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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苏州瑞博生物技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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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  • 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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Abstract

提供了一种抑制RPTOR基因表达水平，调控mTORC1活性的siRNA，含有该siRNA的药物组合物和缀合物。所述siRNA含有正义链和反义链，所述的siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述核苷酸序列I与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述反义链含有核苷酸序列II，所述核苷酸序列II与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异。提供的siRNA、药物组合物和缀合物可以作为mTORC1抑制剂，有效治疗mTORC1激活导致的相关疾病并作为自噬诱导剂治疗自噬相关疾病或者症状。

Description

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 技术领域
本公开涉及一种能够抑制mTORC1活性的核酸和含有该核酸的药物组合物与siRNA缀合物。本公开还涉及这些核酸、药物组合物与siRNA缀合物的制备方法和用途。
背景技术
神经退行性疾病是神经元结构和功能逐渐丧失，包括神经元死亡和胶质细胞平衡，会导致痴呆等认知障碍。包括帕金森病(Parkinson’s disease，PD)、阿尔茨海默病(Alzheimer’s disease，AD)、额颞叶痴呆(Frontotemporal dementia)、亨廷顿氏病(Huntington disease，HD)、肌萎缩侧索硬化症(Amyotrophic lateral sclerosis，ALS，俗称渐冻人症)以及脊髓性肌萎缩症(Spinal muscular atrophy，SMA)等等。AD及PD主要发生于中、老年，随着人口老龄化，AD及PD的发病日益增多，而HD、ALS及SMA等在各个年龄都可能发生。
目前，神经退行性疾病药物治疗以西药为主，但药物治疗需要长期服药，副作用明显，容易影响中枢神经、消化、呼吸等系统，甚至导致内分泌紊乱、情绪改变等。
哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)信号通路是一条调节蛋白质合成、细胞生长、增殖等的信号通路，在细胞内存在mTORC1和mTORC2两种不同的复合体。mTORC1是由RPTOR、Ras蛋白脑组织同源类似物(Ras homolog enriched in brain，RHEB)、含DEP结构域的mTOR相互作用蛋白(DEP domain-containing mTOR-interacting protein，DEPTOR)、哺乳动物酵母同源致命因子Sec13蛋白8(mammalian lethal with SEC13 protein 8，mLST8)和40kDa富脯氨酸AKT底物(Proline rich AKT substrate of 40kDa，PRAS40)组成的复合体，该复合体是自噬调节的关键负调控因子。以往研究表明，阿尔兹海默(Alzheimer’s Disease，AD)患者和动物模型中mTORC1被激活，线粒体自噬被抑制，是导致AD患者老年斑，神经纤维缠结以及认知功能下降的关键原因。文献报道通过抑制RPTOR能抑制mTORC1活性，从而促进细胞自噬和线粒体自噬，进而减少神经退行性疾病患者脑内错误折 叠蛋白。
开发调节RPTOR基因表达水平的新药物，调控mTORC1活性，进而调节与mTORC1激活和自噬功能异常相关的疾病或症状、特别是神经退行性疾病进行治疗，在本领域中存在显著需求。
发明内容
本公开的发明人意外发现，具有本公开提供的如下siRNA及其修饰序列能够特异性地抑制细胞中RPTOR基因的表达，包含本公开的siRNA的药物组合物和siRNA缀合物能够有效地将本公开的siRNA递送至目标组织和/或细胞，从而在神经退行性疾病、特别是阿尔茨海默病的治疗或预防中显示出高的成药潜力。
在一方面，本公开提供了一种能够抑制RPTOR基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分反向互补地形成双链区，其中，所述核苷酸序列I和所述核苷酸序列II选自如下i)-iii)所示序列中的一组：
i)所述核苷酸序列I与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5’-CUGCCAUGGAGUAUCUGAZ a1-3′(SEQ ID NO：1)；
5′-Z a2UCAGAUACUCCAUGGCAG-3′(SEQ ID NO：2)，
其中，Z a1为A，Z a2为U，所述核苷酸序列I中包含位置对应于Z a1的核苷酸Z a3，所述核苷酸序列II中包含位置对应于Z a2的核苷酸Z a4，所述Z a4是所述反义链5′末端的第一个核苷酸；
ii)所述核苷酸序列I与SEQ ID NO：123所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：124所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5′-ACAACAUCAAGUACUACGZ b1-3′(SEQ ID NO：123)；
5′-Z b2CGUAGUACUUGAUGUUGU-3′(SEQ ID NO：124)，
其中，Z b1为A，Z b2为U，所述核苷酸序列I中包含位置对应于Z b1的核苷酸Z b3，所述核苷酸序列II中包含位置对应于Z b2的核苷酸Z b4，所述Z b4是所述反义链5′末端的第一个核苷酸；
iii)所述核苷酸序列I与SEQ ID NO：245所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：246所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5′-CGACUACUACAUCUCCGUZ c1-3′(SEQ ID NO：245)；
5′-Z c2ACGGAGAUGUAGUAGUCG-3′(SEQ ID NO：246)，
其中，Z c1为G，Z c2为C，所述核苷酸序列I中包含位置对应于Z c1的核苷酸Z c3，所述核苷酸序列II中包含位置对应于Z c2的核苷酸Z c4，所述Z c4是所述反义链5′末端的第一个核苷酸。
在另一方面，本公开提供了一种药物组合物，所述药物组合物含有本公开的siRNA和药学上可接受的载体。
在又一方面，本公开提供了一种siRNA缀合物，所述siRNA缀合物含有本公开提供的siRNA以及缀合连接至该siRNA的缀合基团。
在又一方面，本公开提供了本公开的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防与RPTOR功能调节相关的疾病用途。
在又一方面，本公开提供了一种治疗与RPTOR功能调节相关的疾病或症状、例如神经退行性或非酒精性脂肪性肝炎相关的疾病或症状，特别是阿尔茨海默病的方法，所述方法包括向有需要的受试者给予有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物。
在又一方面，本公开提供了一种抑制细胞中RPTOR基因表达的方法，该方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物与所述细胞接触。
此外，本公开还提供了一种试剂盒，所述试剂盒含有本公开的siRNA和/或药物组合物和/或siRNA缀合物。
以引用的方式并入
本说明书中提及的所有出版物、专利以及专利申请均以引用的方式并入本文，其程度与每一单独的出版物、专利或专利申请均专门并且单独地以引用的方式并入本文的程度相同。
有益效果
本公开提供的siRNA、药物组合物和siRNA缀合物具有良好的稳定性，较高的RPTOR mRNA抑制活性，很低的脱靶效应，和/或能显著治疗和缓解与RPTOR功能调节相关的疾病或症状。
例如，本公开提供的siRNA，药物组合物或siRNA缀合物在体外细胞实验中显示出优异的靶基因表达抑制活性。例如，在体外HepG2人肝癌细胞中，在0.5nM的低浓度下，本公开的siRNA显示出至少40.5％RPTOR mRNA抑制率；在5nM的浓度下，本公开的siRNA显示出至少68.3％RPTOR mRNA抑制率；在50nM的浓度下，本公开的siRNA显示出至少71.1％，甚至可达86.8％RPTOR mRNA抑制率，显示出优异的抑制RPTOR基因表达的效果。并且不同长度和修饰方案的本公开siRNA均有很高的抑制率。
又例如，本公开提供的siRNA、药物组合物或siRNA缀合物可在体内具有良好的稳定性和靶mRNA抑制活性。例如，在3mg/kg的浓度下，本公开不同修饰方案的siRNA缀合物在C57BL/6j小鼠体内显示出51.3％或者52.9％RPTOR mRNA表达抑制率，显示出良好的RPTOR mRNA抑制活性。
由此说明，本公开提供的siRNA、药物组合物以及siRNA缀合物能够抑制RPTOR基因的表达，有效治疗与mTORC1激活所致疾病和细胞自噬功能异常相关的疾病，具有良好的应用前景。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
图1是显示了转染50nM浓度的本公开的siRNA和参比siRNA后，体外HepG2人肝癌细胞中的RPTOR mRNA相对表达水平的柱状图。
图2是显示了转染不同浓度的本公开的siRNA后，体外HepG2人肝 癌细胞中的RPTOR mRNA相对表达水平的柱状图。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
定义
在本公开中，RPTOR mRNA是指Genbank注册号为NM_020761.3所示的序列。进一步地，若无特别说明，本公开中所使用的术语“靶基因”是指编码上述RPTOR mRNA的基因，术语“靶mRNA”是指上述RPTOR mRNA。
在上文及下文中，如无特别说明，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接；P1表示该P1右侧相邻的一个核苷酸为5′-磷酸核苷酸或5′-磷酸类似物修饰的核苷酸，在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5′-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5′-磷酸核苷酸。
在上文及下文中，所述“氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被氟取代形成的核苷酸，“非氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。“核苷酸类似物”指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。如异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。所述“甲氧基修饰的核苷酸”指核糖基的2′-羟基被甲氧基取代而形成的核苷酸。
在本文的上下文中，表述“互补”或“反向互补”可互相替代使用，并 具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基各自与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基胸腺嘧啶(T)(或者在RNA中为尿嘧啶(U)相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。
在上文及下文中，如无特别说明，“基本上反向互补”是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配；“实质上反向互补”是指两段核苷酸序列之间存在不多于1个的碱基错配；“完全反向互补”是指两段核苷酸序列之间不存在碱基错配。
在上文及下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方式中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。无碱基核苷酸是指核苷酸中的核酸碱基被其它基团或氢原子代替后形成的单体化合物，该其它基团包括不限于取代或未取代的芳香基或杂芳基。
在上文及下文中，特别是在描述本公开的siRNA、药物组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)是指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，亚磷酰胺固相合成中使用的修饰或未修饰的核苷亚磷酰胺单体(unmodified or modified RNA phosphoramidites，有时RNA phosphoramidites也称为Nucleoside phosphoramidites)。亚磷酰胺固相合成为本领域技术人员所公知的RNA合成中所用的方法。本公开所用的核苷单体均可商购得到。
在本公开的上下文中，除非另有说明，“缀合”是指两个或多个各自 具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。siRNA缀合物应根据上下文，理解为多个siRNA缀合物的总称或者某个化学式所示的siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本公开的siRNA缀合物的特定化合物。
本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或本身不稳定的任何取代或取代模式。
如本文所使用的，“烷基”是指具有指定数量的碳原子的直链和支链，所述数量通常为1至20个碳原子，例如1至10个碳原子，如1至8个或1至6个碳原子。例如，C 1-C 6烷基包含1至6个碳原子的直链和支链烷基。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的二价原子团。
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一分子氢而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1，3-二烯-1-基、丁-1，3-二烯-2-基等等。在某些实施方式中，烯基基团具有2到20个碳原子，而在其他实施方式中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个连接点的残基。
如本文所使用的，“烷氧基”是指通过氧桥连接的指定数量碳原子的烷基，例如，甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、戊氧基、2-戊氧基、异戊氧基、新戊氧基、己氧基、2-己氧基、3-己氧基、3-甲基戊氧基等。烷氧基通常具有1至10个、1至8 个、1至6个，或1至4个通过氧桥连接的碳原子。
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能团对特定的反应条件不敏感，并且可以在分子中的该官能团添加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992，48，2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2，2d ed，John Wiley&Sons，New York，1991中，以引用的方式将上述文献各自整体并入本文。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基氧杂蒽-9-基(Pixyl)和9-(对甲氧基苯基)氧杂蒽-9-基(Mox)。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4，4′-二甲氧基三苯甲基)和TMTr(4，4′，4″-三甲氧基三苯甲基)。
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、兔、绵羊、大鼠和任何种类的家禽。
如本文所使用的，“治疗”指的是获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。
如本文所使用的“预防”指获得有益或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将siRNA、药物组合物或siRNA缀合物给予有罹患特定疾病风险的受试者，或给予报告疾病的一种或多种生理症状的受试者，即便可能该疾病的诊断尚未作出。
本公开的siRNA
在一方面，本公开提供了一种能够抑制RPTOR基因表达的siRNA。
本公开的siRNA含有核苷酸基团作为基本结构单元，本领域技术人员公知，所述核苷酸基团含有磷酸基团、核糖基团和碱基，在此不再赘述。
本公开的siRNA含有正义链和反义链，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。这样，本公开提供的siRNA正义链和反义链的长度比可以是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或23/26。在一些实施方式中，所述siRNA正义链和反义链的长度比为19/21、21/21、21/23或23/25。
按照本公开，所述siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分反向互补形成双链区。
在一些实施方式中，本公开siRNA可以是以下第一种、第二种或者第三种siRNA，在下文中分别对每一种siRNA进行说明。
第一种siRNA
在一些实施方式中，本公开的siRNA是第一种siRNA。其中所述核苷酸序列I与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5′-CUGCCAUGGAGUAUCUGAZ a1-3′(SEQ ID NO：1)；
5′-Z a2UCAGAUACUCCAUGGCAG-3′(SEQ ID NO：2)，
其中，Z a1为A，Z a2为U，所述核苷酸序列I中包含位置对应于Z a1的核苷酸Z a3，所述核苷酸序列II中包含位置对应于Z a2的核苷酸Z a4，所述Z a4是所述反义链5′末端的第一个核苷酸。
在上下文中，“位置对应”是指从核苷酸序列相同端起算，处于核苷酸序列中相同的位置。例如，核苷酸序列I的3′端第一个核苷酸的位置 是对应于SEQ ID NO：1的3′端第1个核苷酸的核苷酸。
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。在一些实施方式中，所述核苷酸序列I与SEQ ID NO：1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述的核苷酸序列II与SEQ ID NO：2所示的核苷酸序列之间不多于1个核苷酸序列差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO：2所示的核苷酸序列之间的核苷酸差异包括Z a4位置处的差异，且Z a4选自A、C或G。在一些实施方式中，其中，Z a3是与Z a4互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有碱基错配。
在一些实施方式中，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸；并且所述核苷酸序列I是SEQ ID NO：3所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO：4所示的核苷酸序列：
5′-CUGCCAUGGAGUAUCUGAZ a3-3′(SEQ ID NO：3)；
5′-Z a4UCAGAUACUCCAUGGCAG-3′(SEQ ID NO：4)，
其中，Z a3选自A、U、G或C，Z a4是与Z a3互补的核苷酸。在一些实施方式中，其中Z a3为A，Z a4为U。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1～4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5′末端，所述核苷酸序列IV连接在所述核苷酸序列II的3′末端。
在一些实施方式中，所述核苷酸序列IV与第二段核苷酸序列实质上 反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO：1表示的核苷酸序列的5′末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。在一些实施方式中，按照5′-3′的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为A，核苷酸序列IV的碱基为U；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为CA，核苷酸序列IV的碱基组成为UG；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为UCA，核苷酸序列IV的碱基组成为UGA；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为GUCA，核苷酸序列IV的碱基组成为UGAC；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。
第二种siRNA
在一些实施方式中，本公开的siRNA是第二种siRNA。其中，所述核苷酸序列I与SEQ ID NO：123所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：124所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5′-ACAACAUCAAGUACUACGZ b1-3′(SEQ ID NO：123)；
5′-Z b2CGUAGUACUUGAUGUUGU-3′(SEQ ID NO：124)，
其中，Z b1为A，Z b2为U，所述核苷酸序列I中包含位置对应于Z b1的核苷酸Z b3，所述核苷酸序列II中包含位置对应于Z b2的核苷酸Z b4，所述Z b4是所述反义链5′末端的第一个核苷酸。
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。在一些实施方式中，所述核苷酸序列I与SEQ ID NO：123所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO：124所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO：124所示的核苷酸序列之间的核苷酸差异包括Z b4位置处的差异，且Z b4选自A、C或G。在一些实施方式中，Z b3是与Z b4互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有碱基错配。
在一些实施方式中，所述核苷酸序列I是SEQ ID NO：125所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO：126所示的核苷酸序列：
5′-ACAACAUCAAGUACUACGZ b3-3′(SEQ ID NO：125)；
5′-Z b4CGUAGUACUUGAUGUUGU-3′(SEQ ID NO：126)，
其中，Z b3选自A、U、G或C，Z b4是与Z b3互补的核苷酸。在一些实施方式中，Z b3为A，Z b4为U。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1-4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5′末端，所述核苷酸序列IV连接在所述核苷酸序列II的3′末端。
在一些实施方式中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO：123表示的核苷酸序列的5′末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。在一些实施方式中，按照5′-3′的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为A，核苷酸序列IV的碱基为U；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为CA，核苷酸序列IV的碱基组成为UG；此时，正义链和反义链的长度比为21/21；或者， 核苷酸序列III和IV的长度均为3个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为UCA，核苷酸序列IV的碱基组成为UGA；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为AUCA，核苷酸序列IV的碱基组成为UGAU；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。
第三种siRNA
在一些实施方式中，本公开的siRNA是第三种siRNA。其中，所述核苷酸序列I与SEQ ID NO：245所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：246所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5′-CGACUACUACAUCUCCGUZ c1-3′(SEQ ID NO：245)；
5′-Z c2ACGGAGAUGUAGUAGUCG-3′(SEQ ID NO：246)，
其中，Z c1为G，Z c2为C，所述核苷酸序列I中包含位置对应于Z c1的核苷酸Z c3，所述核苷酸序列II中包含位置对应于Z c2的核苷酸Z c4，所述Z c4是所述反义链5′末端的第一个核苷酸。
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。在一些实施方式中，所述核苷酸序列I与SEQ ID NO：245所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO：246所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO：246所示的核苷酸序列之间的核苷酸差异包括Z c4位置处的差异，且Z c4选自A、U或G。在一些实施方式中，Z c3是与Z c4互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指 两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有碱基错配。
在一些实施方式中，所述核苷酸序列I是SEQ ID NO：247所示的核苷酸序列，核苷酸序列II是SEQ ID NO：248所示的核苷酸序列：
5′-CGACUACUACAUCUCCGUZ c3-3′(SEQ ID NO：247)；
5′-Z c4ACGGAGAUGUAGUAGUCG-3′(SEQ ID NO：248)，
其中，Z c3选自A、U、G或C，Z c4是与Z c3互补的核苷酸。在一些实施方式中，Z c3为G，Z c4为C。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1-4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5′末端，所述核苷酸序列IV连接在所述核苷酸序列II的3′末端。
在一些实施方式中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO：245表示的核苷酸序列的5′末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。在一些实施方式中，按照5′-3′的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为A，核苷酸序列IV的碱基为U；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为AA，核苷酸序列IV的碱基组成为UU；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为CAA，核苷酸序列IV的碱基组成为UUG；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为GCAA，核苷酸序列IV的碱基组成为UUGC；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补， 因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。
以下，对核苷酸序列V、核苷酸序列VI、核酸序列、siRNA中的核苷酸修饰以及修饰序列的描述适用于上述第一种siRNA、第二种siRNA或第三种siRNA。即如果没有特指，下面对siRNA的描述应视为是对第一种siRNA、第二种siRNA和第三种siRNA逐一进行了描述。例如，如不特别指明具体的siRNA，“所述siRNA还含有核苷酸序列V”的意思是“第一种siRNA、第二种siRNA或第三种siRNA还含有核苷酸序列V”。
在一些实施方式中，所述正义链和所述反义链长度不同，所述反义链还含有核苷酸序列V，核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3′末端，构成反义链的3′突出端。
在一些实施方式中，所述正义链还含有核苷酸序列VI，核苷酸序列VI的长度为1至3个核苷酸，连接在所述正义链的3′末端，构成正义链的3′突出端。
在一些实施方式中，本公开提供的siRNA包括核苷酸序列V，但不包括核苷酸序列VI。由此，本公开提供的siRNA正义链和反义链的长度比可以是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或23/26。在一些实施方式中，本公开提供的siRNA包括核苷酸序列V和VI。在一些实施方式中，核苷酸序列V的长度与核苷酸序列VI的长度相同或不同。由此，本公开提供的siRNA正义链和反义链的长度比可以是(19-26)∶(19-26)。在一些实施方式中，所述核苷酸序列V和/或VI的长度为2个核苷酸，由此，本公开提供的siRNA正义链和反义链的长度比可以是19/21、21/21、21/23、23/23、23/25或25/25。
所述核苷酸序列V中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约成本，在一些实施方式中，所述核苷酸序列V为连续的2个胸腺嘧啶脱氧核糖核苷酸(dTdT)或连续的2个尿嘧啶核糖核苷酸(UU)；或者，为了提高siRNA反义链与靶mRNA的亲和力，核苷酸序列V与靶mRNA的相应位置的核苷酸互补。因此，在一些实施方式中，本公开的siRNA的正义链和反义链长度之比为19/21或21/23，此时，本 公开的siRNA具有更好的mRNA沉默活性。
所述核苷酸序列VI中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约合成成本，在一些实施方式中，所述核苷酸序列VI为连续的两个胸腺嘧啶脱氧核糖核苷酸(dTdT)或连续的两个尿嘧啶核糖核苷酸(UU)；或者，为了提高siRNA正义链与反义链的亲和力，核苷酸序列VI与靶mRNA的相应位置的核苷酸相同。因此，在一些实施方式中，本公开的siRNA包含核苷酸序列V和VI，siRNA正义链和反义链的长度之比为21/21或23/23，此时，本公开的siRNA具有更好的mRMA沉默活性。
靶mRNA的相应位置的核苷酸是指与靶mRNA的一段核苷酸序列在5′末端相邻的核苷酸或核苷酸序列，该段靶mRNA的核苷酸序列是与核苷酸序列II实质上反向互补或完全反向互补，或者与核苷酸序列II和核苷酸序列IV构成的核苷酸序列实质上反向互补或完全反向互补的那段核苷酸序列。
在一些实施方式中，所述siRNA的正义链含有如SEQ ID NO：5所示的核苷酸，所述反义链含有如SEQ ID NO：6所示的核苷酸序列：
5′-CUGCCAUGGAGUAUCUGAZ a3-3′(SEQ ID NO：5)；
5′-Z a4UCAGAUACUCCAUGGCAGUG-3′(SEQ ID NO：6)；
其中，所述Z a4是反义链5′末端的第一个核苷酸，Z a3选自A、U、G或C，并且Z a4是与Z a3互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO：7所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO：8所示的核苷酸序列：
5′-CACUGCCAUGGAGUAUCUGAZ a3-3′(SEQ ID NO：7)；
5′-Z a4UCAGAUACUCCAUGGCAGUGAC-3′(SEQ ID NO：8)；
其中，所述Z a4是反义链5′末端的第一个核苷酸，Z a3选自A、U、G或C，并且Z a4是与Z a3互补的核苷酸。
在一些实施方式中，所述siRNA的正义链含有如SEQ ID NO：127所示的核苷酸序列，所述反义链含有如SEQ ID NO：128所示的核苷酸序列：
5′-ACAACAUCAAGUACUACGZ b3-3′(SEQ ID NO：127)；
5′-Z b4CGUAGUACUUGAUGUUGUUG-3′(SEQ ID NO：128)；
或者，所述siRNA的正义链含有如SEQ ID NO：129所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO：130所示的核苷酸序列：
5′-CAACAACAUCAAGUACUACGZ b3-3′(SEQ ID NO：129)；
5′-Z b4CGUAGUACUUGAUGUUGUUGAU-3′(SEQ ID NO：130)；
其中，所述Z b4是反义链5′末端的第一个核苷酸，Z b3选自A、U、G或C，并且Z b4是与Z b3互补的核苷酸。
在一些实施方式中，所述siRNA的正义链含有如SEQ ID NO：249所示的核苷酸序列，所述反义链含有如SEQ ID NO：250所示的核苷酸序列：
5′-CGACUACUACAUCUCCGUZ c3-3′(SEQ ID NO：249)
5′-Z c4ACGGAGAUGUAGUAGUCGUU-3′(SEQ ID NO：250)
或者，所述siRNA的正义链含有如SEQ ID NO：251所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO：252所示的核苷酸序列：
5′-AACGACUACUACAUCUCCGUZ c3-3′(SEQ ID NO：251)
5′-Z c4ACGGAGAUGUAGUAGUCGUUGC-3′(SEQ ID NO：252)
其中，所述Z c4是反义链5′末端的第一个核苷酸，Z c3选自A、U、G或C，并且Z c4是与Z c3互补的核苷酸。
在一些实施方式中，本公开所述siRNA为表1中列出的siRPTORa1、siRPTORa2、siRPTORa3、siRPTORb1、siRPTORb2、siRPTORb3、siRPTORc1、siRPTORc2和siRPTORc3。
如前所述，本公开的siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸。在一些实施方式中，本公开的siRNA中的核苷酸为未经修饰的核苷酸；在一些实施方式中，本公开的siRNA中的部分或全部核苷酸为修饰的核苷酸，核苷酸基团上的这些修饰不会导致本公开的siRNA抑制RPTOR基因表达的功能明显削弱或丧失。
在一些实施方式中，本公开的siRNA至少含有1个修饰的核苷酸。在本公开的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖 基2′位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者核苷酸上的碱基是经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择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中公开的修饰的核苷酸。
在一些实施方式中，本公开提供的siRNA的正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基；换句话说，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基。
在一些实施方式中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。在一些实施方式中，本公开提供的siRNA的正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。
本公开的发明人惊奇地发现，本公开提供的siRNA在动物实验中获得了血浆中稳定性和基因沉默效率的高度平衡。
在一些实施方式中，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，所述核苷酸序列I中氟代修饰的核苷酸不多于5个，并且，按照5′末端到3′末端的方向，所述核苷酸序列I的第7、8、9位的核苷酸为氟代修饰的核苷酸；所述核苷酸序列II中氟代修饰的核苷酸不多于7个，并且，所述核苷酸序列II的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
在一些实施方式中，按照5′末端到3′末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位或者5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为非氟代修饰的核苷酸；按照5′末端到3′末端的方向，在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为非氟代修饰的核苷酸。
在本公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被氟取代形成的核苷酸，其具有以下式(7)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸、或核苷酸类似物。在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
这些核糖基2′位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2′-烷氧基修饰的核苷酸、2′-经取代的烷氧基修饰的核苷酸、2′-烷基修饰的核苷酸、2′-经取代的烷基修饰的核苷酸、2′-氨基修饰的核苷酸、2′-经取代的氨基修饰的核苷酸、2′-脱氧核苷酸中的一种。
在一些实施方式中，2′-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2′-OMe)，如式(8)所示。在一些实施方式中，2′-经取代的烷氧基修饰的核苷酸，例如可以是2′-O-甲氧基乙基修饰的核苷酸(2′-MOE)，如式(9)所示。在一些实施方式中，2′-氨基修饰的核苷酸(2′-NH 2)如式(10)所示。在一些实施方式中，2′-脱氧核苷酸(DNA)如式(11)所示：
Figure PCTCN2022139942-appb-000001
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方式中，核苷酸类似物可以是异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3′-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2′-、4′-位处以提供一个2′，4′-BNA核苷酸。在一些实施方式中，BNA可以是LNA、ENA、cET BNA等，其中，LNA如式(12) 所示，ENA如式(13)所示，cET BNA如式(14)所示：
Figure PCTCN2022139942-appb-000002
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方式中，无环核苷酸可以是解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(15)所示，GNA如式(16)所示：
Figure PCTCN2022139942-appb-000003
上述式(15)和式(16)中，R选自H、OH或烷氧基(O-烷基)。
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物。在一些实施方式中，异核苷酸可以是碱基从核糖环的1′-位移动至2′-位或3′-位而形成的化合物，如式(17)或(18)所示。
Figure PCTCN2022139942-appb-000004
上述式(17)-式(18)化合物中，Base表示核酸碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸。在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2′-羟基被甲氧基取代而形成的核苷酸。在一些实施方式中，所述非氟代修饰的核苷酸独立地为2′-O-甲氧基修饰或者2′-O-甲氧基乙基修饰的核苷酸。
在上文及下文中，“氟代修饰的核苷酸”指核苷酸的2′-羟基被氟取代， 而形成的具有如式(7)所示结构的化合物；“甲氧基修饰的核苷酸”指核苷酸核糖基团的2′-羟基被甲氧基取代而形成的具有如式(8)所示结构的化合物；“2′-O-甲氧基乙基修饰的核苷酸”，指核苷酸核糖基团的2′-羟基被甲氧基乙基取代而形成的具有如式(9)所示结构的化合物。
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5′末端到3′末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位或者第5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5′末端到3′末端的方向，所述siRNA的正义链中核苷酸序列I的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5′末端到3′末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
或者，按照5′末端到3′末端的方向，所述siRNA的正义链中核苷酸序列I的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5′末端到3′末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
或者，按照5′末端到3′末端的方向，所述siRNA的正义链中核苷酸序列I的第7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5′末端到3′末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸。
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5′末端到3′末端的方向，所述siRNA的正义链中核苷酸序列I的第7、8、9位或者第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸；按照5′末端到3′末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸；并且，按照5′末端到3′末端的方向，所述核苷酸序列II的第3-6个核苷酸中的至少1个为2′-O-甲氧基乙基修饰的核苷酸。在一些实施方式中，所述反义链中第3位或第5位的核苷酸为2′-O-甲氧基乙基修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。在一些实施方式中，按照5′末端到3′末端的方向，所述核苷酸序列II中第3-9个核苷酸中不超过2个核苷酸为2′-O-甲氧基乙基修饰的核苷酸。
在一些实施方式中，本公开提供的siRNA任意地选自以下siRNA中的一种：
siRPTORa1-M1、siRPTORa1-M2、siRPTORa1-M3、siRPTORa2-M1、siRPTORa2-M2、siRPTORa2-M3、siRPTORa3-M1、siRPTORa3-M2、siSRPTORa3-M3、siRPTORb1-M1、siRPTORb1-M2、siRPTORb1-M3、siRPTORb2-M1、siRPTORb2-M2、siRPTORb2-M3、siRPTORb3-M1、siRPTORb3-M2、siSRPTORb3-M3、siRPTORc1-M1、siRPTORc1-M2、siRPTORc1-M3、siRPTORc2-M1、siRPTORc2-M2、siRPTORc2-M3、siRPTORc3-M1、siRPTORc3-M2和siSRPTORc3-M3。
具有上述修饰的siRNA不仅成本低，而且可使血液中的核糖核酸酶不易切割核酸，由此增加核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。同时，上述修饰的siRNA具有较高的抑制靶mRNA的活性。
在一些实施方式中，本公开提供的siRNA的正义链和反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基中的至少一部分为具有修饰基团的磷酸酯基。在一些实施方式中，具有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基；在一些实施方式中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯基：
Figure PCTCN2022139942-appb-000005
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。
在一些实施方式中，本公开提供的siRNA中，硫代磷酸酯基连接存在于由以下位置组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯基连接存在于除正义链5′末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于除正义链3′末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于以下位置中的至少一处：
所述正义链的5′末端第1个核苷酸和第2个核苷酸之间；
所述正义链的5′末端第2个核苷酸和第3个核苷酸之间；
所述正义链的3′末端第1个核苷酸和第2个核苷酸之间；
所述正义链的3′末端第2个核苷酸和第3个核苷酸之间；
所述反义链的5′末端第1个核苷酸和第2个核苷酸之间；
所述反义链的5′末端第2个核苷酸和第3个核苷酸之间；
所述反义链的3′末端第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3′末端第2个核苷酸和第3个核苷酸之间。
在一些实施方式中，本公开提供的siRNA任意地选自以下siRNA中的一种：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、siRPTORa1-T2S、siRPTORb1-M1S、siRPTORb1-M1X、siRPTORb1-M2S、siRPTORb1-M2X、siRPTORb1-M3S、siRPTORb1-M3X、siRPTORb2-M1S、siRPTORb2-M1X、siRPTORb2-M2S、siRPTORb2-M2X、siRPTORb2-M3S、 siRPTORb2-M3X、siRPTORb3-M1S、siRPTORb3-M1X、siRPTORb3-M2S、siRPTORb3-M2X、siRPTORb3-M3S、siRPTORb3-M3X、siRPTORb1-T1S、siRPTORb1-T2S、siRPTORc1-M1S、siRPTORc1-M1X、siRPTORc1-M2S、siRPTORc1-M2X、siRPTORc1-M3S、siRPTORc1-M3X、siRPTORc2-M1S、siRPTORc2-M1X、siRPTORc2-M2S、siRPTORc2-M2X、siRPTORc2-M3S、siRPTORc2-M3X、siRPTORc3-M1S、siRPTORc3-M1X、siRPTORc3-M2S、siRPTORc3-M2X、siRPTORc3-M3S、siRPTORc3-M3X、siRPTORc1-T1S和siRPTORc1-T2S。
在一些实施方式中，所述siRNA反义链的5′末端核苷酸为5′-磷酸核苷酸或5′-磷酸类似物修饰的核苷酸。
常用的所述5′-磷酸核苷酸或5′-磷酸类似物修饰的核苷酸是本领域技术人员所公知的，如5′-磷酸核苷酸可具有如下结构：
Figure PCTCN2022139942-appb-000006
再如，Anastasia Khvorova and Jonathan K.Watts，The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology，2017，35(3)：238-48中公开了如下4种5′-磷酸类似物修饰的核苷酸：
Figure PCTCN2022139942-appb-000007
其中，R选自H、OH、甲氧基、氟；Base表示核酸碱基，选自A、U、C、G或T。
在一些实施方式中，5′-磷酸核苷酸为式(2)所示的含有5′-磷酸修饰的核苷酸，5′-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯(5′-(E)-vinylphosphonate，E-VP)修饰的核苷酸，如式(3)所示，或者 为硫代磷酸酯修饰的核苷酸，如式(5)所示。
在一些实施方式中，本公开提供的siRNA任意地选自以下组中的一种：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-M2XP1、siRPTORa2-M3XP1、siRPTORa3-M1XP1、siRPTORa3-M2XP1、siRPTORa3-M3XP1、siRPTORb1-M1P1、siRPTORb1-M2P1、siRPTORb1-M3P1、siRPTORb2-M1P1、siRPTORb2-M2P1、siRPTORb2-M3P1、siRPTORb3-M1P1、siRPTORb3-M2P1、siRPTORb3-M3P1、siRPTORb1-M1SP1、siRPTORb1-M2SP1、siRPTORb1-M3SP1、siRPTORb2-M1SP1、siRPTORb2-M2SP1、siRPTORb2-M3SP1、siRPTORb3-M1SP1、siRPTORb3-M2SP1、siRPTORb3-M3SP1、siRPTORb1-M1XP1、siRPTORb1-M2XP1、siRPTORb1-M3XP1、siRPTORb2-M1XP1、siRPTORb2-M2XP1、siRPTORb2-M3XP1、siRPTORb3-M1XP1、siRPTORb3-M2XP1、siRPTORb3-M3XP1、siRPTORc1-M1P1、siRPTORc1-M2P1、siRPTORc1-M3P1、siRPTORc2-M1P1、siRPTORc2-M2P1、siRPTORc2-M3P1、siRPTORc3-M1P1、siRPTORc3-M2P1、siRPTORc3-M3P1、siRPTORc1-M1SP1、siRPTORc1-M2SP1、siRPTORc1-M3SP1、siRPTORc2-M1SP1、siRPTORc2-M2SP1、siRPTORc2-M3SP1、siRPTORc3-M1SP1、siRPTORc3-M2SP1、siRPTORc3-M3SP1、siRPTORc1-M1XP1、siRPTORc1-M2XP1、siRPTORc1-M3XP1、siRPTORc2-M1XP1、siRPTORc2-M2XP1、siRPTORc2-M3XP1、siRPTORc3-M1XP1、siRPTORc3-M2XP1和siRPTORc3-M3XP1。
本公开的发明人意外发现，本公开提供的siRNA不仅具有显著增强 的血浆和溶酶体稳定性，还保留很高的基因抑制活性。
本公开提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。
药物组合物
在一方面，本公开提供了一种药物组合物，所述药物组合物含有如上所述的siRNA作为活性成分和药学上可接受的载体。
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe 3O 4或Fe 2O 3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1，2-二油酰基-3-三甲铵丙烷(1，2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N，N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
在一些实施方式中，所述药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，在一些实施方式中，siRNA与药学上可接受的载体的重量比可以为1∶(1-500)，在一些的实施方式中，上述重量比为1∶(1-50)。
在一些实施方式中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH缓冲液、保护剂和渗透压调节剂中的至少一种。
所述pH缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。
所述渗透压调节剂例如可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克(mOsm/kg)。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。在一些实施方式中，所述药物组合物所制成的制剂在给药过程中的剂量会因给药方式的不同而发生调整。
在一些实施方式中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉、脑室内注射或鞘内注射给药，也可以但不限于通过鼻腔给药、口咽吸入、喷雾给药等方式递送所述药物组合物。在一些实施方式中，所述药物组合物用于鞘内注射来递送。在一些实施方式中，将所述药物组合物鞘内注射至脊髓液中可以推注注射形式或经由微型泵来进行，这些微型泵可植入皮肤下方，从而提供将siRNA规律且恒定地递送至脊髓液中。在一些实施方式中，鞘内施用经由手术植入的渗透泵。在一些实施方式中，将渗透泵植入椎管的蛛网膜下腔以促进鞘内施用。关于此鞘内递送系统的更多细节可见于2015年1月28日提交的PCT/US 2015/013253，以引用的方式将该文献的全部内容并入本文。
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于中国专利申请CN103380113A(通过引用的方式将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。
在一些实施方式中，所述有机胺可为中国专利申请CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：
Figure PCTCN2022139942-appb-000008
其中：
X 101和X 102各自独立地是O、S、N-A或C-A，其中A是氢或C 1-C 20烃链；
Y 101和Z 101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO 2；
R 101、R 102、R 103、R 104、R 105、R 106和R 107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R 102是氢；
并且，如果n或m中的至少一个是2，那么R 103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
Figure PCTCN2022139942-appb-000009
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个*N代表式(201)中的氮原子。
在一些实施方式中，R 103是多胺。在其它实施方式中，R 103是缩酮。在一些实施方式中，在式(201)中的R 101和R 102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R 103可以是下述式(204)-式(213)中的任一个：
Figure PCTCN2022139942-appb-000010
Figure PCTCN2022139942-appb-000011
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个*显示R 103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
其中，式(201)所示化合物可以根据中国专利申请CN103380113A中的描述制备。
在一些实施方式中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：
Figure PCTCN2022139942-appb-000012
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
所述聚乙二醇化脂质为1，2-二棕榈酰-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
在一些实施方式中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80)∶(19.7-80)∶(0.3-50)，例如可以为(50-70)∶(20-40)∶(3-20)。
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物中，siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1∶1至约1∶50、从约1∶1至约1∶30、从约1∶3至约1∶20、从约1∶4至约1∶18、从约1∶5至约1∶17、从约1∶5至约1∶15、从约1∶5至约1∶12、从约1∶6至约1∶12或从约1∶6至约1∶10的范围内，例如，本公开的siRNA与全部脂质的重量比为约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或1∶18。
在一些实施方式中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方式中，本公开提供的siRNA与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的siRNA替代现有siRNA即可；在一些实施方式中，可以按照如下方法制备：
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选白药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。
将本公开提供的siRNA溶解于缓冲盐溶液中，得到siRNA水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使siRNA的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸 钾。
将脂质溶液和siRNA水溶液混合，将混合后得到的产物在40-60℃孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和siRNA水溶液的体积比为1∶(2-5)，例如可以为1∶4。
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100KDa条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。
siRNA缀合物
在另一方面，本公开提供了一种siRNA缀合物，所述siRNA缀合物含有上述siRNA以及缀合连接至该siRNA的缀合基团。
一般来说，所述缀合基团包含药学上可接受的至少一个靶向基团和/或递送辅助基团。在一些实施方式中，所述缀合基团还包含接头(1inker)，并且，所述接头和/或所述靶向基团或者所述递送辅助基团依次连接。在一些实施方式中，所述靶向基团为1-6个。在一些实施方式中，所述靶向基团为2-4个。所述siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。siRNA与缀合基团的缀合位点可以在siRNA正义链的3′端或5′端，也可在反义链的5′端，还可以在siRNA的内部序列中。在一些实施方式中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3′末端。在一些实施方式中，所述缀合基团可以连接在核苷酸的磷酸基团、2′-位羟基或者碱基上。在一些实施方式中，所述缀合基团还可以连接在3′-位羟基上，此时核苷酸之间采用2′-5′磷酸二酯键连接。当缀合基团连接在siRNA链的末端时，所述缀合基团通常 连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可以参考文献：Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology，2015，10(5)：1181-7。
在一些实施方式中，所述siRNA与缀合基团间可以通过酸不稳定的、或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，缀合基团可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。
在一些实施方式中，所述药学上可接受的靶向基团可以是siRNA给药领域常规使用的配体。在一些实施方式中，所述的每个配体独立地选自一个能够与细胞表面受体结合的配体。在一些实施方式中，至少一个靶向配体靶向介导向中枢神经系统(CNS)组织递送的受体。这些配体的种类为本领域技术人员所公知，其作用是与靶细胞表面的特异性受体相结合，介导与配体连接的siRNA递送至靶细胞。在一些实施方式中，至少一个所述靶向基团选自能够和表达RPTOR的细胞表面受体结合的配体。在一些实施方式中，至少一个靶向基团是靶向肝实质细胞表面的受体的配体。在一些实施方式中，至少一个或每个靶向基团是靶向肝细胞表面去唾液酸糖蛋白受体的配体。在一些实施方式中，至少一个或每个靶向基团是N-乙酰半乳糖胺(GalNAc)。在一些实施方式中，本公开的缀合物具有CN110959011A中公开的各种siRNA缀合物结构，以引用的方式将其全部公开内容并入本文。
在一些实施方式中，本公开的缀合物具有式(301)所示的结构：
Figure PCTCN2022139942-appb-000013
其中Nu表示本公开的siRNA形成的siRNA基团。在一些实施方式中，所述siRNA缀合物具有式(301)所示的结构，其中Nu具有siRPTORa2-M1S、siRPTORb2-M1S、siRPTORc2-M1S、siRPTORc1-T1S和siRPTORc1-T2S中的一种所对应的序列，所述缀合基团连接至所述Nu中siRNA正义链3′末端核苷酸的核糖3′位，并且所述siRNA缀合物处于钠盐形式。
在一些实施方式中，每个所述靶向基团选自能够和表达RPTOR的细胞表面受体结合的配体。在一些实施方式中，至少一个靶向基团是靶向CNS的靶细胞表面的受体的配体。在一些实施方式中，每个靶向基团是靶向CNS的靶细胞表面的受体的配体。在一些实施方式中，配体可以和siRNA缀合，以实现特异性CNS组织递送，在一些实施方式中，至少一个靶向基团是肽配体。在一些实施方式中，每一个靶向基团是肽配体。在一些实施方式中，靶向配体选自由以下组成的组：血管肽-2、脂蛋白受体相关蛋白(LRP)配体、bEnd.3细胞结合配体、运铁蛋白受体(TfR)配体、甘露糖受体配体、葡萄糖转运蛋白以及LDL受体配体。例如CN112400018A中描述的各种靶向基团(如[0856]段中描述的配体)，以引用的方式将其全部公开内容并入本文。
在一些实施方式中，所述药学上可接受的递送辅助基团可以是亲脂性基团，包含脂肪族化合物或脂环族化合物。在一些实施方式中，亲脂性基团含有直链脂肪族烃、支链脂肪族烃或者类固醇。在一些实施方式中，亲脂性基团含有饱和或不饱和C4-C30烃链。在一些实施例中，亲脂性基团含有饱和或不饱和C6-C18烃链(例如直链C6-C18烷基或烯基)。 在一些实施例中，亲脂性基团含有饱和或不饱和C16烃链(例如直链C16烷基或烯基)。在一些实施例中，亲脂性基团是C6-C30脂肪族酰基，所述C6-C30脂肪族酰基是指C6-C30脂肪酸去掉羟基后剩下的原子团。在一些实施方式中，亲脂性基团以非共价或共价的形式与siRNA缀合。在一些实施方式中，亲脂性基团与siRNA的缀合位点在siRNA正义链的3′端或5′端。在一些实施方式中，亲脂性基团与siRNA的缀合位点在反义链的5′端。在一些实施方式中，亲脂性基团与siRNA的缀合位点还可以在siRNA的内部序列中。在一些实施方式中，亲脂性基团连接在核苷酸的磷酸基团、核糖糖环或者碱基上。在一些实施方式中，亲脂性基团与核苷酸碱基缀合时，优选的位置为不干扰碱基配对所需的氢键相互作用的位置。在一些实施方式中，亲脂性基团连接在核苷酸的磷酸基团、2′-位羟基或者碱基上。在一些实施方式中，亲脂性基团可经由2′-位羟基与核糖糖环缀合。将递送辅助基团连接至siRNA的各种方法是本领域技术人员所公知的，例如CN112400018A中描述的各种制备方法(如[1053]～[1065]段描述的制备方法)，以引用的方式将其全部公开内容并入本文。
在一些实施方式中，所述靶向基团经由一个或多个接头与siRNA缀合。本公开的发明人意外发现，本公开的siRNA缀合物在具有显著提高在血浆中稳定性，还表现出较高的RPTOR mRNA沉默活性。在一些实施方式中，本公开的siRNA可以为表1a、1b和1c中示出的siRNA中的一种。采用这些siRNA，本公开的siRNA缀合物表现出更高的RPTOR mRNA沉默活性。
表1a本公开的第一种siRNA
Figure PCTCN2022139942-appb-000014
Figure PCTCN2022139942-appb-000015
Figure PCTCN2022139942-appb-000016
Figure PCTCN2022139942-appb-000017
表1b本公开的第二种siRNA
Figure PCTCN2022139942-appb-000018
Figure PCTCN2022139942-appb-000019
Figure PCTCN2022139942-appb-000020
表1c本公开的第三种siRNA
Figure PCTCN2022139942-appb-000021
Figure PCTCN2022139942-appb-000022
Figure PCTCN2022139942-appb-000023
Figure PCTCN2022139942-appb-000024
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接；P1表示该P1右侧相邻的一个核苷酸为5′-磷酸核苷酸或5′-磷酸类似物修饰的核苷酸。在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5′-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5′-磷酸核苷酸。字母组合 moe表示在该字母组合 moe左侧相邻的一个核苷酸为具有2′-O-甲氧基乙基修饰的核苷酸。例如，字母组合G moe表示在该字母组合 moe左侧相邻的核苷酸碱基为鸟嘌呤，且具有2′-O-甲氧基乙基修饰的核苷酸。另外，字母组合C moe表示在该字母组合 moe左侧相邻的核苷酸碱基为5-甲基胞嘧啶，且具有2′-O-甲氧基乙基修饰的核苷酸。另外，上述表中所列的序列中的每个U可任意地替换为T，不会对siRNA的活性或脱靶效应产生明显影响。
本公开所述的siRNA、药物组合物或siRNA缀合物中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸 二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子，铵离子NH 4 +，有机铵阳离子中的一种。出于提高溶解性考虑，在一种实施方式中，所述阳离子选自碱金属离子、三级胺形成的铵阳离子和季铵阳离子中的一种或多种。碱金属离子可以是K +和/或Na +，三级胺形成的阳离子可以是三乙胺形成的铵离子和/或N，N-二异丙基乙胺形成的铵离子。因此，本公开所述siRNA或siRNA缀合物可以至少部分以盐的形式存在。在一种方式中，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子至少部分与钠离子结合，本公开所述siRNA或siRNA缀合物以钠盐或部分钠盐的形式存在。
本领域技术人员清楚知晓的是，可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中。制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。
可以采用任意合理的合成路线制备本公开的siRNA缀合物。例如，对于包含靶向基团和可与亚磷酰胺发生反应而形成共价连接的活性反应基团的缀合分子，可先将该缀合分子中的活性基团用保护剂保护后，连接至固相载体，随后通过亚磷酰胺固相合成方法，按照siRNA正义链和反义链的核苷酸种类和顺序，按照3′到5′的方向逐一连接核苷单体，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；分离出siRNA的正义链和反义链，退火，从而获得本公开的siRNA缀合物。
进一步地，siRNA缀合物的制备也可参照已有文献的公开内容进行。例如，WO2019010274A1在实施例1中描述了将具有特定结构的连接基团和靶向配体经反应依次连接至siRNA的方法。以引用的方式将其全部内容并入本文。
本公开的siRNA及含该siRNA的药物组合物及siRNA缀合物的应 用
在一些实施方式中，本公开提供了本公开的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和预防与RPTOR功能调节相关的疾病或症状的药物的用途。在一些实施方式中，所述与RPTOR功能调节相关的疾病或症状是与mTORC1激活所致疾病和细胞自噬功能异常相关的疾病。在一些实施方式中，所述与神经退行性相关的疾病或症状为阿尔兹海默症和/或帕金森病，优选地，所述神经退行性疾病为阿尔兹海默症。在一些实施方式中，所述和细胞自噬功能异常相关的疾病是非酒精性脂肪性肝炎。
在一些实施方式中，本公开提供了一种预防和/或治疗与RPTOR功能调节相关的疾病或症状的方法，该方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物给予有需要的受试者。通过将本公开的siRNA活性成分给予有需要的受试者，可以通过RNA干扰的机制达到预防和/或治疗所引起的疾病的目的。因此，本公开的siRNA和/或药物组合物和/或siRNA缀合物可用于预防和/或治疗与RPTOR功能调节相关的疾病或症状，或用于制备用于预防和/或治疗与RPTOR功能调节相关的疾病或症状的药物。
本文所使用的术语“给药/给予”是指通过使得至少部分地将本公开的siRNA、药物组合物和/或siRNA缀合物定位于期望的位点以产生期望效果的方法或途径，将本公开的siRNA、药物组合物和/或siRNA缀合物放置入受试者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与受试者体循环相比将更多siRNA缀合物递送至特定位点；而全身给药导致将本公开的siRNA、药物组合物和/或siRNA缀合物递送至受试者的基本体循环。考虑到本公开旨在提供预防和/或治疗神经退行性疾病的手段，在一些实施方式中采用将药物递送至中枢神经系统组织的给药方式。在一些实施方式中采用能够将药物递送至鞘内的给药方式。在一些实施方式中，采用将药物注射至脊髓液中的给药方式。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、脑室内给药、鞘内给药、鼻部给药、直 肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每年1次或多次。本公开所述的siRNA、药物组合物或siRNA缀合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD 50(使50％的群体死亡的致死剂量)和ED 50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指能引起50％实验对象出现阳性反应时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。在一些实施方式中，根据给药方式的不同对所述siRNA、药物组合物或siRNA缀合物所制成的制剂在给药过程中的剂量进行调整。
在给予本公开所述的siRNA、药物组合物、和/或siRNA缀合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或30-45g的ob/ob小鼠，以siRNA的量计：(i)对于siRNA缀合物，其siRNA用量可以为0.001-100mg/kg体重，在一些实施方式中为0.01-50mg/kg体重，在一些实施方式中为0.05-20mg/kg体重，另一些实施方式中为0.1-15mg/kg体重，另一些实施方式中为0.1-10mg/kg体重；(ii)对于siRNA与药学上可接受的载体形成的药物组合物，其siRNA用量可以为0.001-50mg/kg体重，在一些实施方式中为0.01-10mg/kg体重，在一些实施方式中为0.05-5mg/kg体重，在一些实施方式中为0.1-3mg/kg体重。
在一些实施方式中，本公开提供了一种抑制细胞中RPTOR基因表达的方法，该方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物与所述细胞接触，将本公开的siRNA和/或药物组合物和/或siRNA缀合物导入所述细胞，通过RNA干扰的机制达到抑制细胞中RPTOR基因表达的目的。
采用本公开提供的方法抑制RPTOR基因在细胞中表达，所提供的修饰的siRNA、药物组合物和/或siRNA缀合物中的siRNA用量一般是这样的量：其足以减少靶基因的表达，并导致在靶细胞表面处1pM至1μM、或0.01nM至100nM、或0.05nM至50nM或0.05nM至约5nM的细胞外浓度。达到该局部浓度所需的量将随各种因素而变化，所述因素包括递送方法、递送部位、在递送部位和靶细胞或组织之间的细胞层的数目、 递送途径(局部还是全身)等。在递送部位处的浓度可以显著高于在靶细胞或组织的表面处的浓度。
试剂盒
在一方面，本公开提供了一种试剂盒，所述试剂盒包含有效量的本公开的siRNA、药物组合物和siRNA缀合物的至少一种。
在一些实施方式中，本文所述的试剂盒可在一个容器中提供siRNA。在一些实施方式中，本文所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方式中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方式中，本文所述的试剂盒可在不同于提供本文所述siRNA的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方式中，所述试剂盒可包含用于将siRNA与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。
在本公开的试剂盒中，所述siRNA和药学上可接受的载体和/或辅料、以及所述修饰的siRNA、药物组合物和/或siRNA缀合物，和/或药学上可接受的载体和/或辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述siRNA和药学上可接受的载体和/或辅料以及所述药物组合物和/或siRNA缀合物的药学上可接受的载体和/或辅料基本上纯净和/或无菌。在一些实施方式中，可在本公开的试剂盒中提供无菌水。
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor LBboratory Press(1989))所记载的方法进行。
若无其它说明，以下提供的试剂比例均按体积比(v/v)计算。数据分析采用Graphpad prism8.0统计分析软件。
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品， 所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor Laboratory Press(1989))所记载的方法进行。
制备例1-5本公开提供的siRNA的合成
通过固相合成方法分别合成表2中所列的siRNA序列，使用DEPC水分别溶解等摩尔的表2中相互互补的正义链和反义链，随后退火得到本公开提供的siRPTORa2-M1X，siRPTORb2-M1X、siRPTORc2-M1X、siRPTORc1-T2S和siRPTORc1-M1S。使用超纯水(Milli-Q超纯水仪，电阻率18.2MΩ*cm(25℃))将siRNA稀释至浓度为0.2g/mL(以siRNA计)后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。实测值与理论值一致，说明所合成的siRNA是目标设计的双链核酸序列。
对比制备例1参比siRNA的合成
通过固相合成方法分别合成了表2中siRNA编号为NC的siRNA对应的正义链和反义链。使用DEPC水溶解等摩尔的正义链和反义链，随后退火得到参比siRNA，编号为NC，NC是作为阴性对照的siRNA，其与RPTOR mRNA不具有序列同源性。
表2 siRNA序列
Figure PCTCN2022139942-appb-000025
Figure PCTCN2022139942-appb-000026
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；大写字母P表示该字母P右侧相邻的一个核苷酸为5′-磷酸核苷酸；字母组合 moe表示在该字母组合 moe左侧相邻的一个核苷酸为具有2′-O-甲氧基乙基修饰的核苷酸。
在上述本公开的siRNA或参比siRNA制备完成后，冻干为固体粉末保存备用。
制备例6-7缀合物1和缀合物2的合成
按照WO2019/105437A1中制备例1中“缀合物1”的方法得到本公开的缀合物1，区别仅在于，在制备正义链和反义链单链时，按照表2中siRPTORc1-T2S的正义链和反义链序列，分别逐一连接对应位置的核苷单体。采用液质联用色谱(LC-MS)进行分子量检测。其结果，正义链理论值：7468.3，正义链实测值：7467.2；反义链理论值：7107.7，反义链实测值：7106.7。实测值与理论值一致，从而确定所合成的缀合物1是目标设计的含有式(301)所示基团的双链核酸序列。缀合物1的结构如下式(301)所示。
Figure PCTCN2022139942-appb-000027
其中，式(301)中的Nu为本公开表1中的siRPTORc1-T2S，缀合基团连接至siRPTORc1-T2S中正义链3′末端的核糖3′位，并且所述缀合物处于钠盐形式。
按照上述方法制备本公开的缀合物2，区别仅在于，在制备正义链和反义链单链时，按照表2中siRPTORc1-T2S的正义链和反义链序列，分别逐一连接对应位置的核苷单体。采用液质联用色谱(LC-MS)进行分子量检测。其结果，正义链理论值：7468.3，正义链实测值：7467.2；反义链理论值：7063.7，反义链实测值：7062.7。实测值与理论值一致，从而确定所合成的缀合物2是目标设计的含有式(301)所示基团的双链核酸序列。缀合物2的结构如式(301)所示。其中，式(301)中的Nu为本公开表1中的siRPTORc1-M1S，缀合基团连接至siRPTORc1-M1S中正义链3′末端的核糖3′位，并且所述缀合物处于钠盐形式。实验例1本公开的siRNA在体外(invitro)的抑制活性。
本实验例考察了本公开的siRPTORa2-M1X，siRPTORb2-M1X和siRPTORc2-M1X在体外HepG2人肝癌细胞中的抑制活性。
使用添加有10％胎牛血清(FBS，RMBIO公司)的DMEM培养基(HyClone公司)，于37℃在含5％CO2/95％空气的培养箱中培养HepG2人肝癌细胞(购自南京科佰生物科技有限公司)。
将HepG2细胞以2.0x10 5细胞/孔接种于12孔板中，每孔1mL细胞液，培养24h后，吸尽培养孔中培养基，向每孔中另外加入500μL Opti-MEM培养基(GIBCO公司)。
用PBS缓冲液将制备例1～3中制备获得的siRPTORa2-M1X，siRPTORb2-M1X和siRPTORc2-M1X分别配制成浓度为20μM的siRNA工作液。
对于每一个siRNA，配制1A1溶液，每份1A1溶液含有48.5μL Opti-MEM培养基和1.5μL的20μM siRNA工作液。
配制1B溶液，每份1B溶液含有49μL Opti-MEM培养基和1μL的Lipofectamine TM 2000(Invitrogen公司)。
对于每一个siRNA，分别将1份1B溶液与1份1A1溶液室温下孵 育20min，分别得到转染复合物1Xa，1Xb或1Xc。
将1份1B溶液与50μL Opti-MEM培养基混合，室温下孵育20min，得到转染复合物1X。
在两个培养孔(均为上述含有HepG2细胞和500μL Opti-MEM培养基的培养孔，下同)中，分别加入转染复合物1Xa，均匀混合，加入量为100μL/孔，得到终浓度为50nM的转染混合物，记为测试组1。
在两个培养孔(均为上述含有HepG2细胞和500μL Opti-MEM培养基的培养孔，下同)中，分别加入转染复合物1Xb，均匀混合，加入量为100μL/孔，得到终浓度为50nM的转染混合物，记为测试组2。
在两个培养孔(均为上述含有HepG2细胞和500μL Opti-MEM培养基的培养孔，下同)中，分别加入转染复合物1Xc，均匀混合，加入量为100μL/孔，得到终浓度为50nM的转染混合物，记为测试组3。
在两个培养孔中，分别加入转染复合物1X，均匀混合，加入量为100μL/孔，得到不含siRNA的转染混合物，记为空白对照组。
将上述测试组1～3和空白对照组在培养孔中培养4h后，吸去各培养孔中的上清液，向每孔中加入1mL Opti-MEM培养基。将12孔板置于CO 2培养箱中继续在37℃下培养24h。
使用生工UNIQ-10柱式总RNA抽提试剂盒(购自生工生物，货号：TC13KA4109)根据说明书记载的方法分别提取各孔细胞中的总RNA。
对于每孔细胞，分别取1μg总RNA，使用反转录试剂盒Goldenstar TM RT6cDNA Synthesis Kit(北京擎科新业生物技术有限公司)提供的试剂，其中选取Goldenstar TM Oligo(dT) 17作为引物，按试剂盒说明书中反转录操作步骤配置反转录反应体系20μL，对各孔细胞的总RNA进行反转录。反转录的条件为：对于每一反转录反应体系，将反转录反应体系置于50℃孵育50min，然后在85℃孵育5min，最后4℃孵育30s，反应结束后，向反转录反应体系中加入DEPC水80μL，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μL做模板，使用
Figure PCTCN2022139942-appb-000028
SYBR qPCR SuperMix Plus试剂盒(购自近岸蛋白质科 技有限公司，货号E096-01B)提供的试剂配置qPCR反应体系20μL，其中，用于扩增目标基因RPTOR和内参基因GAPDH的PCR引物序列如表3所示，每条引物的终浓度为0.25μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为95℃预变性10min，然后95℃变性30s，60℃退火30s，72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因RPTOR和内参基因GAPDH的产物W1。产物W1随即依次经过95℃15s，60℃1min，95℃15s的孵育，实时荧光定量PCR仪分别收集产物W1中目标基因和内参基因GAPDH的溶解曲线，得到目标基因RPTOR和内参基因GAPDH的Ct值。
表3引物信息
Figure PCTCN2022139942-appb-000029
采用比较Ct(ΔΔCt)法，对各测试组中目标基因RPTOR进行相对定量计算，计算方法如下：
ΔCt(测试组)＝Ct(测试组目标基因)-Ct(测试组内参基因)
ΔCt(对照组)＝Ct(对照组目标基因)-Ct(对照组内参基因)
ΔΔCt(测试组)＝ΔCt(测试组)-ΔCt(对照组平均)
ΔΔCt(对照组)＝ΔCt(对照组)-ΔCt(对照组平均)
其中，ΔCt(对照组平均)是对照组两个培养孔各自的ΔCt(对照组)的算术平均值。从而，测试组和对照组的每一培养孔均对应一个ΔΔCt值。
以对照组平均值为基准，对测试组RPTOR mRNA的表达水平进行归一化，定义空白对照组RPTOR mRNA表达水平平均值为100％，
测试组RPTOR mRNA相对表达水平＝2 -ΔΔCt(测试组)×100％
测试组RPTOR mRNA抑制率＝(1-测试组RPTOR mRNA相对表达水平)×100％
实验结果如图1所示。
对比实验例1参比siRNA NC在体外(in vitro)的抑制活性
按照实验例1的方法同时考察了阴性参比siRNA NC在体外HepG2人肝癌细胞的抑制活性，区别仅在于，以对比制备例制备获得的参比siRNA NC代替siRPTORa2-M1X，siRPTORb2-M1X或者siRPTORc2-M1X配制成浓度为20μM的siRNA工作液进行测试。结果如图1所示。
图1是显示了转染50nM的本公开的siRNA和参比siRNA NC后，体外HepG2人肝癌细胞中的RPTOR mRNA相对表达水平的柱状图，图1中NC代表参比siRNA NC。图1的结果表明，在体外HepG2人肝癌细胞中，siRPTORa2-M1X在50nM浓度时RPTOR mRNA抑制率为76.8％；siRPTORb2-M1X在50nM浓度时RPTOR mRNA抑制率为86.8％；siRPTORc2-M1X在50nM浓度时RPTOR mRNA抑制率为78.8％；表现出优异的RPTOR mRNA抑制活性，显示出优异的抑制RPTOR基因表达的效果。
实验例2本公开的siRNA在体外(in vitro)的靶mRNA抑制活性
本实验例考察了不同浓度的本公开的siRPTORa2-M1X，siRPTORb2-M1X和siRPTORc2-M1X在体外HepG2人肝癌细胞中的RPTOR mRNA抑制活性。
使用添加有10％胎牛血清(FBS，RMBIO公司)的DMEM培养基(HyClone公司)，于37℃在含5％CO2/95％空气的培养箱中培养HepG2人肝癌细胞(购自南京科佰生物科技有限公司)。
将HepG2细胞以2.0x10 5细胞/孔接种于12孔板中，每孔1mL细胞液，培养24h后，吸尽培养孔中培养基，向每孔中另外加入500μL Opti-MEM培养基(GIBCO公司)。
对于每一个siRNA，用PBS缓冲液将制备例1～3中制备获得的siRPTORa2-M1X，siRPTORb2-M1X和siRPTORc2-M1X分别配制成浓度为20μM、2μM和0.2μM的siRNA工作液。
对于每一个siRNA，配制2A1溶液，每份2A1溶液含有48.5μL Opti-MEM培养基和1.5μL浓度为20μM的siRNA工作液，分别得到2A1a、 2A1b或2A1c的siRNA工作液。
对于每一个siRNA，配制2A2溶液，每份2A2溶液含有48.5μL Opti-MEM培养基和1.5μL浓度为2μM的siRNA工作液，分别得到2A2a、2A2b或2A2c的siRNA工作液。
对于每一个siRNA，配制2A3溶液，每份2A3溶液含有48.5μL Opti-MEM培养基和1.5μL浓度为0.2μM的siRNA工作液，分别得到2A3a、2A3b或2A3c的siRNA工作液。
配制2B溶液，每份2B溶液含有49μL Opti-MEM培养基和1μL的Lipofectamine TM 2000(Invitrogen公司)。
配制2X0溶液，将1份2B溶液与50μL Opti-MEM培养基混合，室温下孵育20min，得到空白转染复合物2X0。
配制2X1溶液，分别将1份2B溶液与1份2A1a溶液混合、1份2A2a溶液混合、1份2A3a溶液混合，室温下孵育20min，分别得到转染复合物2Xa1、2Xa2或2Xa3。
配制2X2溶液，分别将1份2B溶液与1份2A1b溶液混合，1份2A2b溶液混合、1份2A3b溶液混合，室温下孵育20min，分别得到转染复合物2Xb1、2Xb2或2Xb3。
配制2X3溶液，分别将1份2B溶液与1份2A1c溶液混合，1份2A2c溶液混合、1份2A3c溶液混合，室温下孵育20min，分别得到转染复合物2Xc1、2Xc2或2Xc3。
对于每一个siRNA，在两个培养孔(均为上述含有HepG2细胞和500μL Opti-MEM培养基的培养孔，下同)中，分别加入转染复合物2Xa1、2Xa2或2Xa3，均匀混合，加入量为100μL/孔，得到终浓度为50nM、5nM或0.5nM含siRPTORa2-M1X的转染混合物，记为测试组1。
对于每一个siRNA，在两个培养孔(均为上述含有HepG2细胞和500μL Opti-MEM培养基的培养孔，下同)中，分别加入转染复合物2Xb1、2Xb2或2Xb3，均匀混合，加入量为100μL/孔，得到终浓度为50nM、5nM或0.5nM含siRPTORb2-M1X的转染混合物，记为测试组2。
对于每一个siRNA，在两个培养孔(均为上述含有HepG2细胞和500μL Opti-MEM培养基的培养孔，下同)中，分别加入转染复合物2Xc1、2Xc2或2Xc3，均匀混合，加入量为100μL/孔，得到终浓度为50nM、5nM或0.5nM含siRPTORc2-M1X的转染混合物，记为测试组3。
在两个培养孔中，分别加入空白转染复合物2X0，均匀混合，加入量为100μL/孔，得到不含siRNA的转染混合物，记为空白对照组。
将上述测试组1～3和空白对照组在培养孔中培养4h后，吸去各培养孔中的上清液，向每孔中加入1mL Opti-MEM培养基。将12孔板置于CO 2培养箱中继续在37℃下培养24h。
采用和实验例1中相同的方法提取各孔细胞中的总RNA和反转录并对各测试组中目标基因RPTOR mRNA进行相对定量计算。结果如图2所示。
图2是转染不同浓度的本公开的siRNA后，体外HepG2人肝癌细胞中的RPTOR mRNA相对表达水平的柱状图，图2的结果表明体外HepG2人肝癌细胞中，在0.5nM的低浓度下，siRPTORc2-M1X显示出至少40.5％的RPTOR mRNA抑制率；在5nM的浓度下siRPTORa2-M1X显示出至少68.3％的RPTOR mRNA抑制率，并且在50nM的浓度下显示出至少71.1％RPTOR mRNA抑制率；在50nM的浓度下，siRPTORb2-M1X甚至可达86.4％的RPTOR mRNA抑制率，显示出优异的抑制RPTOR基因表达的效果。
实验例3siRNA缀合物在小鼠体内的抑制活性
将制备例6-7中制备获得的缀合物1和缀合物2分别用PBS溶解为3mg/ml的siRNA缀合物溶液(以siRNA计)。将C57BL/6小鼠(雌性，16-18g重，6-8周龄，购自于斯贝福公司)随机分为3组，每组6只小鼠，分别编号。以颈背部皮下注射的方式，向第一组每只小鼠分别给予上述siRNA缀合物1溶液，给药前称重并记录体重，按体重给药，给药体积均为5mL/kg，作为测试组1；向第二组每只小鼠分别给予上述siRNA缀合物2溶液，给药前称重并记录体重，按体重给药，给药体积均为5mL/kg，作为测试组2；另外向一组小鼠中的每只分别给予PBS，给药体积均为 5mL/kg，作为空白对照组。
以给药时间点作为第1天计算，在第8天，取测试组和空白对照组每只小鼠的肝组织，用RNAlater保存。
采用和实验例1中相同的方法提取各孔细胞中的总RNA和反转录并对各测试组中目标基因RPTOR进行相对定量计算，使用的引物信息如上表3所示，以空白对照组的结果归一化。结果如表4所示。
表4 siRNA缀合物在小鼠体内抑制活性。
组别 测试组1 测试组2
RPTOR mRNA抑制率％ 52.9％ 51.3％
结果可见，在3mg/kg的浓度下，相对于空白对照组的结果，本公开的不同修饰方案的siRNA缀合物在C57BL/6i小鼠体内，在给药后一周的时间内，仍显示出超过50％的体内RPTOR mRNA抑制率，显示出良好的RPTOR mRNA抑制活性和较长时间的体内稳定性。
根据上述实验结果可知，本公开的siRNA能有效抑制细胞中RPTOR基因表达，因此在制备用于治疗和/或预防与mTORC1异常激活和细胞自噬相关疾病，例如NASH或神经退行性疾病或者相关的症状的药物方面表现出良好的潜力。
以上详细描述了本公开的优选实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合。为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (40)

  1. 一种siRNA，所述siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分反向互补形成双链区，其中，所述核苷酸序列I和所述核苷酸序列II选自如下i)-iii)所示序列中的一组：
    i)所述核苷酸序列I与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列II与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5′-CUGCCAUGGAGUAUCUGAZ a1-3′(SEQ ID NO：1)；
    5′-Z a2UCAGAUACUCCAUGGCAG-3′(SEQ ID NO：2)，
    其中，Z a1为A，Z a2为U，所述核苷酸序列I中包含位置对应于Z a1的核苷酸Z a3，所述核苷酸序列II中包含位置对应于Z a2的核苷酸Z a4，所述Z a4是所述反义链5′末端的第一个核苷酸；
    ii)所述核苷酸序列I与SEQ ID NO：123所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：124所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5′-ACAACAUCAAGUACUACGZ b1-3′(SEQ ID NO：123)；
    5′-Z b2CGUAGUACUUGAUGUUGU-3′(SEQ ID NO：124)，
    其中，Z b1为A，Z b2为U，所述核苷酸序列I中包含位置对应于Z b1的核苷酸Z b3，所述核苷酸序列II中包含位置对应于Z b2的核苷酸Z b4，所述Z b4是所述反义链5′末端的第一个核苷酸；
    iii)所述核苷酸序列I与SEQ ID NO：245所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO：246所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5′-CGACUACUACAUCUCCGUZ c1-3′(SEQ ID NO：245)；
    5′-Z c2ACGGAGAUGUAGUAGUCG-3′(SEQ ID NO：246)，
    其中，Z c1为G，Z c2为C，所述核苷酸序列I中包含位置对应于Z c1的核苷酸Z c3，所述核苷酸序列II中包含位置对应于Z c2的核苷酸Z c4，所述Z c4是所述反义链5′末端的第一个核苷酸。
  2. 如权利要求1所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO：1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO：2所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO：123所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO：124所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO：245所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO：246所示的核苷酸序列之间不多于1个核苷酸差异。
  3. 如权利要求1或2所述的siRNA，其中，所述核苷酸序列II与SEQ ID NO：2所示的核苷酸序列之间的核苷酸差异包括Z a4位置处的差异，且Z a4选自A、C或G；
    或者，所述核苷酸序列II与SEQ ID NO：124所示的核苷酸序列之间的核苷酸差异包括Z b4位置处的差异，且Z b4选自A、C或G；
    或者，所述核苷酸序列II与SEQ ID NO：246所示的核苷酸序列之间的核苷酸差异包括Z c4位置处的差异，且Z c4选自A、U或G。
  4. 如权利要求1-3中任一项所述的siRNA，其中，Z a3是与Z a4互补的核苷酸；或者，Z b3是与Z b4互补的核苷酸；或者，Z c3是与Z c4互补的核苷酸。
  5. 如权利要求1-4中任一项所述的siRNA，其中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
  6. 如权利要求1-5中任一项所述的siRNA，其中，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的 长度为19-26个核苷酸；并且所述核苷酸序列I是SEQ ID NO：3所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO：4所示的核苷酸序列：
    5′-CUGCCAUGGAGUAUCUGAZ a3-3′(SEQ ID NO：3)；
    5′-Z a4UCAGAUACUCCAUGGCAG-3′(SEQ ID NO：4)，
    其中，Z a3选自A、U、G或C，Z a4是与Z a3互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO：125所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO：126所示的核苷酸序列：
    5′-ACAACAUCAAGUACUACGZ b3-3′(SEQ ID NO：125)；
    5′-Z b4CGUAGUACUUGAUGUUGU-3′(SEQ ID NO：126)，
    其中，Z b3选自A、U、G或C，Z b4是与Z b3互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO：247所示的核苷酸序列，核苷酸序列II是SEQ ID NO：248所示的核苷酸序列：
    5′-CGACUACUACAUCUCCGUZ c3-3′(SEQ ID NO：247)；
    5′-Z c4ACGGAGAUGUAGUAGUCG-3′(SEQ ID NO：248)，
    其中，Z c3选自A、U、G或C，Z c4是与Z c3互补的核苷酸。
  7. 如权利要求6所述的siRNA，其中Z a3为A，Z a4为U；或者，Z b3为A，Z b4为U；或者，Z c3为G，Z c4为C。
  8. 如权利要求1-7中任一项所述的siRNA，其中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV的长度各自独立地为1-4个核苷酸，所述核苷酸序列III连接在核苷酸序列I的5′末端，核苷酸序列IV连接在核苷酸序列II的3′末端，所述核苷酸序列III和所述核苷酸序列IV长度相等，并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
  9. 如权利要求8所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，并且，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III 的碱基为A；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为CA；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为UCA；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为GUCA；
    或者，所述核苷酸序列I与SEQ ID NO：123所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，并且，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为A；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为CA；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为UCA；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为AUCA；
    或者，所述核苷酸序列I与SEQ ID NO：245所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，并且，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为A；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为AA；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为CAA；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，按照5′末端到3′末端的方向，核苷酸序列III的碱基组成为GCAA。
  10. 如权利要求1-9中任一项所述的siRNA，其中，所述反义链还含有核苷酸序列V，核苷酸序列V的长度为1指3个核苷酸，连接在所述反义链的3′末端，构成反义链的3′突出端；和/或所述正义链还含有核苷酸序列VI，核苷酸序列VI的长度为1至3个核苷酸，连接在所述正义链的3′末端，构成正义链的3′突出端。
  11. 如权利要求10所述的siRNA，其中，所述核苷酸序列V和/或核苷酸序列VI的长度为2个核苷酸。
  12. 如权利要求10或11所述的siRNA，其中，所述核苷酸序列V和/或VI为连续的两个胸腺嘧啶脱氧核糖核苷酸或连续两个尿嘧啶核糖核苷酸；或者所述核苷酸序列V与靶mRNA相应位置的核苷酸互补，和/或所述核苷酸序列VI与靶mRNA相应位置的核苷酸相同。
  13. 如权利要求1-12中任一项所述的siRNA，其中，所述siRNA的正义链含有如SEQ ID NO：5所示的核苷酸，所述反义链含有如SEQ ID NO：6所示的核苷酸序列：
    5′-CUGCCAUGGAGUAUCUGAZ a3-3′(SEQ ID NO：5)；
    5′-Z a4UCAGAUACUCCAUGGCAGUG-3′(SEQ ID NO：6)；
    其中，所述Z a4是反义链5′末端的第一个核苷酸，Z a3选自A、U、G或C，并且Z a4是与Z a3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO：7所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO：8所示的核苷酸序列：
    5′-CACUGCCAUGGAGUAUCUGAZ a3-3′(SEQ ID NO：7)；
    5′-Z a4UCAGAUACUCCAUGGCAGUGAC-3′(SEQ ID NO：8)；
    其中，所述Z a4是反义链5′末端的第一个核苷酸，Z a3选自A、U、G或C，并且Z a4是与Z a3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO：127所示的核苷酸序列，所述反义链含有如SEQ ID NO：128所示的核苷酸序列：
    5′-ACAACAUCAAGUACUACGZ b3-3′(SEQ ID NO：127)；
    5′-Z b4CGUAGUACUUGAUGUUGUUG-3′(SEQ ID NO：128)；
    或者，所述siRNA的正义链含有如SEQ ID NO：129所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO：130所示的核苷酸序列：
    5′-CAACAACAUCAAGUACUACGZb3-3′(SEQ ID NO：129)；
    5′-Zb4CGUAGUACUUGAUGUUGUUGAU-3′(SEQ ID NO：130)；
    其中，所述Z b4是反义链5′末端的第一个核苷酸，Z b3选自A、U、G或C，并且Z b4是与Z b3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO：249所示的核苷酸序列，所述反义链含有如SEQ ID NO：250所示的核苷酸序列：
    5′-CGACUACUACAUCUCCGUZ c3-3′(SEQ ID NO：249)
    5′-Z c4ACGGAGAUGUAGUAGUCGUU-3′(SEQ ID NO：250)
    或者，所述siRNA的正义链含有如SEQ ID NO：251所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO：252所示的核苷酸序列：
    5′-AACGACUACUACAUCUCCGUZ c3-3′(SEQ ID NO：251)
    5′-Z c4ACGGAGAUGUAGUAGUCGUUGC-3′(SEQ ID NO：252)
    其中，所述Z c4是反义链5′末端的第一个核苷酸，Z c3选自A、U、G或C，并且Z c4是与Z c3互补的核苷酸。
  14. 如权利要求1-13中任一项所述的siRNA，其中，所述siRNA为siRPTORa1、siRPTORa2、siRPTORa3、siRPTORb1、siRPTORb2、siRPTORb3、siRPTORc1、siRPTORc2和siRPTORc3中的一种。
  15. 如权利要求1-14中任一项所述的siRNA，其中，所述正义链和所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基。
  16. 如权利要求1-15中任一项所述的siRNA，其中，所述正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。
  17. 如权利要求16所述的siRNA，其中，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，并且，按照5′末端到3′末端的方向，所述核苷酸序列I的至少第7、8、9位的核苷酸为氟代修饰的核苷酸；按照5′末端到3′末端的方向，所述核苷酸序列II的至少第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
  18. 如权利要求16或17所述的siRNA，其中，所述非氟代修饰的核苷酸为2′-O-甲氧基修饰或者2′-O-甲氧基乙基修饰的核苷酸。
  19. 如权利要求18所述的siRNA，其中，按照5′末端到3′末端的方 向，所述核苷酸序列II的第3-6个核苷酸中的至少1个为2′-O-甲氧基乙基修饰的核苷酸。
  20. 如权利要求19所述的siRNA，其中，所述核苷酸序列II中的第3个或第5个核苷酸为2′-O-甲氧基乙基修饰的核苷酸。
  21. 如权利要求19所述的siRNA，其中，按照5′末端到3′末端的方向，所述核苷酸序列II中第3-9个核苷酸中不超过2个核苷酸为2′-O-甲氧基乙基修饰的核苷酸。
  22. 如权利要求17-21任一项所述的siRNA，其中，所述正义链和所述反义链中的其它核苷酸为2′-O-甲氧基修饰的核苷酸。
  23. 如权利要求1-22中任一项所述的siRNA，其中，所述siRNA为siRPTORa1-M1、siRPTORa1-M2、siRPTORa1-M3、siRPTORa2-M1、siRPTORa2-M2、siRPTORa2-M3、siRPTORa3-M1、siRPTORa3-M2、siSRPTORa3-M3、siRPTORb1-M1、siRPTORb1-M2、siRPTORb1-M3、siRPTORb2-M1、siRPTORb2-M2、siRPTORb2-M3、siRPTORb3-M1、siRPTORb3-M2、siSRPTORb3-M3、siRPTORc1-M1、siRPTORc1-M2、siRPTORc1-M3、siRPTORc2-M1、siRPTORc2-M2、siRPTORc2-M3、siRPTORc3-M1、siRPTORc3-M2和siSRPTORc3-M3中的一种。
  24. 如权利要求15所述的siRNA，其中，所述具有修饰基团的磷酸酯基为磷酸酯基的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基。
  25. 如权利要求1-22中任一项所述的siRNA，其中，所述siRNA为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、siRPTORa1-T2S、siRPTORb1-M1S、siRPTORb1-M1X、siRPTORb1-M2S、siRPTORb1-M2X、siRPTORb1-M3S、siRPTORb1-M3X、siRPTORb2-M1S、siRPTORb2-M1X、siRPTORb2-M2S、siRPTORb2-M2X、siRPTORb2-M3S、siRPTORb2-M3X、siRPTORb3-M1S、siRPTORb3-M1X、siRPTORb3-M2S、siRPTORb3-M2X、siRPTORb3-M3S、siRPTORb3-M3X、siRPTORb1-T1S、siRPTORb1-T2S、siRPTORc1-M1S、siRPTORc1-M1X、siRPTORc1-M2S、siRPTORc1-M2X、siRPTORc1-M3S、siRPTORc1-M3X、siRPTORc2-M1S、siRPTORc2-M1X、siRPTORc2-M2S、siRPTORc2-M2X、siRPTORc2-M3S、siRPTORc2-M3X、siRPTORc3-M1S、siRPTORc3-M1X、siRPTORc3-M2S、siRPTORc3-M2X、siRPTORc3-M3S、siRPTORc3-M3X、siRPTORc1-T1S和siRPTORc1-T2S中的一种。
  26. 如权利要求15所述的siRNA，其中，所述反义链的5′末端核苷酸为5′-磷酸核苷酸或5′-磷酸类似物修饰的核苷酸。
  27. 如权利要求26所述的siRNA，其中，所述siRNA为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-M2XP1、siRPTORa2-M3XP1、siRPTORa3-M1XP1、siRPTORa3-M2XP1、siRPTORa3-M3XP1、siRPTORb1-M1P1、siRPTORb1-M2P1、siRPTORb1-M3P1、siRPTORb2-M1P1、siRPTORb2-M2P1、siRPTORb2-M3P1、siRPTORb3-M1P1、siRPTORb3-M2P1、siRPTORb3-M3P1、siRPTORb1-M1SP1、siRPTORb1-M2SP1、siRPTORb1-M3SP1、siRPTORb2-M1SP1、siRPTORb2-M2SP1、siRPTORb2-M3SP1、 siRPTORb3-M1SP1、siRPTORb3-M2SP1、siRPTORb3-M3SP1、siRPTORb1-M1XP1、siRPTORb1-M2XP1、siRPTORb1-M3XP1、siRPTORb2-M1XP1、siRPTORb2-M2XP1、siRPTORb2-M3XP1、siRPTORb3-M1XP1、siRPTORb3-M2XP1、siRPTORb3-M3XP1、siRPTORc1-M1P1、siRPTORc1-M2P1、siRPTORc1-M3P1、siRPTORc2-M1P1、siRPTORc2-M2P1、siRPTORc2-M3P1、siRPTORc3-M1P1、siRPTORc3-M2P1、siRPTORc3-M3P1、siRPTORc1-M1SP1、siRPTORc1-M2SP1、siRPTORc1-M3SP1、siRPTORc2-M1SP1、siRPTORc2-M2SP1、siRPTORc2-M3SP1、siRPTORc3-M1SP1、siRPTORc3-M2SP1、siRPTORc3-M3SP1、siRPTORc1-M1XP1、siRPTORc1-M2XP1、siRPTORc1-M3XP1、siRPTORc2-M1XP1、siRPTORc2-M2XP1、siRPTORc2-M3XP1、siRPTORc3-M1XP1、siRPTORc3-M2XP1和siRPTORc3-M3XP1中的一种。
  28. 一种药物组合物，所述药物组合物含有权利要求1-27中任意一项所述的siRNA和药学上可接受的载体。
  29. 如权利要求28所述的药物组合物，其中，所述siRNA与药学上可接受的载体的重量比为1∶(1-500)。
  30. 如权利要求28所述的药物组合物，其中，所述siRNA与药学上可接受的载体的重量比为1∶(1-50)。
  31. 一种siRNA缀合物，所述siRNA缀合物含有权利要求1-27中任意一项所述的siRNA以及缀合连接至该siRNA的缀合基团，所述缀合基团包含药学上可接受的至少一个靶向基团和/或递送辅助基团；可选地，所述缀合基团还包含接头，并且，所述接头和/或所述靶向基团或者所述递送辅助基团依次连接；可选地，每个所述靶向基团选自能够和细胞表面受体结合的配体，和/或每个递送辅助基团选自能够增加所述siRNA缀合物在递送目标器官或组织中的生物相容性的基团。
  32. 如权利要求31所述的siRNA缀合物，其中，所述siRNA缀合物具有式(301)所示的结构，其中Nu具有siRPTORa2-M1S、siRPTORb2-M1S、siRPTORc2-M1S、siRPTORc1-T1S和siRPTORc1-T2S中的一种所对应的序列，所述缀合基团连接至所述Nu中siRNA正义链3′末端核苷酸的核糖3′位，并且所述siRNA缀合物处于钠盐形式。
  33. 如权利要求1-27中任意一项所述的siRNA和/或权利要求28-30中任意一项所述的药物组合物和/或权利要求31或32所述的siRNA缀合物在制备用于抑制细胞中RPTOR基因表达的药物中的用途。
  34. 如权利要求1-27中任意一项所述的siRNA和/或权利要求28-30中任意一项所述的药物组合物和/或权利要求31或32所述的siRNA缀合物在制备用于治疗与RPTOR功能调节相关的疾病或症状的药物中的用途。
  35. 如权利要求34所述的用途，其中，所述与RPTOR功能调节相关的疾病或症状是与mTORC1激活所致疾病和细胞自噬功能异常相关的疾病。
  36. 如权利要求35所述的用途，其中，所述与RPTOR功能调节相关的疾病或症状是神经退行性疾病或非酒精性脂肪性肝炎。
  37. 如权利要求36所述的用途，所述神经退行性疾病为阿尔兹海默症，肌萎缩侧索硬化、额颞叶痴呆、路易体痴呆，亨廷顿病，雷士综合征和帕金森病中的一种或多种，优选地，所述神经退行性疾病为阿尔兹海默症。
  38. 一种治疗和/或预防与RPTOR功能调节相关的疾病或症状的方法，所述方法包括向有需要的受试者给予有效量的如权利要求1-27中任意一项所述的siRNA和/或权利要求28-30中任意一项所述的药物组合物和/或权利要求31或32所述的siRNA缀合物。
  39. 一种抑制细胞中RPTOR基因表达的方法，该方法包括将有效量的权利要求1-27中任意一项所述的siRNA和/或权利要求28-30中任意一项所述的药物组合物和/或权利要求31或32所述的siRNA缀合物与所述细胞接触。
  40. 一种试剂盒，该试剂盒包含权利要求1-27中任意一项所述的siRNA和/或权利要求28-30中任意一项所述的药物组合物和/或权利要求31或32中任意一项所述的siRNA缀合物。
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