotides:
  • Z c1 is G
  • Z c2 is C
  • the nucleotide sequence I contains a nucleotide Z c3 corresponding to Z c1
  • the nucleotide sequence II contains a nucleoside corresponding to Z c2 acid Z c4 , said Z c4 being the first nucleotide at the 5' end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 246 includes a difference at the Z c4 position, and Z c4 is selected from A, U or g.
  • Zc3 is a nucleotide complementary to Zc4 .
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two core There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementarity refers to no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity It means that there is no base mismatch between two nucleotide sequences.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 247
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 248:
  • Z c3 is selected from A, U, G or C, and Z c4 is a nucleotide complementary to Z c3 .
  • Z c3 is G and Z c4 is C.
  • the sense strand further comprises nucleotide sequence III
  • the antisense strand further comprises nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each 1-4 nuclei Nucleotide
  • the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary or completely reverse complementary
  • the nucleotide sequence III is connected to 5 of the nucleotide sequence I ' end
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence
  • the second nucleotide sequence refers to the expression in the target mRNA that is identified by SEQ ID A nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by NO: 245 and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the core The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides, according to the 5' end In the direction to the 3' end, the base composition of the nucleotide sequence III is AA, and the base composition of the nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, Both nucleotide sequences III and IV are 3 nucleotides in length, according to the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CAA, and the base composition of the
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • nucleotide sequence V nucleotide sequence VI
  • nucleic acid sequence nucleotide modification in siRNA, and modified sequence
  • first siRNA second siRNA or third siRNA. That is, if there is no specific description, the following description of siRNA should be regarded as a description of the first siRNA, the second siRNA and the third siRNA one by one.
  • siRNA further contains the nucleotide sequence V
  • the siRNA further contains the nucleotide sequence V
  • the specific siRNA is specified.
  • the sense strand and the antisense strand are different in length, and the antisense strand also contains a nucleotide sequence V, the length of the nucleotide sequence V is 1 to 3 nucleotides, linked at The 3' end of the antisense strand constitutes the 3' overhang of the antisense strand.
  • the sense strand further contains nucleotide sequence VI, the length of nucleotide sequence VI is 1 to 3 nucleotides, connected to the 3' end of the sense strand, constituting the 3' end of the sense strand. ' overhang.
  • the siRNA provided by the present disclosure includes nucleotide sequence V but does not include nucleotide sequence VI.
  • the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23 , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25, or 23/26.
  • the siRNA provided by the present disclosure includes nucleotide sequences V and VI.
  • the length of nucleotide sequence V is the same or different from the length of nucleotide sequence VI.
  • the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure may be (19-26):(19-26).
  • the length of the nucleotide sequence V and/or VI is 2 nucleotides, thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/21, 21 /21, 21/23, 23/23, 23/25, or 25/25.
  • each nucleotide in the nucleotide sequence V can be any nucleotide.
  • the nucleotide sequence V is two consecutive thymine deoxy Ribonucleotide (dTdT) or 2 consecutive uracil ribonucleotides (UU); or, in order to improve the affinity of the siRNA antisense strand and the target mRNA, the nucleotide sequence V and the corresponding position of the target mRNA acid complementary. Therefore, in some embodiments, the ratio of the lengths of the sense strand and the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, at this time, the siRNA of the present disclosure has better mRNA silencing activity.
  • Each nucleotide in the nucleotide sequence VI can be any nucleotide, in order to facilitate synthesis and save synthesis costs, in some embodiments, the nucleotide sequence VI is two consecutive thymines Deoxyribonucleotide (dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to improve the affinity of siRNA sense strand and antisense strand, the core of nucleotide sequence VI and the corresponding position of target mRNA nucleotides are the same.
  • dTdT Deoxyribonucleotide
  • UU uracil ribonucleotides
  • the siRNA of the present disclosure comprises nucleotide sequences V and VI, and the length ratio of the sense strand and the antisense strand of the siRNA is 21/21 or 23/23. At this time, the siRNA of the present disclosure has more Good mRMA silencing activity.
  • the nucleotide at the corresponding position of the target mRNA refers to the nucleotide or nucleotide sequence adjacent to the 5' end of a nucleotide sequence of the target mRNA, and the nucleotide sequence of the target mRNA is the same as the nucleotide sequence Sequence II is substantially reverse-complementary or completely reverse-complementary, or a nucleotide sequence that is substantially reverse-complementary or completely reverse-complementary to the nucleotide sequence composed of nucleotide sequence II and nucleotide sequence IV.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5
  • the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 6:
  • said Z a4 is the first nucleotide at the 5' end of the antisense strand
  • Z a3 is selected from A, U, G or C
  • Z a4 is a nucleotide complementary to Z a3 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 8:
  • Z a4 is the first nucleotide at the 5' end of the antisense strand
  • Z a3 is selected from A, U, G or C
  • Z a4 is a nucleotide complementary to Z a3 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 127
  • the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 128:
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 129
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 130:
  • said Z b4 is the first nucleotide at the 5' end of the antisense strand
  • Z b3 is selected from A, U, G or C
  • Z b4 is a nucleotide complementary to Z b3 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 249, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 250:
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 251
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 252:
  • said Z c4 is the first nucleotide at the 5' end of the antisense strand
  • Z c3 is selected from A, U, G or C
  • Z c4 is a nucleotide complementary to Z c3 .
  • the siRNAs described in the present disclosure are siRPTORa1, siRPTORa2, siRPTORa3, siRPTORb1, siRPTORb2, siRPTORb3, siRPTORc1, siRPTORc2, and siRPTORc3 listed in Table 1.
  • the nucleotides in the siRNAs of the present disclosure are each independently modified or unmodified nucleotides.
  • the nucleotides in the siRNA of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, and the core
  • the siRNA of the present disclosure contains at least one modified nucleotide.
  • modified nucleotide refers to a nucleotide or nucleotide analogue, or a nucleoside, in which the 2' hydroxyl group of the ribose group of a nucleotide is substituted by other groups.
  • the base on the acid is the nucleotide of the modified base.
  • the modified nucleotides will not cause obvious weakening or loss of the function of siRNA to inhibit gene expression. For example, modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 can be selected.
  • At least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate with a modification group
  • at least a part of the phosphate group and/or the ribose group in the phosphate-sugar backbone of at least one single chain in the sense strand and the antisense strand is a phosphate group with a modification group and/or Or a ribose group with a modifying group.
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
  • the inventors of the present disclosure surprisingly found that the siRNA provided by the present disclosure achieved a high balance of stability in plasma and gene silencing efficiency in animal experiments.
  • the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and the fluorinated modified nucleotides in the nucleotide sequence I are not more than 5, And, according to the direction from the 5' end to the 3' end, the 7th, 8th, and 9th nucleotides of the nucleotide sequence I are fluorine-modified nucleotides; in the nucleotide sequence II, the fluorine There are no more than 7 nucleotides modified by fluorine substitution, and the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorine-modified nucleotides.
  • the core at the 7th, 8th, and 9th or 5th, 7th, 8th, and 9th positions of the nucleotide sequence I
  • the nucleotides are fluorinated modified nucleotides, and the nucleotides in the remaining positions in the sense strand are non-fluorinated modified nucleotides; according to the direction from the 5' end to the 3' end, in the antisense strand , the 2nd, 6th, 14th, 16th or 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides, and in the antisense strand Nucleotides at the remaining positions are non-fluorinated modified nucleotides.
  • fluorine-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, which has the structure shown in the following formula (7).
  • Non-fluorine-modified nucleotide refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is substituted by a non-fluorine group A sort of.
  • Nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by a non-fluorine group are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'- Substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.
  • the 2'-alkoxy-modified nucleotides are methoxy-modified nucleotides (2'-OMe), as shown in formula (8).
  • the 2'-substituted alkoxy modified nucleotide for example, can be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), such as formula (9 ) shown.
  • the 2′-amino modified nucleotide (2′-NH 2 ) is represented by formula (10).
  • the 2'-deoxynucleotide (DNA) is shown in formula (11):
  • Nucleotide analogs are capable of replacing nucleotides in nucleic acids, but are structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymidine ribonucleotides group of ribonucleotides.
  • the nucleotide analogs can be isonucleotides, bridged nucleic acid (BNA for short) or acyclic nucleotides.
  • BNA refers to constrained or inaccessible nucleotides. BNAs may contain five-membered, six-membered, or seven-membered ring bridged structures with "fixed" C3'-endosugar constrictions. Typically the bridge is incorporated at the 2'-,4'-position of the ribose to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA etc., wherein, LNA is as shown in formula (12), ENA is as shown in formula (13), and cET BNA is as shown in formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide can be unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is shown in formula (15), and GNA is shown in formula (16):
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotide refers to a compound formed by changing the position of the base in the nucleotide on the ribose ring.
  • the isonucleotide can be a compound formed by moving the base from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18).
  • Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA.
  • each non-fluorinated modified nucleotide is a methoxy modified nucleotide.
  • the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is substituted with a methoxy group.
  • the non-fluorinated modified nucleotides are independently 2'-O-methoxy modified or 2'-O-methoxyethyl modified nucleotides.
  • fluorine-modified nucleotide refers to a compound with the structure shown in formula (7) formed by replacing the 2'-hydroxyl group of the nucleotide with fluorine;
  • methoxy-modified Nucleotide refers to a compound with the structure shown in formula (8) formed by substituting the 2'-hydroxyl group of the nucleotide ribose group with a methoxy group;
  • 2'-O-methoxyethyl modified core Nucleotide refers to a compound having the structure shown in formula (9) formed by substituting the 2'-hydroxyl group of the nucleotide ribose group with a methoxyethyl group.
  • the siRNA of the present disclosure is an siRNA with the following modifications: according to the direction from the 5' end to the 3' end, in the sense strand, the 7th, 8th, and 9th positions of the nucleotide sequence I Or the 5th, 7th, 8th, and 9th nucleotides are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand Among them, the 2nd, 6th, 14th, 16th or 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides, and the antisense Nucleotides at the remaining positions in the chain are methoxy-modified nucleotides.
  • the siRNA of the present disclosure is an siRNA with the following modifications: according to the direction from the 5' end to the 3' end, the 5th, 7th, 8th and 9th positions of the nucleotide sequence I in the sense strand of the siRNA
  • the nucleotides are fluorine-modified nucleotides
  • the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides
  • the siRNA The 2nd, 6th, 14th, and 16th or 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the nucleotide sequence II in the antisense strand are fluorinated modified nucleotides
  • the antisense strand of siRNA Nucleotides at other positions are methoxy-modified nucleotides;
  • the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the sense of the siRNA
  • the nucleotides at the remaining positions of the chain are methoxy-modified nucleotides, and, according to the direction from the 5' end to the 3' end, the 2nd, 6th, and 14th positions of the nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at position 16 and 16 are fluorine-modified nucleotides, and the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides;
  • the 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the sense strand of the siRNA
  • the nucleotides at the remaining positions are methoxy-modified nucleotides, and, according to the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th positions of the nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at the position are fluorine-modified nucleotides, and the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides.
  • the siRNA of the present disclosure is an siRNA with the following modifications: according to the direction from the 5' end to the 3' end, the 7th, 8th, 9th or 5th position of the nucleotide sequence I in the sense strand of the siRNA is , the nucleotides at positions 7, 8 and 9 are fluorine-modified nucleotides, and the nucleotides at the rest of the sense strand of the siRNA are methoxy-modified nucleotides; direction, the 2nd, 6th, 14th, and 16th or 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the nucleotide sequence II in the antisense strand of the siRNA are fluorinated modified nucleotides and, according to the direction from the 5' end to the 3' end, at least one of the 3-6 nucleotides of the nucleotide sequence II is a 2'-O-methoxyethyl modified nucleoside
  • the 3rd or 5th nucleotide in the antisense strand is a 2′-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions in the antisense strand are Nucleotides are methoxy-modified nucleotides.
  • nucleotides in the 3rd to 9th nucleotides in the nucleotide sequence II are 2'-O-methoxy Ethyl-modified nucleotides.
  • the siRNA provided by the present disclosure is arbitrarily selected from one of the following siRNAs:
  • the siRNA with the above modification is not only low in cost, but also makes it difficult for ribonuclease in the blood to cut nucleic acid, thereby increasing the stability of nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.
  • the modified siRNA has higher activity of inhibiting target mRNA.
  • the phosphate group with the modification group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the The phosphate group with modification group is a phosphorothioate group with structure shown in formula (1):
  • This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.
  • the phosphorothioate linkage is present at at least one of the following positions: the first and second cores at either end of the sense strand or the antisense strand between the second and third nucleotides at either end of the sense or antisense strand; or any combination of the above. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 5' end of the sense strand. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present in at least one of the following positions:
  • the siRNA provided by the present disclosure is arbitrarily selected from one of the following siRNAs: siRPTORa1-M1S, siRPTORa1-M1X, siRPTORa1-M2S, siRPTORa1-M2X, siRPTORa1-M3S, siRPTORa1-M3X, siRPTORa2-M1S, siRPTORa2-M1X, siRPTORa2-M2S, siRPTORa2-M2X, siRPTORa2-M3S, siRPTORa2-M3X, siRPTORa3-M1S, siRPTORa3-M1X, siRPTORa3-M2S, siRPTORa3-M2X, siRPTORa3-M3S, siRPTORa3-M3X, siRPTORa1-T1S, si RPTORa1- T2S, siRPTORb1-M1S, siRPTORb1-M1X, siRPTORb1-M2S, siRPTORa1-M
  • the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides or 5'-phosphate analog modified nucleotides are well known to those skilled in the art, for example, the 5'-phosphate nucleotides may have the following structure:
  • R is selected from H, OH, methoxy, fluorine;
  • Base represents a nucleic acid base, selected from A, U, C, G or T.
  • the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification shown in formula (2)
  • the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate ( 5'-(E)-vinylphosphonate, E-VP) modified nucleotides, as shown in formula (3), or phosphorothioate modified nucleotides, as shown in formula (5).
  • the siRNA provided by the present disclosure is arbitrarily selected from one of the following groups: siRPTORa1-M1P1, siRPTORa1-M2P1, siRPTORa1-M3P1, siRPTORa2-M1P1, siRPTORa2-M2P1, siRPTORa2-M3P1, siRPTORa3-M1P1, siRPTORa3-M2P1, siRPTORa3-M3P1, siRPTORa1-M1SP1, siRPTORa1-M2SP1, siRPTORa1-M3SP1, siRPTORa2-M1SP1, siRPTORa2-M2SP1, siRPTORa2-M3SP1, siRPTORa3-M1SP1, siRPTORa3-M2SP1, siRPTORa3-M3SP1, siRPTORa1-M1XP1, siRPTORa1- M2XP1, siRPTORa1-M3XP1, siRPTORa2-M1XP1, siRPTORa2-M1XP1, siRPTORa2-M1
  • the inventors of the present disclosure unexpectedly found that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains high gene inhibitory activity.
  • the siRNA provided in the present disclosure can be obtained by conventional siRNA preparation methods in the art (such as solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis has commercialized customized services.
  • a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a correspondingly modified nucleoside monomer, a method for preparing a correspondingly modified nucleoside monomer and introducing a modified nucleotide group Methods of siRNA are also well known to those skilled in the art.
  • the pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (magnetic nanoparticles, such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon nanotubes ( carbon nanotubes), mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), polylysine acid (poly(L-lysine), PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly D Type or L-type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid)copolymer, PLGA), poly(aminoethylethylene phosphate) (poly(2-
  • the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1:( 1-500), in some embodiments, the above weight ratio is 1:(1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds routinely used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent and an osmotic pressure regulator.
  • the pH buffer can be a tris hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5 buffer.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator can be, for example, sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmol/kg (mOsm/kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
  • the dosage of the preparation made from the pharmaceutical composition will be adjusted due to different administration methods during administration.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation can be used for subcutaneous, intramuscular, intracerebroventricular injection or intrathecal injection, but not limited to, and can also deliver the pharmaceutical composition through nasal administration, oropharyngeal inhalation, spray administration and the like.
  • the pharmaceutical composition is delivered by intrathecal injection.
  • the intrathecal injection of the pharmaceutical composition into the spinal fluid can be performed as a bolus injection or via minipumps that can be implanted under the skin to provide regular and constant delivery of siRNA to in the spinal fluid.
  • a surgically implanted osmotic pump is administered intrathecally.
  • an osmotic pump is implanted in the subarachnoid space of the spinal canal to facilitate intrathecal administration. Further details regarding this intrathecal delivery system can be found in PCT/US 2015/013253, filed January 28, 2015, which is hereby incorporated by reference in its entirety.
  • the pharmaceutical composition may be in the form of a liposomal formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipid.
  • the organic amine, helper lipid and pegylated lipid can be selected from the amine-containing transfection compounds described in Chinese patent application CN103380113A (which is incorporated herein by reference in its entirety) or its One or more of pharmaceutically acceptable salts or derivatives, helper lipids and pegylated lipids.
  • the organic amine can be a compound represented by formula (201) or a pharmaceutically acceptable salt thereof described in Chinese patent application CN103380113A:
  • X 101 and X 102 are each independently O, S, NA or CA, wherein A is hydrogen or C 1 -C 20 hydrocarbon chain;
  • R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 are each independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic Group, cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, substituted or unsubstituted, branched or straight chain acyl group, substituted or unsubstituted Substituted, branched or linear aryl, substituted or unsubstituted, branched or linear heteroaryl;
  • x is an integer of 1-10;
  • R 103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl, the alkyl A radical or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
  • R 103 can be any of the following formulas (204)-(213):
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R 103 is the same as in formula (201) Possible points of attachment of the nitrogen atom in , where each H at any * position can be replaced to achieve attachment to the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.
  • the organic amine is an organic amine shown in formula (214) and/or an organic amine shown in formula (215):
  • the helper lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives
  • the pegylated lipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxyl (polyethylene glycol)]-2000.
  • the molar ratio among the organic amine, the helper lipid and the pegylated lipid is (19.7-80):(19.7-80 ):(0.3-50), such as (50-70):(20-40):(3-20).
  • the particles of the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, usually about 40 nm to about 135 nm, more typically, the liposome
  • the average diameter of the particles is from about 50 nm to about 120 nm, from about 50 nm to about 100 nm, from about 60 nm to about 90 nm, or from about 70 nm to about 90 nm, for example, the liposome particles have an average diameter of about 30, 40, 50, 60, 70 nm , 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160nm.
  • the weight of siRNA and total lipid is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1: 18.
  • the weight ratio of the disclosed siRNA to total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1 :11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.
  • each component of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used.
  • the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, only the siRNA provided by the present disclosure can be used to replace the existing siRNA; in some In the embodiment, it can be prepared according to the following method:
  • the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid around room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more of, for example, can be ethanol.
  • the siRNA provided by the present disclosure is dissolved in a buffered saline solution to obtain an aqueous siRNA solution.
  • concentration of the buffered saline solution is 0.05-0.5M, such as 0.1-0.2M
  • the pH of the buffered saline solution is adjusted to 4.0-5.5, such as 5.0-5.2
  • amount of the buffered saline solution is such that the concentration of siRNA does not exceed 0.6mg /mL, for example, can be 0.2-0.4 mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and/or potassium acetate.
  • the lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation.
  • the volume ratio of lipid solution and siRNA aqueous solution is 1:(2-5), for example, it can be 1:4.
  • the incubated liposome preparation Concentrate or dilute the incubated liposome preparation, remove impurities, and sterilize to obtain the pharmaceutical composition provided by the present disclosure, whose physical and chemical parameters are pH 6.5-8, encapsulation efficiency not less than 80%, particle size 40-200nm, polydispersity index not higher than 0.30, osmotic pressure 250-400mOsm/kg; for example, physical and chemical parameters can be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100nm, more The dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • concentration or dilution can be performed before, after or simultaneously with the removal of impurities.
  • Various existing methods can be used to remove impurities, for example, a tangential flow system, a hollow fiber column, and ultrafiltration at 100KDa can be used, and the ultrafiltration exchange solution is phosphate buffer saline (PBS) with a pH of 7.4.
  • PBS phosphate buffer saline
  • Various existing methods can be used for the sterilization method, for example, filtration sterilization on a 0.22 ⁇ m filter can be used.
  • the present disclosure provides an siRNA conjugate comprising the siRNA described above and a conjugation group conjugated to the siRNA.
  • the conjugating group comprises at least one targeting group and/or delivery aiding group that is pharmaceutically acceptable.
  • the conjugation group further comprises a linker, and the linker and/or the targeting group or the delivery auxiliary group are connected in sequence.
  • the siRNA molecule may be non-covalently or covalently conjugated to the conjugating group, for example may be covalently conjugated to the conjugating group.
  • the conjugation site between the siRNA and the conjugating group can be at the 3' end or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA.
  • the conjugation site between the siRNA and the conjugating group is at the 3' end of the sense strand of the siRNA.
  • the conjugate group can be attached to the phosphate group, the 2'-position hydroxyl group or the base of the nucleotide.
  • the conjugate group can also be connected to the 3'-position hydroxyl, and in this case, the nucleotides are connected by 2'-5' phosphodiester bonds.
  • the conjugation group When the conjugation group is connected to the end of the siRNA chain, the conjugation group is usually connected to the phosphate group of the nucleotide; when the conjugation group is connected to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose sugar ring or base.
  • the literature Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleotides elicit robust gene silencing in vivo in hepatitis.ACS Chemical biology, 2015, 10(5):1181-7.
  • the siRNA and the conjugated group may be linked by acid-labile or reducible chemical bonds, and these chemical bonds can be degraded in the acidic environment of the endosome of the cell, thereby making the siRNA a free state.
  • the conjugation group can be attached to the sense strand of siRNA, so as to minimize the impact of conjugation on siRNA activity.
  • the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA administration.
  • each ligand is independently selected from a ligand capable of binding to a cell surface receptor.
  • at least one targeting ligand targets a receptor that mediates delivery to central nervous system (CNS) tissue.
  • CNS central nervous system
  • the types of these ligands are well known to those skilled in the art, and their role is to bind to specific receptors on the surface of target cells and mediate delivery of siRNA linked to ligands to target cells.
  • at least one targeting group is selected from ligands capable of binding to cell surface receptors expressing RPTOR.
  • At least one targeting moiety is a ligand targeting a receptor on the surface of a hepatic parenchymal cell. In some embodiments, at least one or each targeting moiety is a ligand targeting the asialoglycoprotein receptor on the surface of hepatocytes. In some embodiments, at least one or each targeting group is N-acetylgalactosamine (GalNAc). In some embodiments, the conjugates of the present disclosure have various siRNA conjugate structures disclosed in CN110959011A, the entire disclosure of which is incorporated herein by reference.
  • the conjugates of the present disclosure have the structure shown in formula (301):
  • Nu represents the siRNA group formed by the siRNA of the present disclosure.
  • the siRNA conjugate has a structure shown in formula (301), wherein Nu has a corresponding one of siRPTORa2-M1S, siRPTORb2-M1S, siRPTORc2-M1S, siRPTORc1-T1S and siRPTORc1-T2S
  • the sequence of the conjugate group is attached to the ribose 3' position of the 3' terminal nucleotide of the sense strand of the siRNA in the Nu, and the siRNA conjugate is in the form of a sodium salt.
  • each targeting group is selected from ligands capable of binding to cell surface receptors expressing RPTOR.
  • at least one targeting moiety is a ligand targeting a receptor on the surface of a target cell of the CNS.
  • each targeting group is a ligand that targets a receptor on the surface of a target cell in the CNS.
  • the ligand can be conjugated to the siRNA to achieve specific CNS tissue delivery, and in some embodiments, at least one targeting moiety is a peptide ligand. In some embodiments, each targeting group is a peptide ligand.
  • the targeting ligand is selected from the group consisting of angiopeptide-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) Ligands, mannose receptor ligands, glucose transporters, and LDL receptor ligands.
  • LRP lipoprotein receptor-related protein
  • TfR transferrin receptor
  • the pharmaceutically acceptable delivery adjuvant group may be a lipophilic group comprising an aliphatic compound or an alicyclic compound.
  • the lipophilic group contains a linear aliphatic hydrocarbon, a branched aliphatic hydrocarbon, or a steroid.
  • the lipophilic group contains a saturated or unsaturated C4-C30 hydrocarbon chain.
  • the lipophilic group contains a saturated or unsaturated C6-C18 hydrocarbon chain (eg, a linear C6-C18 alkyl or alkenyl group).
  • lipophilic groups contain saturated or unsaturated C16 hydrocarbon chains (eg, straight chain C16 alkyl or alkenyl).
  • the lipophilic group is a C6-C30 aliphatic acyl group, and the C6-C30 aliphatic acyl group refers to the remaining atomic group after the hydroxyl group is removed from a C6-C30 fatty acid.
  • the lipophilic group is non-covalently or covalently conjugated to the siRNA.
  • the conjugation site of the lipophilic group to the siRNA is at the 3' end or the 5' end of the sense strand of the siRNA. In some embodiments, the conjugation site of the lipophilic group to the siRNA is at the 5' end of the antisense strand.
  • the conjugation site of the lipophilic group to the siRNA can also be in the internal sequence of the siRNA.
  • the lipophilic group is attached to the phosphate group, ribose sugar ring, or base of a nucleotide.
  • preferred locations are those that do not interfere with the hydrogen bonding interactions required for base pairing.
  • the lipophilic group is attached to the phosphate group, the 2'-hydroxyl group, or the base of the nucleotide.
  • the lipophilic group can be conjugated to the ribose sugar ring via the 2'-position hydroxyl group.
  • the targeting group is conjugated to the siRNA via one or more linkers.
  • the inventors of the present disclosure unexpectedly found that the siRNA conjugates of the present disclosure have significantly improved stability in plasma, and also exhibited higher RPTOR mRNA silencing activity.
  • the siRNA of the present disclosure may be one of the siRNAs shown in Tables 1a, 1b, and 1c. Using these siRNAs, the siRNA conjugates of the present disclosure exhibit higher RPTOR mRNA silencing activity.
  • the uppercase letters C, G, U, and A indicate the base composition of nucleotides;
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates The adjacent nucleotide on the left side of the letter f is a fluorine-modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by a phosphorothioate group;
  • P1 indicates that the right side of the P1 is connected
  • the adjacent one nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • P1 is VP, Ps or P representing a specific modification
  • the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is vinyl phosphate (5'-(E)- Vinylphosphonate, E-VP) modified nucleotides
  • the letter combination Ps indicates that the adjacent nucleotide on the right side of the letter combination Ps is a phosphorothioate modified nucleotide
  • the capital letter P indicates that the right side of the letter P is the same
  • the adjacent nucleotide is a 5'-phosphate nucleotide.
  • the letter combination moe indicates that the adjacent nucleotide to the left of the letter combination moe is a nucleotide with 2'-O-methoxyethyl modification.
  • the letter combination G moe means that the nucleotide base adjacent to the left side of the letter combination moe is guanine and has a 2'-O-methoxyethyl modified nucleotide.
  • the letter combination C moe indicates that the nucleotide base adjacent to the left side of the letter combination moe is 5-methylcytosine and has a 2'-O-methoxyethyl modified nucleotide.
  • each U in the sequences listed in the above table can be arbitrarily replaced with T, which will not significantly affect the activity or off-target effect of the siRNA.
  • each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, and the phosphodiester bond or phosphorothioate
  • the non-bridging oxygen atom or sulfur atom in the bond has a negative charge, which can exist in the form of hydroxyl or mercapto, and the hydrogen ions in the hydroxyl or mercapto can also be partially or completely replaced by cations.
  • the cation may be any cation, such as one of metal cations, ammonium ions NH 4 + , and organic ammonium cations.
  • the cation is selected from one or more of alkali metal ions, ammonium cations formed from tertiary amines, and quaternary ammonium cations.
  • the alkali metal ions may be K + and/or Na +
  • the cations formed by tertiary amines may be ammonium ions formed by triethylamine and/or ammonium ions formed by N,N-diisopropylethylamine.
  • the siRNA or siRNA conjugates described herein may exist at least in part in the form of a salt.
  • the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond is at least partially combined with a sodium ion, and the siRNA or siRNA conjugate described in the present disclosure is a sodium salt or a partial sodium salt form exists.
  • modified nucleotide groups can be introduced into the siRNAs described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are either commercially available or prepared using known methods.
  • the siRNA conjugates of the present disclosure can be prepared using any reasonable synthetic route.
  • the reactive group in the conjugated molecule can be protected with a protecting agent first, and then linked To the solid-phase carrier, and then through the phosphoramidite solid-phase synthesis method, according to the nucleotide type and sequence of the siRNA sense strand and antisense strand, the nucleoside monomers are connected one by one in the direction from 3' to 5', and each nucleoside
  • the linking of monomers includes four steps of deprotection, coupling, capping, oxidation or sulfuration; the sense and antisense strands of siRNA are separated and annealed to obtain the siRNA conjugate of the present disclosure.
  • siRNA conjugates can also be carried out with reference to the disclosure content of existing literature.
  • WO2019010274A1 described in Example 1 a method of sequentially linking a linking group with a specific structure and a targeting ligand to siRNA through reactions. Its entire contents are incorporated herein by reference.
  • siRNA of the present disclosure and the pharmaceutical composition containing the siRNA and the siRNA conjugate use
  • the present disclosure provides the use of the siRNA and/or the pharmaceutical composition and/or the siRNA conjugate of the present disclosure in the preparation of a medicament for the treatment and prevention of diseases or symptoms related to the regulation of RPTOR function.
  • the diseases or symptoms related to the regulation of RPTOR function are diseases related to diseases caused by activation of mTORC1 and abnormal autophagy function.
  • the neurodegenerative disease or symptom is Alzheimer's disease and/or Parkinson's disease, preferably, the neurodegenerative disease is Alzheimer's disease.
  • the disease associated with abnormal autophagy function is non-alcoholic steatohepatitis.
  • the present disclosure provides a method for preventing and/or treating diseases or symptoms associated with RPTOR function regulation, the method comprising introducing an effective amount of the disclosed siRNA and/or pharmaceutical composition and/or siRNA The conjugate is administered to a subject in need thereof.
  • the siRNA active ingredient of the present disclosure By administering the siRNA active ingredient of the present disclosure to a subject in need, the purpose of preventing and/or treating the resulting disease can be achieved through the mechanism of RNA interference. Therefore, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure can be used for the prevention and/or treatment of diseases or symptoms related to the regulation of RPTOR function, or for the preparation of A drug that modulates an associated disease or condition.
  • the term "administration/administration” refers to a method or approach that at least partially localizes the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure at a desired site to produce a desired effect,
  • the siRNAs, pharmaceutical compositions and/or siRNA conjugates of the present disclosure are placed into a subject.
  • Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, local administration results in delivery of more of the siRNA conjugate to a specific site compared to the subject's systemic circulation; whereas systemic administration results in delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to the subject's basic systemic circulation.
  • the present disclosure is intended to provide means for the prevention and/or treatment of neurodegenerative diseases
  • delivery of the drug to central nervous system tissue is employed.
  • a mode of administration that delivers the drug intrathecally is employed.
  • the drug is administered by injection into the spinal fluid.
  • Administration to a subject may be by any suitable route known in the art, including, but not limited to: oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal Drugs, airway (aerosol), intracerebroventricular, intrathecal, nasal, rectal, and topical (including buccal and sublingual).
  • oral or parenteral routes such as intravenous, intramuscular, subcutaneous, transdermal Drugs, airway (aerosol), intracerebroventricular, intrathecal, nasal, rectal, and topical (including buccal and sublingual).
  • the frequency of administration can be one or more times per day, every week, every two weeks, every three weeks, every month or every year.
  • the dosage of the siRNA, the pharmaceutical composition or the siRNA conjugate described in the present disclosure can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially the age, body weight and sex of the subject.
  • Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., by determining the LD50 (the lethal dose causing 50% of the population to die) and the ED50 (in dose response, the dose eliciting 50% of the maximum response intensity, In the qualitative response, it refers to the dose that can cause 50% of the test subjects to have a positive response).
  • a range of dosage for use in humans can be derived based on the data obtained from cell culture assays and animal studies.
  • the dose of the siRNA, the pharmaceutical composition or the preparation made of the siRNA conjugate is adjusted during the administration according to the different administration methods.
  • the amount of siRNA can be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in some embodiments 0.05-20 mg/kg body weight, 0.1-15 mg/kg body weight in other embodiments, 0.1-10 mg/kg body weight in other embodiments;
  • the amount of siRNA can be 0.001-50 mg/kg body weight, in some embodiments 0.01-10 mg/kg body weight, in some embodiments 0.05-5 mg/kg body weight, in some embodiments 0.1-3 mg/kg weight.
  • the present disclosure provides a method of inhibiting RPTOR gene expression in a cell, the method comprising contacting an effective amount of the disclosed siRNA and/or pharmaceutical composition and/or siRNA conjugate with the cell , introducing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure into the cell, and achieve the purpose of inhibiting the expression of RPTOR gene in the cell through the mechanism of RNA interference.
  • the amount of siRNA in the provided modified siRNA, pharmaceutical composition and/or siRNA conjugate is generally such an amount: it is sufficient to reduce the expression of the target gene, and This results in an extracellular concentration of 1 pM to 1 ⁇ M, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or 0.05 nM to about 5 nM at the target cell surface.
  • the amount necessary to achieve this local concentration will vary depending on various factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, the route of delivery (local or systemic), etc. .
  • the concentration at the site of delivery can be significantly higher than the concentration at the surface of the target cell or tissue.
  • the present disclosure provides a kit comprising an effective amount of at least one of an siRNA of the present disclosure, a pharmaceutical composition, and a siRNA conjugate.
  • kits described herein can provide siRNA in one container.
  • a kit described herein may comprise a container providing a pharmaceutically acceptable excipient.
  • the kit may also contain other components, such as stabilizers or preservatives.
  • the kits described herein can comprise at least one additional therapeutic agent in a container other than the container in which the siRNA described herein is provided.
  • the kit can comprise instructions for mixing the siRNA with a pharmaceutically acceptable carrier and/or excipients or other ingredients, if any.
  • the siRNA and pharmaceutically acceptable carrier and/or adjuvant, and the modified siRNA, pharmaceutical composition and/or siRNA conjugate, and/or pharmaceutically acceptable carrier And/or excipients may be provided in any form, such as liquid form, dry form or lyophilized form.
  • the siRNA and the pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or the pharmaceutically acceptable carrier and/or adjuvant of the siRNA conjugate are substantially pure and/or sterile.
  • sterile water can be provided in kits of the present disclosure.
  • reagents and medium used in the following examples are commercially available, and the operations such as nucleic acid electrophoresis and real-time PCR used are all referred to in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)). method to proceed.
  • the reagents and culture media used in the following examples are all commercially available, and the operations such as nucleic acid electrophoresis and real-time PCR used are all referred to in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). method to proceed.
  • siRNA sequences listed in Table 2 by solid-phase synthesis method, respectively dissolve equimolar sense strand and antisense strand in Table 2 using DEPC water, and then anneal to obtain siRPTORa2-M1X and siRPTORb2- M1X, siRPTORc2-M1X, siRPTORc1-T2S, and siRPTORc1-M1S.
  • the sense and antisense strands corresponding to the siRNAs whose siRNA numbers are NC in Table 2 were synthesized by solid-phase synthesis method. Use DEPC water to dissolve equimolar sense strand and antisense strand, and then anneal to obtain a reference siRNA, numbered NC, NC is the siRNA used as a negative control, which has no sequence homology with RPTOR mRNA.
  • the uppercase letters C, G, U, and A indicate the base composition of nucleotides;
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates The nucleotide next to the left side of the letter f is a fluorine-modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are linked by phosphorothioate groups;
  • the uppercase letter P indicates the letter A nucleotide adjacent to the right side of P is a 5'-phosphate nucleotide;
  • the letter combination moe indicates that a nucleotide adjacent to the left side of the letter combination moe has a 2'-O-methoxyethyl modification of nucleotides.
  • siRNA or reference siRNA of the present disclosure is prepared, freeze-dry and store as solid powder for future use.
  • the conjugate 1 of the present disclosure was obtained, the only difference is that when preparing the sense strand and the antisense strand single strand, according to the siRPTORc1-T2S in Table 2
  • the sequences of the sense strand and the antisense strand are connected to the nucleoside monomers at the corresponding positions one by one.
  • Molecular weight detection was performed by liquid chromatography-mass chromatography (LC-MS).
  • Nu in formula (301) is siRPTORc1-T2S in Table 1 of the present disclosure, the conjugating group is connected to the ribose 3' position at the 3' end of the sense strand in siRPTORc1-T2S, and the conjugate is in the sodium salt form.
  • siRPTORa2-M1X, siRPTORb2-M1X and siRPTORc2-M1X of the present disclosure in HepG2 human liver cancer cells in vitro was investigated.
  • HepG2 human liver cancer cells purchased from Nanjing Kebai Company were cultured at 37°C in an incubator containing 5% CO2/95% air. Biological Technology Co., Ltd).
  • HepG2 cells were seeded in a 12-well plate at 2.0 ⁇ 105 cells/well, 1 mL of cell liquid per well, and after 24 h of culture, the medium in the culture wells was aspirated, and an additional 500 ⁇ L of Opti-MEM medium (GIBCO, Inc. ).
  • siRPTORa2-M1X, siRPTORb2-M1X and siRPTORc2-M1X prepared in Preparation Examples 1-3 were prepared with PBS buffer solution to prepare siRNA working solutions with a concentration of 20 ⁇ M.
  • each 1A1 solution contains 48.5 ⁇ L Opti-MEM medium and 1.5 ⁇ L of 20 ⁇ M siRNA working solution.
  • each 1B solution contains 49 ⁇ L of Opti-MEM medium and 1 ⁇ L of Lipofectamine TM 2000 (Invitrogen).
  • test group 1 In the two culture wells (the above-mentioned culture wells containing HepG2 cells and 500 ⁇ L Opti-MEM medium, the same below), respectively add the transfection complex 1Xa, mix evenly, and the addition amount is 100 ⁇ L/well to obtain a final concentration of The 50nM transfection mixture was designated as test group 1.
  • transfection complex 1Xb In the two culture wells (the above culture wells containing HepG2 cells and 500 ⁇ L Opti-MEM medium, the same below), respectively add transfection complex 1Xb, mix evenly, and the addition amount is 100 ⁇ L/well, and the final concentration is The transfection mixture at 50 nM was designated as test group 2.
  • transfection complex 1Xc the addition amount is 100 ⁇ L/well to obtain a final concentration of The transfection mixture at 50 nM was designated as test group 3.
  • transfection complex 1X In the two culture wells, respectively add transfection complex 1X, mix evenly, the addition amount is 100 ⁇ L/well, obtain the transfection mixture without siRNA, record as the blank control group.
  • test groups 1-3 and the blank control group were cultured in the culture wells for 4 hours, the supernatant in each culture well was sucked off, and 1 mL of Opti-MEM medium was added to each well.
  • the 12-well plate was placed in a CO 2 incubator to continue culturing at 37 °C for 24 h.
  • each reverse transcription reaction system For each reverse transcription reaction system, take 5 ⁇ L of the above cDNA-containing solution as a template, use SYBR qPCR SuperMix Plus Kit (purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B) provides 20 ⁇ L of reagents to configure the qPCR reaction system.
  • the PCR primer sequences used to amplify the target gene RPTOR and the internal reference gene GAPDH are shown in Table 3 As indicated, the final concentration of each primer was 0.25 ⁇ M.
  • Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument, and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 30s, and extension at 72°C for 30s. After repeating the above-mentioned denaturation, annealing and extension process 40 times in total, the product W1 containing amplified target gene RPTOR and internal reference gene GAPDH was obtained. The product W1 was then incubated at 95°C for 15s, 60°C for 1min, and 95°C for 15s. The real-time fluorescent quantitative PCR instrument collected the melting curves of the target gene and the internal reference gene GAPDH in the product W1 respectively, and obtained the Ct values of the target gene RPTOR and the internal reference gene GAPDH. .
  • the comparative Ct ( ⁇ Ct) method was used to perform relative quantitative calculation of the target gene RPTOR in each test group, and the calculation method was as follows:
  • ⁇ Ct (test group) Ct (target gene of test group) - Ct (internal reference gene of test group)
  • ⁇ Ct (control group) Ct (control group target gene) - Ct (control group internal reference gene)
  • ⁇ Ct (test group) ⁇ Ct (test group) - ⁇ Ct (average of the control group)
  • ⁇ Ct (control group) ⁇ Ct (control group) - ⁇ Ct (control group average)
  • ⁇ Ct average of control group
  • ⁇ Ct control group
  • the expression level of RPTOR mRNA in the test group was normalized, and the average value of the expression level of RPTOR mRNA in the blank control group was defined as 100%.
  • Test group RPTOR mRNA inhibition rate (1- test group RPTOR mRNA relative expression level) ⁇ 100%
  • Figure 1 is a bar graph showing the relative expression level of RPTOR mRNA in HepG2 human liver cancer cells in vitro after transfection of 50nM siRNA of the present disclosure and reference siRNA NC, where NC in Figure 1 represents the reference siRNA NC.
  • the results in Figure 1 show that in HepG2 human liver cancer cells in vitro, the RPTOR mRNA inhibition rate of siRPTORa2-M1X was 76.8% at 50nM concentration; the RPTOR mRNA inhibition rate of siRPTORb2-M1X was 86.8% at 50nM concentration; When the RPTOR mRNA inhibition rate was 78.8%, it showed excellent RPTOR mRNA inhibitory activity, showing an excellent effect of inhibiting RPTOR gene expression.
  • This experimental example investigated the RPTOR mRNA inhibitory activity of different concentrations of siRPTORa2-M1X, siRPTORb2-M1X and siRPTORc2-M1X of the present disclosure in HepG2 human liver cancer cells in vitro.
  • HepG2 human liver cancer cells purchased from Nanjing Kebai Company were cultured at 37°C in an incubator containing 5% CO2/95% air. Biological Technology Co., Ltd).
  • HepG2 cells were seeded in a 12-well plate at 2.0 ⁇ 105 cells/well, 1 mL of cell liquid per well, and after 24 h of culture, the medium in the culture wells was aspirated, and an additional 500 ⁇ L of Opti-MEM medium (GIBCO, Inc. ).
  • siRPTORa2-M1X, siRPTORb2-M1X and siRPTORc2-M1X prepared in Preparation Examples 1-3 were prepared into siRNA working solutions with concentrations of 20 ⁇ M, 2 ⁇ M and 0.2 ⁇ M, respectively, with PBS buffer.
  • each 2A1 solution contains 48.5 ⁇ L Opti-MEM medium and 1.5 ⁇ L siRNA working solution with a concentration of 20 ⁇ M to obtain siRNA working solution of 2A1a, 2A1b or 2A1c, respectively.
  • each 2A2 solution contains 48.5 ⁇ L Opti-MEM medium and 1.5 ⁇ L siRNA working solution with a concentration of 2 ⁇ M, to obtain siRNA working solution of 2A2a, 2A2b or 2A2c, respectively.
  • each 2A3 solution contains 48.5 ⁇ L Opti-MEM medium and 1.5 ⁇ L siRNA working solution with a concentration of 0.2 ⁇ M to obtain siRNA working solution of 2A3a, 2A3b or 2A3c, respectively.
  • each 2B solution contains 49 ⁇ L of Opti-MEM medium and 1 ⁇ L of Lipofectamine TM 2000 (Invitrogen).
  • each siRNA For each siRNA, add the transfection complex 2Xa1, 2Xa2 or 2Xa3 respectively into two culture wells (the above culture wells containing HepG2 cells and 500 ⁇ L Opti-MEM medium, the same below), and mix evenly. 100 ⁇ L/well to obtain a transfection mixture containing siRPTORa2-M1X at a final concentration of 50 nM, 5 nM or 0.5 nM, which was designated as test group 1.
  • each siRNA For each siRNA, add the transfection complex 2Xb1, 2Xb2 or 2Xb3 respectively into two culture wells (the above culture wells containing HepG2 cells and 500 ⁇ L Opti-MEM medium, the same below), and mix evenly. 100 ⁇ L/well to obtain a transfection mixture containing siRPTORb2-M1X at a final concentration of 50 nM, 5 nM or 0.5 nM, which was designated as test group 2.
  • each siRNA For each siRNA, add the transfection complex 2Xc1, 2Xc2 or 2Xc3 respectively into two culture wells (the above culture wells containing HepG2 cells and 500 ⁇ L Opti-MEM medium, the same below), and mix evenly. 100 ⁇ L/well to obtain a transfection mixture containing siRPTORc2-M1X at a final concentration of 50 nM, 5 nM or 0.5 nM, which was designated as test group 3.
  • test groups 1-3 and the blank control group were cultured in the culture wells for 4 hours, the supernatant in each culture well was sucked off, and 1 mL of Opti-MEM medium was added to each well.
  • the 12-well plate was placed in a CO 2 incubator to continue culturing at 37 °C for 24 h.
  • Fig. 2 is a histogram of the relative expression level of RPTOR mRNA in HepG2 human liver cancer cells in vitro after transfecting different concentrations of siRNA of the present disclosure.
  • the results of Fig. 2 show that in HepG2 human liver cancer cells in vitro, at a low concentration of 0.5nM siRPTORc2-M1X showed at least 40.5% RPTOR mRNA inhibition rate; siRPTORa2-M1X showed at least 68.3% RPTOR mRNA inhibition rate at a concentration of 5nM, and at least 71.1% RPTOR mRNA inhibition rate at a concentration of 50nM; At a concentration of 50nM, siRPTORb2-M1X can even achieve 86.4% RPTOR mRNA inhibition rate, showing an excellent effect of inhibiting RPTOR gene expression.
  • Conjugate 1 and Conjugate 2 prepared in Preparation Example 6-7 were respectively dissolved in PBS to form a 3 mg/ml siRNA conjugate solution (calculated as siRNA).
  • C57BL/6 mice female, 16-18 g in weight, 6-8 weeks old, purchased from Speiford Co., Ltd.
  • the above-mentioned siRNA conjugate 1 solution was administered to each mouse in the first group by subcutaneous injection on the back of the neck, weighed and recorded body weight before administration, and administered according to body weight.
  • the administration volume was 5 mL/kg, as Test group 1: Each mouse in the second group was given the above-mentioned siRNA conjugate 2 solution, weighed and recorded body weight before administration, administered according to body weight, and the administration volume was 5mL/kg, as test group 2; In addition, PBS was administered to each of a group of mice, and the administration volume was 5 mL/kg, which was used as a blank control group.
  • the administration time point was regarded as the first day, and on the eighth day, the liver tissues of each mouse in the test group and the blank control group were collected and preserved with RNAlater.
  • Test group 1 Test group 2 RPTOR mRNA inhibition rate % 52.9% 51.3%
  • the results show that, at a concentration of 3 mg/kg, compared with the results of the blank control group, the siRNA conjugates of different modification schemes of the present disclosure still show the same effect within one week after administration in C57BL/6i mice. More than 50% RPTOR mRNA inhibition rate in vivo, showing good RPTOR mRNA inhibition activity and long-term stability in vivo.
  • the siRNA disclosed in the present disclosure can effectively inhibit the expression of RPTOR gene in cells, so it can be used in the treatment and/or prevention of diseases related to mTORC1 abnormal activation and autophagy, such as NASH or neurodegenerative diseases or related diseases.
  • diseases related to mTORC1 abnormal activation and autophagy such as NASH or neurodegenerative diseases or related diseases.
  • the drug side of symptoms shows good potential.

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