Nucleic acids, compositions and complexes containing the same, and methods of preparation and use

Abstract

The present disclosure provides an siRNA that suppresses the expression level of the RPTOR gene and regulates mTORC1 activity, and a pharmaceutical composition and complex containing the siRNA. The siRNA comprises a sense strand and an antisense strand, and the nucleotides in the siRNA are each independently modified or unmodified nucleotides, the sense strand comprises nucleotide sequence I, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and have no more than three nucleotide differences, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 are equal in length and have no more than three nucleotide differences. The siRNA, pharmaceutical composition and complex provided by the present disclosure can effectively treat diseases associated with the activation of mTORC1 as an mTORC1 inhibitor, and can treat autophagy-related diseases or symptoms as an autophagy inducer.

Description

[Technical field]

[0001] The present disclosure relates to nucleic acids capable of suppressing the activity of mTORC1, pharmaceutical compositions and siRNA complexes comprising the nucleic acids. The present disclosure further relates to methods for preparing and using these nucleic acids, pharmaceutical compositions and siRNA complexes. [Background technology]

[0002] Neurodegenerative diseases are the progressive loss of neuronal structure and function, including neuronal death and glial cell balance, which leads to cognitive impairment such as dementia. They include Parkinson's disease (PD), Alzheimer's disease (AD), frontotemporal dementia, Huntington disease (HD), amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA). AD and PD mainly occur in middle-aged and elderly people, and as the population ages, the incidence of AD and PD is increasing, while HD, ALS, and SMA can occur in all age groups.

[0003] At present, drug treatment for neurodegenerative diseases is mainly based on Western medicine, but drug treatment requires long-term medication and has obvious side effects, is prone to affecting the central nervous system, digestive system, respiratory system, etc., and can also cause endocrine disruption, mood changes, etc.

[0004] The mammalian target of rapamycin (mTOR) signaling pathway is a signaling pathway that regulates protein synthesis, cell growth, proliferation, etc., and in cells, two distinct complexes, mTORC1 and mTORC2, exist. mTORC1 is a complex consisting of RPTOR, Ras homolog enriched in brain (RHEB), DEP domain-containing mTOR-interacting protein (DEPTOR), mammalian lethal with SEC13 protein 8 (mLST 8), and proline rich AKT substrate of 40 kDa (PRAS40), and is a key negative regulator of autophagy regulation. Previous studies have revealed that mTORC1 is activated and mitochondrial autophagy is suppressed in Alzheimer's Disease (AD) patients and animal models, which is the main cause of senile plaques, neurofibrillary tangles, and cognitive decline in AD patients. It has been reported that mTORC1 activity can be suppressed by inhibiting RPTOR, thereby promoting cellular and mitochondrial autophagy and reducing misfolded proteins in the brains of patients with neurodegenerative diseases.

[0005] There is a growing need in the art to develop new drugs that modulate RPTOR gene expression levels, thereby regulating mTORC1 activity and further regulating and treating diseases or conditions associated with mTORC1 activation and abnormal autophagy function, particularly neurodegenerative diseases. Summary of the Invention [Problem to be solved by the invention]

[0006] The inventors of the present disclosure have unexpectedly discovered that the following siRNAs and modified sequences thereof provided by the present disclosure can specifically suppress RPTOR gene expression in cells, and that pharmaceutical compositions and siRNA complexes comprising the siRNAs of the present disclosure can effectively deliver the siRNAs of the present disclosure to target tissues and / or cells, thereby exhibiting high druggability in the treatment or prevention of neurodegenerative diseases, in particular Alzheimer's disease. [Means for solving the problem]

[0007] In one aspect, the present disclosure provides an siRNA capable of suppressing expression of the RPTOR gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently modified or unmodified, the sense strand comprising nucleotide sequence I, the antisense strand comprising nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II at least partially forming a double-stranded region in a reverse complementary manner, the nucleotide sequence I and the nucleotide sequence II being a pair selected from the sequences shown in i) to iii), i) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 are equal in length and have no more than three nucleotide differences; 5'-CUGCCAUGGAGUAUCUGAZ a1 -3' (SEQ ID NO: 1), 5'-Z a2 UCAGAUACUCCAUGGCAG-3' (SEQ ID NO: 2) However, Z a1 is A and Z a2 is U, and in said nucleotide sequence I, position Z a1 Nucleotide Z corresponding to a3 and in said nucleotide sequence II, position Z a2 Nucleotide Z corresponding to a4 wherein Z a4is the first nucleotide at the 5' end of the antisense strand; or ii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 123 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 124 are equal in length and have no more than three nucleotide differences; 5'-ACAACAUCAAGUACUACGZ b1 -3' (SEQ ID NO: 123), 5'-Z b2 CGUAGUACUUGAUGUUGU-3' (SEQ ID NO: 124) However, Z b1 is A and Z b2 is U, and in said nucleotide sequence I, position Z b1 Nucleotide Z corresponding to b3 and in said nucleotide sequence II, position Z b2 Nucleotide Z corresponding to b4 wherein Z b4 is the first nucleotide at the 5' end of the antisense strand; or iii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 245 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 246 are equal in length and have no more than three nucleotide differences; 5'-CGACUACUACAUCUCCGUZ c1 -3' (SEQ ID NO: 245), 5'-Z c2 ACGGAGAUGUAGUAGUCG-3' (SEQ ID NO: 246) However, Z c1 is G and Z c2 is C, and in said nucleotide sequence I, position Z c1 Nucleotide Z corresponding to c3 and in said nucleotide sequence II, position Z c2 Nucleotide Z corresponding to c4 wherein Z c4is the first nucleotide at the 5' end of the antisense strand.

[0008] In another aspect, the present disclosure provides a pharmaceutical composition, the pharmaceutical composition comprising a siRNA of the present disclosure and a pharma- ceutically acceptable carrier.

[0009] In yet another embodiment, the present disclosure provides a siRNA complex, the siRNA complex comprising an siRNA provided by the present disclosure and a conjugation group conjugated and bound to the siRNA.

[0010] In yet another aspect, the present disclosure provides a use of the siRNA, and / or pharmaceutical composition, and / or siRNA complex of the present disclosure in preparing a medicament for treating and / or preventing a disease associated with modulation of RPTOR function.

[0011] In yet another aspect, the present disclosure provides a method for treating a disease or condition associated with modulation of RPTOR function, such as a neurodegenerative disease or a disease or condition associated with non-alcoholic steatohepatitis, particularly Alzheimer's disease, the method comprising administering an effective amount of a siRNA, and / or pharmaceutical composition, and / or siRNA complex of the present disclosure to a subject in need thereof.

[0012] In yet another aspect, the present disclosure provides a method for suppressing RPTOR gene expression in a cell, the method comprising contacting the cell with an effective amount of an siRNA, and / or pharmaceutical composition, and / or siRNA complex of the present disclosure.

[0013] Additionally, the present disclosure further provides a kit, the kit comprising the siRNA, and / or pharmaceutical composition, and / or siRNA complex of the present disclosure.

[0014] [Incorporation by reference] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Effect of the Invention

[0015] The siRNAs, pharmaceutical compositions and siRNA complexes provided by the present disclosure have excellent stability, high RPTOR mRNA suppression activity, reduced off-target effects, and / or can significantly treat and alleviate diseases or symptoms associated with modulation of RPTOR function.

[0016] For example, the siRNA, pharmaceutical composition or siRNA complex provided by the present disclosure shows excellent target gene expression suppression activity in in vitro cell experiments.For example, the siRNA of the present disclosure shows at least 40.5% RPTOR mRNA suppression rate at a low concentration of 0.5nM in in vitro HepG2 human liver cancer cells, at least 68.3% RPTOR mRNA suppression rate at a concentration of 5nM, and at least 71.1% and even 86.8% RPTOR mRNA suppression rate at a concentration of 50nM, showing excellent RPTOR gene expression suppression effect.In addition, the siRNAs of the present disclosure with different lengths and modification schemes all have high suppression rates.

[0017] In addition, for example, the siRNA, pharmaceutical composition, or siRNA complex provided by the present disclosure has higher stability and target mRNA suppression activity in vivo. For example, at a concentration of 3 mg / kg, the siRNA complexes with different modification schemes of the present disclosure exhibit 51.3% or 52.9% RPTOR mRNA expression suppression rate in the body of C57BL / 6j mice, showing excellent RPTOR mRNA suppression activity.

[0018] Thus, the siRNA, pharmaceutical composition, and siRNA complex provided by the present disclosure can suppress the expression of the RPTOR gene and effectively treat diseases caused by mTORC1 activation and diseases associated with abnormalities in cellular autophagy function, and have bright prospects for application.

[0019] Other features and advantages of the present disclosure are described in detail in the Detailed Description section that follows. [Brief description of the drawings]

[0020]

Figure 1

Figure 2

[0021] Hereinafter, the embodiment of the present disclosure will be described in detail. It should be understood that the embodiment of the present disclosure described herein is merely for explaining and interpreting the present disclosure, and is not intended to limit the present disclosure.

[0022] <Definition> In this disclosure, RPTOR mRNA refers to the sequence set forth in Genbank Accession No. NM_020761.3. Furthermore, unless otherwise specified, the term "target gene" as used in this disclosure refers to a gene encoding the RPTOR mRNA, and the term "target mRNA" refers to the RPTOR mRNA.

[0023] Unless otherwise specified in the context, capital letters C, G, U, and A represent the base sequence of nucleotides, lower case letter m represents a nucleotide adjacent to the left side of the letter m that is a methoxy-modified nucleotide, lower case letter f represents a nucleotide adjacent to the left side of the letter f that is a fluoro-modified nucleotide, lower case letter s represents that two nucleotides adjacent to the left and right sides of the letter s are linked by a thiophosphate group, and P1 represents that one nucleotide adjacent to the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. In some embodiments, P1 represents VP, Ps, or P with a specified modification, the combination letter VP represents that one nucleotide adjacent to the right of the combination letter VP is a vinyl phosphate (5'-(E)-vinylphosphonate (E-VP)) modified nucleotide, the combination letter Ps represents that one nucleotide adjacent to the right of the combination letter Ps is a thiophosphate modified nucleotide, and the capital letter P represents that one nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0024] In this context, the "fluoro-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl of the ribose group of the nucleotide is replaced with fluorine, and the "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the 2'-hydroxyl of the ribose group of the nucleotide is replaced with a non-fluorine group. The "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide, such as an isonucleotide, bridged nucleotide (abbreviated as BNA) or acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl of the ribose group is replaced with methoxy.

[0025] In the context of this specification, the terms "complementary" or "reverse complementary" may be used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, bases on one strand are paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair contains one purine and one pyrimidine. If adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, it is considered that both strands are complementary and that the sequence of the strand can be inferred from the sequence of the complementary strand. Accordingly, in the art, the term "mismatch" means that bases at corresponding positions in a double-stranded nucleic acid are not present in a complementary pairing.

[0026] Unless otherwise specified in context, "essentially reverse complementary" refers to no more than three base mismatches between two related nucleotide sequences, "substantially reverse complementary" refers to no more than one base mismatch between two nucleotide sequences, and "fully reverse complementary" refers to no base mismatches between two nucleotide sequences.

[0027] In this context, the presence of a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter, for example, when one 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 that a nucleotide difference exists between the two nucleotide sequences at that position. In some embodiments, a nucleotide difference is also considered to have occurred at a position when an abasic nucleotide or its equivalent is used instead of the nucleotide at the original position. An abasic nucleotide is a monomeric compound in which the nucleobase of a nucleotide is replaced with another group or a hydrogen atom, including, but not limited to, a substituted or unsubstituted aromatic or heteroaryl group.

[0028] In the context, particularly in describing the preparation method of the siRNA, pharmaceutical composition or siRNA complex of the present disclosure, unless otherwise specified, the nucleoside monomer refers to modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, RNA phosphoramidites are sometimes referred to as nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis according to the type and order of nucleotides in the siRNA or siRNA complex to be prepared. Phosphoramidite solid-phase synthesis is a method used for RNA synthesis known to those skilled in the art. All of the nucleoside monomers used in the present disclosure are commercially available.

[0029] In the context of the present disclosure, unless otherwise specified, "conjugate" refers to two or more chemical moieties each having a specific function being covalently bonded to each other, and accordingly, "complex" refers to a compound formed by covalently bonding the respective chemical moieties. Furthermore, "siRNA complex" refers to a compound formed by covalently bonding one or more chemical moieties having a specific function to siRNA. Depending on the context, the siRNA complex should be understood as a collective term for multiple siRNA complexes, or an siRNA complex represented by a certain chemical formula. In the context of the present disclosure, a "complex molecule" should be understood as a specific compound that can be reacted to be conjugated to siRNA and ultimately form the siRNA complex of the present disclosure.

[0030] It will be understood by those of skill in the art that with respect to any group that contains one or more substituents, it is not intended that these groups introduce any substitutions or substitution patterns that are sterically impractical, synthetically impractical, and / or inherently unstable.

[0031] As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C 1 -C 6 Alkyl is straight and branched chain alkyl containing 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to include all branched and straight chain forms having that number of carbons. Thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl, and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to a divalent group similar to alkyl, but having two points of attachment.

[0032] As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl having at least one carbon-carbon double bond, which is obtained by removing one hydrogen molecule from adjacent carbon atoms of a parent alkyl. The group may 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), prop-2-en-2-yl, and butenyl such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, and buta-1,3-dien-2-yl. In some embodiments, alkenyl groups have from 2 to 20 carbon atoms, while in others, they have from 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl, referring to residues similar to alkenyl, but that have two points of attachment.

[0033] As used herein, "alkoxy" refers to an alkyl having the specified number of carbon atoms attached through an oxygen bridge, such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, etc. An alkoxy typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.

[0034] A variety of hydroxy protecting groups can be used in the present disclosure. Generally, a protecting group can render a chemical functional group insensitive to certain reaction conditions and can be added to and removed from that functional group in a molecule without substantially damaging the remainder of the molecule. Representative hydroxy 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, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive illustrative examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive illustrative examples of hydroxy protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4''-trimethoxytrityl).

[0035] The term "subject" as used herein refers to any animal, e.g., a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any type of poultry.

[0036] As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including, but not limited to, a therapeutic effect. A "therapeutic effect" refers to eradicating or ameliorating the underlying disorder being treated. A therapeutic effect is also obtained by observing an improvement in a subject by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, even though the subject may still be afflicted by the underlying disorder.

[0037] As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a prophylactic effect. To obtain a "prophylactic effect," a siRNA, pharmaceutical composition, or siRNA complex may be administered to a subject who may not have been diagnosed with a particular disease, but who is at risk of suffering from the disease, or who has been reported to have one or more physiological symptoms of the disease.

[0038] siRNA of the present disclosure In one aspect, the present disclosure provides siRNAs capable of suppressing RPTOR gene expression.

[0039] The siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group, and a base, which is known to those skilled in the art, and therefore will not be described here.

[0040] The siRNA of the present disclosure comprises a sense strand and an antisense strand, the sense strand and the antisense strand being the same or different in length, the sense strand being 19 to 23 nucleotides in length, and the antisense strand being 19 to 26 nucleotides in length. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure may 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. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 21, 21 / 23, or 23 / 25.

[0041] According to the present disclosure, the siRNA comprises a sense strand and an antisense strand, each of the nucleotides in the siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part.

[0042] In some embodiments, the siRNA of the present disclosure may be a first, second or third type of siRNA as described below, each of which is described below.

[0043] First type of siRNA In some embodiments, the siRNA of the present disclosure is a first type of siRNA. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 are equal in length and have no more than three nucleotide differences; 5'-CUGCCAUGGAGUAUCUGAZ a1 -3' (SEQ ID NO: 1), 5'-Z a2 UCAGAUACUCCAUGGCAG-3' (SEQ ID NO: 2) However, Z a1 is A and Z a2 is U, and in said nucleotide sequence I, position Z a1 Nucleotide Z corresponding to a3 and in said nucleotide sequence II, position Z a2 Nucleotide Z corresponding to a4 wherein Z a4 is the first nucleotide at the 5' end of the antisense strand.

[0044] In this context, "corresponding in position" refers to being at the same position in a nucleotide sequence from the same end of the nucleotide sequence. For example, the first nucleotide at the 3' end of nucleotide sequence I is the nucleotide that corresponds in position to the first nucleotide at the 3' end of SEQ ID NO:1.

[0045] In some embodiments, the sense strand only comprises nucleotide sequence I and the antisense strand only comprises nucleotide sequence II. In some embodiments, nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:1 have no more than one nucleotide difference and / or nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:2 have no more than one nucleotide sequence difference.

[0046] In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:2 is Z a4 including the difference at the position of Z a4 is selected from A, C, or G. In some embodiments, Z a3 is Z a4The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0047] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are basically reverse complementary, substantially reverse complementary, or completely reverse complementary, where basically reverse complementary means that there are no more than three base mismatches between the two nucleotide sequences, substantially reverse complementary means that there are no more than one base mismatch between the two nucleotide sequences, and completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0048] In some embodiments, the sense strand and the antisense strand are the same or different in length, the sense strand is 19 to 23 nucleotides in length, and the antisense strand is 19 to 26 nucleotides in length, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4, 5'-CUGCCAUGGAGUAUCUGAZ a3 -3' (SEQ ID NO: 3), 5'-Z a4 UCAGAUACUCCAUGGCAG-3' (SEQ ID NO: 4) However, Z a3 is selected from A, U, G or C; Z a4 is Z a3 In some embodiments, Z a3 is A and Z a4 is U.

[0049] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, and the nucleotide sequence III is linked to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II.

[0050] In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially or completely reverse-complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 in the target mRNA and having the same length as the nucleotide sequence IV. In some embodiments, from 5' to 3', the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of nucleotide sequence III is A and the base of nucleotide sequence IV is U, and the length ratio of the sense strand to the antisense strand is 20 / 20; or, the nucleotide sequences III and IV are both 2 nucleotides in length, from 5' to 3', the base sequence of nucleotide sequence III is CA and the base sequence of nucleotide sequence IV is UG, and the length ratio of the sense strand to the antisense strand is 21 / 21. or nucleotide sequences III and IV are both 3 nucleotides in length, the base sequence of nucleotide sequence III is UCA and the base sequence of nucleotide sequence IV is UGA, from the 5' end to the 3' end, with the length ratio of the sense strand to the antisense strand being 22 / 22; or nucleotide sequences III and IV are both 4 nucleotides in length, the base sequence of nucleotide sequence III is GUCA and the base sequence of nucleotide sequence IV is UGAC, from the 5' end to the 3' end, with the length ratio of the sense strand to the antisense strand being 23 / 23.

[0051] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0052] Second type of siRNA In some embodiments, the siRNA of the present disclosure is a second type of siRNA. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 123 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 124 are equal in length and have no more than three nucleotide differences; 5'-ACAACAUCAAGUACUACGZ b1 -3' (SEQ ID NO: 123), 5'-Z b2 CGUAGUACUUGAUGUUGU-3' (SEQ ID NO: 124) However, Z b1 is A and Z b2 is U, and in said nucleotide sequence I, position Z b1 Nucleotide Z corresponding to b3 and in said nucleotide sequence II, position Z b2 Nucleotide Z corresponding to b4 wherein Z b4 is the first nucleotide at the 5' end of the antisense strand.

[0053] In some embodiments, the sense strand only comprises nucleotide sequence I and the antisense strand only comprises nucleotide sequence II. In some embodiments, nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:123 have no more than one nucleotide difference and / or nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:124 have no more than one nucleotide difference.

[0054] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 124 is Zb4 including the difference at the position of Z b4 is selected from A, C, or G. In some embodiments, Z b3 is Z b4 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0055] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are basically reverse complementary, substantially reverse complementary, or completely reverse complementary, where basically reverse complementary means that there are no more than three base mismatches between the two nucleotide sequences, substantially reverse complementary means that there are no more than one base mismatch between the two nucleotide sequences, and completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0056] In some embodiments, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 125, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 126; 5'-ACAACAUCAAGUACUACGZ b3 -3' (SEQ ID NO: 125), 5'-Z b4 CGUAGUACUUGAUGUUGU-3' (SEQ ID NO: 126) However, Z b3 is selected from A, U, G or C; Z b4 is Z b3 In some embodiments, Z b3 is A and Z b4 is U.

[0057] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, and the nucleotide sequence III is linked to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II.

[0058] In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially or completely reverse-complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO: 123 in the target mRNA and having the same length as the nucleotide sequence IV. In some embodiments, from 5' to 3', the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of nucleotide sequence III is A and the base of nucleotide sequence IV is U, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, from 5' to 3', the base sequence of nucleotide sequence III is CA and the base sequence of nucleotide sequence IV is UG, and the length ratio of the sense strand to the antisense strand is 21 / 21. or nucleotide sequences III and IV are both 3 nucleotides in length, the base sequence of nucleotide sequence III is UCA and the base sequence of nucleotide sequence IV is UGA, from the 5' to the 3' end, with the length ratio of the sense strand to the antisense strand being 22 / 22; or nucleotide sequences III and IV are both 4 nucleotides in length, the base sequence of nucleotide sequence III is AUCA and the base sequence of nucleotide sequence IV is UGAU, from the 5' to the 3' end, with the length ratio of the sense strand to the antisense strand being 23 / 23.

[0059] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0060] The third type of siRNA In some embodiments, the siRNA of the present disclosure is a third type of siRNA. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 245 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 246 are equal in length and have no more than three nucleotide differences; 5'-CGACUACUACAUCUCCGUZ c1 -3' (SEQ ID NO: 245), 5'-Z c2 ACGGAGAUGUAGUAGUCG-3' (SEQ ID NO: 246) However, Z c1 is G and Z c2 is C, and in said nucleotide sequence I, position Z c1 Nucleotide Z corresponding to c3 and in said nucleotide sequence II, position Z c2 Nucleotide Z corresponding to c4 wherein Z c4 is the first nucleotide at the 5' end of the antisense strand.

[0061] In some embodiments, the sense strand only comprises nucleotide sequence I and the antisense strand only comprises nucleotide sequence II. In some embodiments, nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:245 have no more than one nucleotide difference and / or nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:246 have no more than one nucleotide difference.

[0062] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 246 is Zc4 including the difference at the position of Z c4 is selected from A, U, or G. In some embodiments, Z c3 is Z c4 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0063] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are basically reverse complementary, substantially reverse complementary, or completely reverse complementary, where basically reverse complementary means that there are no more than three base mismatches between the two nucleotide sequences, substantially reverse complementary means that there are no more than one base mismatch between the two nucleotide sequences, and completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0064] In some embodiments, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 247, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 248; 5'-CGACUACUACAUCUCCGUZ c3 -3' (SEQ ID NO: 247), 5'-Z c4 ACGGAGAUGUAGUAGUCG-3' (SEQ ID NO: 248) However, Z c3 is selected from A, U, G or C; Z c4 is Z c3 In some embodiments, Z c3 is G and Z c4 is C.

[0065] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, and the nucleotide sequence III is linked to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II.

[0066] In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially or completely reverse-complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO: 245 in the target mRNA and having the same length as the nucleotide sequence IV. In some embodiments, from 5' to 3', the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of nucleotide sequence III is A and the base of nucleotide sequence IV is U, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, from 5' to 3', the base sequence of nucleotide sequence III is AA and the base sequence of nucleotide sequence IV is UU, and the length ratio of the sense strand to the antisense strand is 21 / 21. or nucleotide sequences III and IV are both 3 nucleotides in length, the base sequence of nucleotide sequence III is CAA and the base sequence of nucleotide sequence IV is UUG, from the 5' end to the 3' end, and the length ratio of the sense strand to the antisense strand is 22 / 22; or nucleotide sequences III and IV are both 4 nucleotides in length, the base sequence of nucleotide sequence III is GCAA and the base sequence of nucleotide sequence IV is UUGC, from the 5' end to the 3' end, and the length ratio of the sense strand to the antisense strand is 23 / 23.

[0067] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0068] Hereinafter, the description of nucleotide sequence V, nucleotide sequence VI, nucleic acid sequence, nucleotide modification in siRNA and modified sequence applies to the above-mentioned first type siRNA, second type siRNA or third type siRNA. That is, unless otherwise specified, the description of siRNA should be considered as the description of the first type siRNA, the second type siRNA and the third type siRNA one by one. For example, if no specific siRNA is specified, "the siRNA further comprises nucleotide sequence V" means "the first type siRNA, the second type siRNA or the third type siRNA further comprises nucleotide sequence V".

[0069] In some embodiments, the sense strand and the antisense strand have different lengths, and the antisense strand further comprises a nucleotide sequence V, which is 1 to 3 nucleotides in length and is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand.

[0070] In some embodiments, the sense strand further comprises a nucleotide sequence VI, which is 1-3 nucleotides in length and is attached to the 3' end of the sense strand, constituting a 3' overhang of the sense strand.

[0071] In some embodiments, the siRNA provided by the present disclosure comprises nucleotide sequence V but does not comprise nucleotide sequence VI. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure may 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. In some embodiments, the siRNA provided by the present disclosure comprises nucleotide sequences V and VI. In some embodiments, the nucleotide sequence V and the nucleotide sequence VI are the same or different in length. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure may be (19-26):(19-26). In some embodiments, the nucleotide sequences V and / or VI are 2 nucleotides in length, such that the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure may be 19 / 21, 21 / 21, 21 / 23, 23 / 23, 23 / 25 or 25 / 25.

[0072] Each nucleotide in the nucleotide sequence V can be any nucleotide, in order to facilitate synthesis and save synthesis cost, in some embodiments, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to improve the affinity between the antisense strand of siRNA and the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Thus, in some embodiments, the length ratio of the sense strand and the antisense strand of the siRNA of the present disclosure is 19 / 21 or 21 / 23, and the siRNA of the present disclosure has higher mRNA silencing activity.

[0073] Each nucleotide in the nucleotide sequence VI can be any nucleotide, in some embodiments, in order to facilitate synthesis and save synthesis cost, the nucleotide sequence VI is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to improve the affinity between the sense strand and the antisense strand of siRNA, the nucleotide sequence VI is the same as the nucleotide at the corresponding position of target mRNA.Therefore, in some embodiments, 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 siRNA is 21 / 21 or 23 / 23, and the siRNA of the present disclosure has higher mRNA silencing activity.

[0074] The nucleotide at the corresponding position of the target mRNA refers to a nucleotide or nucleotide sequence adjacent to the nucleotide sequence of the target mRNA at the 5' end, which is a nucleotide sequence that is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, 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.

[0075] In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:5, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:6; 5'-CUGCCAUGGAGUAUCUGAZ a3 -3' (SEQ ID NO:5), 5'-Z a4 UCAGAUACUCCAUGGCAGUG-3' (SEQ ID NO: 6) However, the above Z a4 is the first nucleotide at the 5' end of the antisense strand, and Z a3 is selected from A, U, G or C; Z a4 is Z a3 is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 8; 5'-CACUGCCAUGGAGUAUCUGAZ a3 -3' (SEQ ID NO: 7), 5'-Z a4 UCAGAUACUCCAUGGCAGUGAC-3' (SEQ ID NO: 8) However, the above Z a4 is the first nucleotide at the 5' end of the antisense strand, and Z a3 is selected from A, U, G or C; Z a4 is Z a3 is a nucleotide complementary to

[0076] In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 127, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 128; 5'-ACAACAUCAAGUACUACGZ b3 -3' (SEQ ID NO: 127), 5'-Z b4 CGUAGUACUUGAUGUUGUUG-3' (SEQ ID NO: 128) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 129, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 130; 5'-CAACAACAUCAAGUACUACGZ b3 -3' (SEQ ID NO: 129), 5'-Z b4 CGUAGUACUUGAUGUUGUUGAU-3' (SEQ ID NO: 130) However, the above Z b4 is the first nucleotide at the 5' end of the antisense strand, and Z b3 is selected from A, U, G or C; Z b4 is Z b3 is a nucleotide complementary to

[0077] In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 249, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 250; 5'-CGACUACUACAUCUCCGUZ c3 -3' (SEQ ID NO: 249), 5'-Z c4 ACGGAGAUGUAGUAGUCGUU-3' (SEQ ID NO: 250) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 251, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 252; 5'-AACGACUACUACAUCUCCGUZ c3 -3' (SEQ ID NO: 251), 5'-Z c4 ACGGAGAUGUAGUAGUCGUUGC-3' (SEQ ID NO: 252) However, the above Z c4 is the first nucleotide at the 5' end of the antisense strand, and Z c3 is selected from A, U, G or C; Z c4 is Z c3 is a nucleotide complementary to

[0078] In some embodiments, the siRNA described in this disclosure is siRPTORa1, siRPTORa2, siRPTORa3, siRPTORb1, siRPTORb2, siRPTORb3, siRPTORc1, siRPTORc2, and siRPTORc3 listed in Table 1.

[0079] As mentioned above, each nucleotide in the siRNA of the present disclosure is independently modified or unmodified nucleotide. In some embodiments, the nucleotide in the siRNA of the present disclosure is unmodified nucleotide, and in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, and these modifications on the nucleotide group do not obviously weaken or eliminate the function of the siRNA of the present disclosure to suppress RPTOR gene expression.

[0080] In some embodiments, the siRNA of the present disclosure comprises at least one modified nucleotide. In the context of the present disclosure, the term "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 with another group, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide does not obviously weaken or lose the function of siRNA to suppress gene expression. For example, the modified nucleotide disclosed in JK Watts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 may be selected.

[0081] In some embodiments, the sense strand or the antisense strand of the siRNA provided by the present disclosure has at least one nucleotide that is a modified nucleotide and / or at least one phosphate group that is a phosphate group having a modified group. In other words, in the sense strand and the antisense strand, at least a portion of the phosphate groups and / or ribose groups in the phosphate-sugar backbone of at least one single strand is a phosphate group having a modified group and / or a ribose group having a modified group.

[0082] In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

[0083] The inventors of the present disclosure have surprisingly found that the siRNAs provided by the present disclosure provide a high balance between plasma stability and gene silencing efficiency in animal studies.

[0084] In some embodiments, the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, wherein there are no more than five fluoro-modified nucleotides in nucleotide sequence I, and from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence I are fluoro-modified nucleotides, there are no more than seven fluoro-modified nucleotides in nucleotide sequence II, and the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II are fluoro-modified nucleotides.

[0085] In some embodiments, from the 5' to the 3' end, in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of nucleotide sequence I are fluoro-modified nucleotides and the nucleotides at the remaining positions are non-fluoro-modified nucleotides; and from the 5' to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of nucleotide sequence II are fluoro-modified nucleotides and the nucleotides at the remaining positions are non-fluoro-modified nucleotides.

[0086] In the context of this disclosure, "fluoro-modified nucleotide" refers to a nucleotide having the structure shown in the following formula (7) in which the 2'-position hydroxyl of the ribose group of the nucleotide is replaced with fluorine. "Non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the 2'-position hydroxyl of the ribose group of the nucleotide is replaced with a non-fluorine group. In some embodiments, each non-fluoro-modified nucleotide is one independently selected from nucleotides or nucleotide analogs in which the 2'-position hydroxyl of the ribose group of the nucleotide is replaced with a non-fluorine group.

[0087] These nucleotides in which the hydroxy at the 2' position of the ribose group is substituted with a non-fluorine group are known to those skilled in the art, and these nucleotides may be one 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'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0088] In some embodiments, the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe) as shown in formula (8). In some embodiments, the 2'-substituted alkoxy modified nucleotide may be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (9). In some embodiments, the 2'-amino modified nucleotide (2'-NH 2 ) is shown in formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (11).

[0089] [ka]

[0090] A nucleotide analog refers to a group that can substitute for a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, a nucleotide analog can be an isonucleotide, a bridged nucleotide (abbreviated as BNA), or an acyclic nucleotide.

[0091] BNA refers to a constrained or inaccessible nucleotide. BNAs may include five-, six-, or seven-membered bridged structures with a "fixed" C3'-endo sugar puckering. Typically, the bridge is introduced at the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, the BNA may be an LNA as shown in formula (12), an ENA as shown in formula (13), a cET BNA as shown in formula (14), or the like.

[0092] [ka]

[0093] An acyclic nucleotide is a nucleotide in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA) as shown in formula (15) or a glycerol nucleic acid (GNA) as shown in formula (16).

[0094] [ka]

[0095] In the above formula (15) and formula (16), R is selected from H, OH, or alkoxy (O-alkyl).

[0096] An isonucleotide refers to a compound in which the position of the base in the ribose ring in a nucleotide is changed. In some embodiments, an isonucleotide may be a compound in which the base is shifted from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18).

[0097] [ka]

[0098] In the compounds of formula (17) and formula (18) above, Base represents a nucleic acid base such as A, U, G, C or T, and R is selected from H, OH, F or the non-fluorine groups described above. In some embodiments, the nucleotide analog is one selected from isonucleotide, LNA, ENA, cET, UNA and GNA. In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide. In this context, the methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxy of the ribose group is replaced with methoxy. In some embodiments, the non-fluoro modified nucleotide is independently a 2'-O-methoxy modified or a 2'-O-methoxyethyl modified nucleotide.

[0099] In this context, a "fluoro-modified nucleotide" refers to a compound formed by replacing the 2'-hydroxy of a nucleotide with fluorine and having the structure shown in formula (7), a "methoxy-modified nucleotide" refers to a compound formed by replacing the 2'-hydroxy of a ribose group of a nucleotide with methoxy and having the structure shown in formula (8), and a "2'-O-methoxyethyl-modified nucleotide" refers to a compound formed by replacing the 2'-hydroxy of a ribose group of a nucleotide with methoxyethyl and having the structure shown in formula (9).

[0100] In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications: from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence I are fluoro-modified nucleotides, and the nucleotides at the remaining positions are methoxy-modified nucleotides, and in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluoro-modified nucleotides, and the nucleotides at the remaining positions are methoxy-modified nucleotides.

[0101] In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications: from the 5' to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, and the remaining nucleotides in the sense strand of the siRNA are methoxy-modified nucleotides, from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 or 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, and the remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides; Alternatively, from the 5' to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides; Alternatively, from the 5' to the 3' end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and, from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides.

[0102] In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications: from the 5'-end to the 3'-end, the nucleotides at positions 7, 8, and 9 or 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, and the remaining nucleotides in the sense strand of the siRNA are methoxy-modified nucleotides; from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 14, and 16 or 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides; and from the 5'-end to the 3'-end, at least one of the nucleotides at positions 3 to 6 of the nucleotide sequence II is a 2'-O-methoxyethyl-modified nucleotide. In some embodiments, in the antisense strand, the nucleotide at position 3 or 5 is a 2'-O-methoxyethyl-modified nucleotide, and the remaining nucleotides are methoxy-modified nucleotides. In some embodiments, no more than two of the nucleotides at positions 3 to 9 of nucleotide sequence II, from the 5' to the 3' terminus, are 2'-O-methoxyethyl modified nucleotides.

[0103] In some embodiments, the siRNA provided by the present disclosure optionally comprises: 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, It is one selected from siRPTORb2-M3, siRPTORb3-M1, siRPTORb3-M2, siSRPTORb3-M3, siRPTORc1-M1, siRPTORc1-M2, siRPTORc1-M3, siRPTORc2-M1, siRPTORc2-M2, siRPTORc2-M3, siRPTORc3-M1, siRPTORc3-M2 and siSRPTORc3-M3.

[0104] The siRNA having the above modification is not only low-cost, but also can make nucleic acid difficult to be cut by ribonuclease in blood, thereby improving the stability of nucleic acid and further enhancing the property of nucleic acid resistance to nuclease hydrolysis.In addition, the above modified siRNA has high target mRNA suppression activity.

[0105] In some embodiments, in the sense strand and antisense strand of the siRNA provided by the present disclosure, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand is a phosphate ester group having a modification group.In some embodiments, the phosphate ester group having a modification group is a thiophosphate ester group in which at least one oxygen atom in the phosphodiester bond in the phosphate ester group is replaced with a sulfur atom.In some embodiments, the phosphate ester group having a modification group is a thiophosphate ester group having the structure shown in formula (1).

[0106] [ka]

[0107] Such modifications stabilize the double-stranded structure of siRNA and enable high specificity and high affinity base pairing to be maintained.

[0108] In some embodiments, in the siRNA provided by the present disclosure, the thiophosphate group is present at at least one position selected from the group consisting of between the 1st and 2nd nucleotides at any one end of the sense strand or antisense strand, between the 2nd and 3rd nucleotides at any one end of the sense strand or antisense strand, and any combination thereof. In some embodiments, the thiophosphate group is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the thiophosphate group is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the thiophosphate group is present at Between the first and second nucleotides from the 5' end of the sense strand, Between the second and third nucleotides from the 5' end of the sense strand, Between the first and second nucleotides from the 3' end of the sense strand, Between the second and third nucleotides from the 3' end of the sense strand, Between the first and second nucleotides from the 5' end of the antisense strand, Between the second and third nucleotides from the 5' end of the antisense strand, Between the first and second nucleotides from the 3' end of the antisense strand, and It is bound to at least one site between the second and third nucleotides from the 3' end of the antisense strand.

[0109] In some embodiments, the siRNA provided by the present disclosure is optionally selected from 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 ...X, siRPTORa1-M3S, siRPTORa1-M3X, siRPTORa3-M1S, siRPTORa1-M1X, siRPTORa1-M2X, siRPTORa1-M3S, siRPTORa1-M3X, siRPTORa3-M3X, siRPTORa3-M1S, siRPTORa1-M1X, siRPTORa1-M2X, siRPTORa1-M3S, siRPTORa1-M3X, siRPTORa3-M3X, siRPTORa3-M3X, siRPTORa3-M3X, siRPTORa3-M3X, siRPTORa3-M3X, TORa3-M1X, siRPTORa3-M2S, siRPTORa3-M2X, siRPTORa3-M3S, siRPTORa3-M3X, siRPTORa1-T1S, siRPTORa1-T2S, siRPTORb1-M1S, siR PTORb1-M1X, siRPTORb1-M2S, siRPTORb1-M2X, siRPTORb1-M3S, siRPTORb1-M3X, siRPTORb2-M1S, siRPTORb2-M1X, siRPTORb2-M2S, si RPTORb2-M2X, siRPTORb2-M3S, siRPTORb2-M3X, siRPTORb3-M1S, siRPTORb3-M1X, siRPTORb3-M2S, siRPTORb3-M2X, siRPTORb3-M3S, s iRPTORb3-M3X, siRPTORb1-T1S, siRPTORb1-T2S, siRPTORc1-M1S, siRPTORc1-M1X, siRPTORc1-M2S, siRPTORc1-M2X, siRPTORc1-M3S, It is one selected from 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 and siRPTORc1-T2S.

[0110] In some embodiments, the 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

[0111] The commonly used 5'-phosphate nucleotides or 5'-phosphate analog modified nucleotides are known to those skilled in the art. For example, the 5'-phosphate nucleotide may have the following structure:

[0112] [ka]

[0113] Furthermore, for example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four types of 5'-phosphate analog modified nucleotides:

[0114] [ka]

[0115] wherein R is selected from H, OH, methoxy, and fluorine; and Base represents a nucleic acid base selected from A, U, C, G, or T.

[0116] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate (E-VP)) modification as shown in formula (3), or a thiophosphate modified nucleotide as shown in formula (5).

[0117] In some embodiments, the siRNA provided by the present disclosure is optionally selected from 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-M 2SP1, siRPTORa2-M3SP1, siRPTORa3-M1SP1, siRPTORa3-M2SP1, siRPTORa3-M3SP1, siRPTORa1-M1XP1, siRPTORa1-M2XP1, siRPTORa1-M3XP1, siRPTO Ra2-M1XP1, siRPTORa2-M2XP1, siRPTORa2-M3XP1, siRPTORa3-M1XP1, siRPTORa3-M2XP1, siRPTORa3-M3XP1, siRPTORb1-M1P1, siRPTORb1-M2P1, siRP TORb1-M3P1, siRPTORb2-M1P1, siRPTORb2-M2P1, siRPTORb2-M3P1, siRPTORb3-M1P1, siRPTORb3-M2P1, siRPTORb3-M3P1, siRPTORb1-M1SP1, siRPTO Rb1-M2SP1, siRPTORb1-M3SP1, siRPTORb2-M1SP1, siRPTORb2-M2SP1, siRPTORb2-M3SP1, siRPTORb3-M1SP1, siRPTORb3-M2SP1, siRPTORb3-M3SP1, si RPTORb1-M1XP1, siRPTORb1-M2XP1, siRPTORb1-M3XP1, siRPTORb2-M1XP1, siRPTORb2-M2XP1, siRPTORb2-M3XP1, siRPTORb3-M1XP1, siRPTORb3-M2X P1, siRPTORb3-M3XP1, siRPTORc1-M1P1, siRPTORc1-M2P1, siRPTORc1-M3P1, siRPTORc2-M1P1, siRPTORc2-M2P1, siRPTORc2-M3P1, siRPTORc3-M1P1,It is one selected from 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 and siRPTORc3-M3XP1.

[0118] The inventors of the present disclosure have unexpectedly discovered that the siRNAs provided by the present disclosure not only have significantly improved plasma and lysosomal stability, but also maintain extremely high gene silencing activity.

[0119] The siRNA provided by the present disclosure can be obtained by the usual siRNA preparation method in the field (for example, solid-phase synthesis method and liquid-phase synthesis method).Solid-phase synthesis is already available as a commercial customization service.The modified nucleotide can be introduced into the siRNA described in the present disclosure by using the nucleoside monomer having the corresponding modification, and the method of preparing the nucleoside monomer having the corresponding modification and the method of introducing the modified nucleotide into the siRNA are also well known to those skilled in the art.

[0120] Pharmaceutical Compositions In one aspect, the present disclosure provides a pharmaceutical composition, the pharmaceutical composition comprising the above-described siRNA as an active ingredient and a pharma- ceutically acceptable carrier.

[0121] The pharma- ceutical acceptable carrier may be a carrier commonly used in the field of siRNA administration, for example, magnetic nanoparticles (e.g., Fe 3 O 4 or Fe2 O 3 Nanoparticles based on cellulose acetate, carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(N,N-dimethylaminoethyl methacrylate) (PPEEA). methacrylate, PDMAEMA) and one or more of their derivatives.

[0122] In some embodiments, there are no particular requirements for the contents of siRNA and pharma- ceutically acceptable carrier in the pharmaceutical composition, and in some embodiments, the weight ratio of siRNA to pharma- ceutically acceptable carrier may be 1:(1 to 500), and in some embodiments, the weight ratio is 1:(1 to 50).

[0123] In some embodiments, the pharmaceutical composition may include other pharma- ceutically acceptable additives, which may be one or more of various agents or compounds commonly used in the art. For example, the other pharma-ceutically acceptable additives may include at least one of a pH buffer, a protective agent, and an osmolality adjusting agent.

[0124] The pH buffer solution may be a trishydroxymethylaminomethane hydrochloride buffer solution having a pH of 7.5 to 8.5 and / or a phosphate buffer solution having a pH of 5.5 to 8.5, for example, a phosphate buffer solution having a pH of 5.5 to 8.5.

[0125] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent may be 0.01 to 30% by weight based on the total weight of the pharmaceutical composition.

[0126] The osmotic pressure regulator may be, for example, sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is determined so that the osmotic pressure of the pharmaceutical composition is 200 to 700 milliosmoles per kilogram (mOsm / kg). Depending on the desired osmotic pressure, a person skilled in the art can easily determine the content of the osmotic pressure regulator. In some embodiments, the dosage during the administration of the formulation produced from the pharmaceutical composition is adjusted according to the administration method.

[0127] In some embodiments, the pharmaceutical composition may be a liquid formulation such as an injection solution, or may be a lyophilized powder injection that is mixed with a liquid additive at the time of administration to form a liquid formulation. The liquid formulation may be used for subcutaneous, intramuscular, intracerebroventricular or intrathecal injection administration, but is not limited thereto, and the pharmaceutical composition may be delivered by nasal administration, oral inhalation, spray administration, but is not limited thereto. In some embodiments, the pharmaceutical composition is delivered by intrathecal injection. In some embodiments, the intrathecal injection of the pharmaceutical composition into the spinal fluid may be performed in the form of a bolus injection or via a micropump, and these micropumps may be implanted subcutaneously to deliver siRNA to the spinal fluid regularly and constantly. In some embodiments, the intrathecal administration is performed via an osmotic pump implanted by surgery. In some embodiments, to facilitate intrathecal administration, an osmotic pump is implanted in the subarachnoid space of the spinal canal. Further details of this intrathecal delivery system are described in PCT / US2015 / 013253, filed January 28, 2015, the contents of which are incorporated herein by reference in their entirety.

[0128] In some embodiments, the pharmaceutical composition may be in the form of a liposome formulation. In some embodiments, the pharma- ceutical acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as organic amine), an auxiliary lipid, and / or a polyethylene glycolated lipid. The organic amine, auxiliary lipid, and polyethylene glycolated lipid may be one or more selected from the amine-containing transfection compound or its pharma- ceutical acceptable salt or derivative, auxiliary lipid, and polyethylene glycolated lipid described in Chinese Patent Publication No. 103380113 (incorporated herein in its entirety by reference).

[0129] In some embodiments, the organic amine may be a compound represented by formula (201) or a pharma- ceutically acceptable salt thereof, as described in Chinese Patent Publication No. 103380113:

[0130] [ka]

[0131] However, X 101 and X 102 are each independently O, S, NA or CA, and A is hydrogen or C 1 -C 20 is a hydrocarbon chain, Y 101 and Z 101 are each independently C=O, C=S, S=O, CH-OH or SO 2 and R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, a substituted or unsubstituted, branched or straight chain acyl, a substituted or unsubstituted, branched or straight chain aryl, or a substituted or unsubstituted, branched or straight chain heteroaryl; x is an integer from 1 to 10; n is an integer of 1 to 3, m is an integer of 0 to 20, and p is 0 or 1, where m=p=0, R 102 is hydrogen, When at least one of n and m is 2, R 103 and the nitrogen in formula (201) form a structure represented by formula (202) or formula (203).

[0132] [ka]

[0133] In the above, g, e, and f are each independently an integer of 1 to 6, and "HCC" represents a hydrocarbon chain. * N represents a nitrogen atom in formula (201).

[0134] In some embodiments, R 103 is a polyamine. In another embodiment, R 103 In some embodiments, R in formula (201) is a ketal. 101 and R 102 each is independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl, said alkyl or alkenyl having 3 to about 20 carbon atoms, e.g., 8 to about 18 carbon atoms, and 0 to 4 double bonds, e.g., 0 to 2 double bonds.

[0135] In some embodiments, when each of n and m is independently 1 or 3, R 103 may be any one of the following formulas (204) to (213).

[0136] [ka]

[0137] In formulae (204) to (213), g, e, and f each independently represent an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, * is R 103 and the nitrogen atom in formula (201), and * Each H on position may be substituted to provide a bond with the nitrogen atom in formula (201).

[0138] The compound of formula (201) may be prepared according to the description in Chinese Patent Publication No. 103380113.

[0139] In some embodiments, the organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215).

[0140] [ka]

[0141] the co-lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative; The polyethylene glycolated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0142] In some embodiments, the molar ratio of the organic amine, the colipid, and the polyethylene glycolated lipid in the pharmaceutical composition is (19.7 to 80):(19.7 to 80):(0.3 to 50), and may be, for example, (50 to 70):(20 to 40):(3 to 20).

[0143] In some embodiments, the particles of the pharmaceutical composition formed by the siRNA of the present disclosure and the amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, e.g., the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0144] In some embodiments, in a pharmaceutical composition formed by an siRNA of the disclosure and the amine-containing transfection reagent, the weight ratio (weight / weight ratio) of siRNA to total lipids (e.g., organic amine, auxiliary lipid, and / or pegylated lipid) is within the range of about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12, or about 1:6 to about 1:10, e.g., the weight ratio of siRNA of the disclosure to total lipids 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.

[0145] In some embodiments, the pharmaceutical composition may be marketed with each component being independent, or may be used as a liquid formulation.In some embodiments, the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutical acceptable carrier may be prepared according to various known methods, and the siRNA provided by the present disclosure may be used instead of conventional siRNA.In some embodiments, it may be prepared according to the following method.

[0146] The organic amine, auxiliary lipid and polyethylene glycolated lipid are suspended in alcohol in the above molar ratio and mixed uniformly to obtain a lipid solution. The amount of alcohol is determined so that the total mass concentration of the obtained lipid solution is 2 to 25 mg / mL, for example, 8 to 18 mg / mL. The alcohol is one or more selected from pharma- ceutically acceptable alcohols, such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, and other alcohols that are liquid at around room temperature, and may be, for example, ethanol.

[0147] The siRNA provided by the present disclosure is dissolved in a buffer salt solution to obtain an siRNA aqueous solution. The concentration of the buffer salt solution is 0.05 to 0.5 M, and may be, for example, 0.1 to 0.2 M, the pH of the buffer salt solution is adjusted to 4.0 to 5.5, and may be, for example, 5.0 to 5.2, and the amount of the buffer salt solution is determined so that the concentration of the siRNA is 0.6 mg / mL or less, and may be, for example, 0.2 to 0.4 mg / mL. The buffer salt is one or more selected from soluble acetates and soluble citrates, and may be, for example, sodium acetate and / or potassium acetate.

[0148] After mixing the lipid solution and the siRNA aqueous solution, the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome formulation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1:(2-5), and may be, for example, 1:4.

[0149] The incubated liposome preparation is concentrated or diluted, impurities are removed, and bacteria are removed to obtain a pharmaceutical composition provided by the present disclosure. Its physicochemical parameters are pH 6.5-8, encapsulation efficiency 80% or more, particle size 40-200 nm, polydispersity index 0.30 or less, and osmotic pressure 250-400 mOsm / kg. For example, the physicochemical parameters may be pH 7.2-7.6, encapsulation efficiency 90% or more, particle size 60-100 nm, polydispersity index 0.20 or less, and osmotic pressure 300-400 mOsm / kg.

[0150] Concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously with removing impurities. As a method for removing impurities, various conventional methods may be used, for example, ultrafiltration may be performed under conditions of 100 KDa using a tangential flow system and a hollow fiber column, and the ultrafiltration exchange solution may be a phosphate buffer solution (PBS) of pH 7.4. As a method for sterilization, various conventional methods may be used, for example, sterilization may be performed by filtering through a 0.22 μm filter.

[0151] siRNA complex In yet another aspect, the present disclosure provides a siRNA complex, the siRNA complex comprising the siRNA described above and a conjugation group conjugated and bound to the siRNA.

[0152] Typically, the conjugated group comprises at least one pharma- ceutically acceptable targeting group and / or delivery-assisting group. In some embodiments, the conjugated group further comprises a linker, and the linker and / or the targeting group or the delivery-assisting group are linked in sequence. In some embodiments, the number of targeting groups is 1 to 6. In some embodiments, the number of targeting groups is 2 to 4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugated group, for example, covalently conjugated to the conjugated group. The conjugation site between the siRNA and the conjugated group may be at the 3'-end or 5'-end of the sense strand of the siRNA, at the 5'-end of the antisense strand, or in an internal sequence of the siRNA. In some embodiments, the conjugated site between the siRNA and the conjugated group is at the 3'-end of the sense strand of the siRNA. In some embodiments, the conjugated group may be linked to the phosphate group, hydroxyl at the 2'-position, or base of the nucleotide. In some embodiments, the conjugated group may be attached to the 3'-hydroxyl, in which case the nucleotides are linked by a 2'-5' phosphodiester bond. When the conjugated group is attached to the end of the siRNA strand, it is usually attached to the phosphate group of the nucleotide, and when the conjugated group is attached to the internal sequence of the siRNA, it is usually attached to the ribose sugar ring or base. For various binding methods, see 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.

[0153] In some embodiments, the siRNA and the conjugation group can be linked by acid-labile or reducible chemical bonds, and these chemical bonds can be degraded in the acidic environment of cell endosome, and the siRNA can be released.For non-degradable conjugation methods, the conjugation group is bound to the sense strand of siRNA, so that the influence of conjugation on siRNA activity can be minimized.

[0154] In some embodiments, the pharma- ceutically acceptable targeting group may be a ligand commonly used in the field of siRNA administration. In some embodiments, each of the ligands is independently selected from ligands capable of binding to cell surface receptors. In some embodiments, at least one targeting ligand targets a receptor that mediates delivery to central nervous system (CNS) tissue. These types of ligands are known to those skilled in the art, and their function is generally to bind to a specific receptor on the surface of a target cell and mediate delivery of the siRNA bound to the ligand to the target cell. In some embodiments, at least one of the targeting groups is selected from ligands capable of binding to a cell surface receptor expressing the RPTOR gene. In some embodiments, at least one targeting group is a ligand that targets a receptor on the surface of liver parenchymal cells. In some embodiments, at least one or each targeting group is a ligand that targets an asialoglycoprotein receptor on the surface of liver cells. In some embodiments, at least one or each targeting group is N-acetylgalactosamine (GalNAc). In some embodiments, the complexes of the present disclosure have various siRNA complex structures disclosed in CN110959011A, the disclosure of which is incorporated herein by reference in its entirety.

[0155] In some embodiments, the conjugate of the present disclosure has a structure shown in formula (301):

[0156] [ka]

[0157] In some embodiments, the siRNA complex has a structure shown in formula (301), where Nu has a sequence corresponding to one of siRPTORa2-M1S, siRPTORb2-M1S, siRPTORc2-M1S, siRPTORc1-T1S, and siRPTORc1-T2S, the conjugate group is attached to the 3' position of the ribose group of the 3'-terminal nucleotide of the siRNA sense strand in Nu, and the siRNA complex is in sodium salt form.

[0158] In some embodiments, each of the targeting groups is selected from ligands that can bind to cell surface receptors expressing the RPTOR gene. In some embodiments, at least one of the targeting groups is a ligand that targets a receptor on the surface of a target cell in the CNS. In some embodiments, each of the targeting groups is a ligand that targets a receptor on the surface of a target cell in the CNS. In some embodiments, the ligand can be conjugated with siRNA to achieve delivery to a specific CNS tissue, and in some embodiments, at least one of the targeting groups is a peptide ligand. In some embodiments, each of the targeting groups is a peptide ligand. In some embodiments, the targeting ligand is selected from the group consisting of angiotensin 2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter, and LDL receptor ligand. For example, there are various targeting groups described in the specification of Chinese Patent Publication No. 112400018 (e.g., the ligands described in paragraph

[0856] ), the disclosure of which is incorporated herein in its entirety by reference.

[0159] In some embodiments, the pharma- ceutically acceptable delivery-assisting group may be a lipophilic group comprising an aliphatic compound or an alicyclic compound. In some embodiments, the lipophilic group comprises a straight chain aliphatic hydrocarbon, a branched chain aliphatic hydrocarbon, or a steroid. In some embodiments, the lipophilic group comprises a saturated or unsaturated C4-C30 hydrocarbon chain. In some examples, the lipophilic group comprises a saturated or unsaturated C 6 -C 18 Hydrocarbon chains (e.g., linear C 6 -C 18 In some embodiments, the lipophilic group comprises a saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C 16 In some embodiments, the lipophilic group is a C6-C30 fatty acyl, which refers to the remaining atomic group after removing the hydroxyl from a C6-C30 fatty acid. In some embodiments, the lipophilic group is non-covalently or covalently conjugated to the siRNA. In some embodiments, the conjugation site between the lipophilic group and the siRNA is at the 3'-end or 5'-end of the sense strand of the siRNA. In some embodiments, the conjugation site between the lipophilic group and the siRNA is at the 5'-end of the antisense strand. In some embodiments, the conjugation site between the lipophilic group and the siRNA may be in an internal sequence of the siRNA. In some embodiments, the lipophilic group is attached to the phosphate group, ribose sugar ring, or base of the nucleotide. In some embodiments, when the lipophilic group is conjugated to the base of the nucleotide, the preferred position is one that does not interfere with the hydrogen bonding interactions required for base pairing. In some embodiments, the lipophilic group is attached to the phosphate group, 2'-position hydroxyl, or base of the nucleotide. In some embodiments, the lipophilic group may be conjugated to the ribose sugar ring via the 2'-hydroxyl position.Various methods for binding delivery-assisting groups to siRNA are well known to those skilled in the art, such as the various preparation methods described in Chinese Patent Publication No. 112400018 (e.g., the preparation methods described in paragraphs

[1053] to

[1065] ), the disclosure of which is incorporated herein in its entirety by reference.

[0160] In some embodiments, the targeting group is conjugated to siRNA via one or more linkers.The inventors of the present disclosure have unexpectedly found that the siRNA complex of the present disclosure has significantly improved plasma stability and also exhibits high RPTOR mRNA silencing activity.In some embodiments, the siRNA of the present disclosure can be one of the siRNAs shown in Table 1a, 1b and 1c.Using these siRNAs, the siRNA complex of the present disclosure exhibits higher RPTOR mRNA silencing activity.

[0161] Table 1a. First class siRNA of the present disclosure [Table 1] TIFF2024546667000015.tif230170TIFF2024546667000016.tif111170

[0162] Table 1b. Second class siRNA of the present disclosure [Table 2] TIFF2024546667000018.tif228170TIFF2024546667000019.tif111170

[0163] Table 1c. Third siRNA of the present disclosure [Table 3] TIFF2024546667000021.tif228170TIFF2024546667000022.tif111170

[0164] Here, capital letters C, G, U, and A represent the base sequence of nucleotides, lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, lower case letter s represents that the two nucleotides on the left and right sides of the letter are bonded by a thiophosphate group, and P1 represents that one nucleotide adjacent to the right side of the P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. In some embodiments, P1 represents VP, Ps, or P with a specified modification, the combination letter VP represents that one nucleotide adjacent to the right side of the combination letter VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide, the combination letter Ps represents that one nucleotide adjacent to the right side of the combination letter Ps is a thiophosphate-modified nucleotide, and capital letter P represents that one nucleotide adjacent to the right side of the letter P is a 5'-phosphate nucleotide. Combination letters moe is the combination character moe represents a nucleotide with a 2'-O-methoxyethyl modification to the left of the combination letter G. moe is the combination character moe The nucleotide adjacent to the left of is a nucleotide with the base guanine and a 2'-O-methoxyethyl modification. moe is the combination character moe The nucleotide adjacent to the left of represents a nucleotide having a 5-methylcytosine base and a 2'-O-methoxyethyl modification. In addition, each U in the sequence shown in the above table may be optionally replaced with T, without any obvious effect on the activity or off-target effect of the siRNA.

[0165] In the siRNA, pharmaceutical composition or siRNA complex described in the present disclosure, adjacent nucleotides are linked by a phosphodiester bond or a thiophosphodiester bond, and the non-bridging oxygen or sulfur atom in the phosphodiester or thiophosphodiester bond may be negatively charged and exist as a hydroxyl or sulfhydryl, and the hydrogen ions in the hydroxyl or sulfhydryl may be partially or completely replaced by a cation. The cation may be any cation, for example, a metal cation, an ammonium ion NH 4 + , or one of organic ammonium cations. In consideration of improving solubility, in one embodiment, the cation is one or more selected from an alkali metal ion, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation. The alkali metal ion is K + And / or Na + and the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and / or an ammonium ion formed by N,N-diisopropylethylamine. Thus, the siRNA or siRNA complex described in the present disclosure may exist at least partially as a salt. In one embodiment, the non-bridging oxygen or sulfur atom in the phosphodiester bond or thiophosphodiester bond is at least partially bound to a sodium ion, and the siRNA or siRNA complex described in the present disclosure exists as a sodium salt or partial sodium salt.

[0166] As those skilled in the art are clearly aware, modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications.The method of preparing nucleoside monomers with corresponding modifications and the method of introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.All modified nucleoside monomers can be purchased commercially or prepared by known methods.

[0167] The siRNA complex of the present disclosure can be prepared by any reasonable synthetic route.For example, in the case of a complex molecule that contains targeting group and the active reactive group that can react with phosphoramidite to form covalent bond, first, the active group in the complex molecule is protected with a protecting agent, and then is bound to solid support; then, using phosphoramidite solid-phase synthesis method, according to the type and order of nucleotides of the sense strand and antisense strand of siRNA, nucleoside monomers are bound one by one from 3' to 5', and the binding of each nucleoside monomer includes four reactions of deprotection, coupling, capping, oxidation or sulfurization; then, the sense strand and antisense strand of siRNA are isolated and annealed, thereby obtaining the siRNA complex of the present disclosure.

[0168] Furthermore, the preparation of siRNA complex can also be carried out by referring to the disclosures of existing documents.For example, in Example 1 of International Publication No. 2019010274, a method is described in which a linking group having a specific structure and a targeting ligand are reacted and sequentially bound to siRNA.The disclosures thereof are incorporated herein in their entirety by reference.

[0169] Use of the siRNA of the present disclosure, pharmaceutical compositions containing the siRNA, and siRNA complexes In some embodiments, the present disclosure provides the use of the siRNA, and / or pharmaceutical composition, and / or siRNA complex of the present disclosure in the preparation of a medicament for treating and preventing a disease or condition associated with RPTOR function regulation. In some embodiments, the disease or condition associated with RPTOR function regulation is a disease caused by activation of mTORC1 and a disease associated with cellular autophagy dysfunction. In some embodiments, the disease or condition associated with neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease, preferably, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the disease associated with cellular autophagy dysfunction is non-alcoholic steatohepatitis.

[0170] In some embodiments, the present disclosure provides a method for preventing and / or treating a disease or condition associated with RPTOR functional regulation, the method comprising administering an effective amount of the siRNA and / or pharmaceutical composition and / or siRNA complex of the present disclosure to a subject in need thereof. By administering the siRNA active ingredient of the present disclosure to a subject in need thereof, the purpose of preventing and / or treating a disease by RNA interference mechanism can be achieved. Thus, the siRNA and / or pharmaceutical composition and / or siRNA complex of the present disclosure may be used for preventing and / or treating a disease or condition associated with RPTOR functional regulation, or may be used for preparing a drug for preventing and / or treating a disease or condition associated with RPTOR functional regulation.

[0171] The term "drug administration / administration" as used herein refers to placing the siRNA, pharmaceutical composition and / or siRNA complex of the present disclosure into the body of a subject by a method or route that allows at least a portion of the siRNA, pharmaceutical composition and / or siRNA complex of the present disclosure to localize at a desired site to produce a desired effect. Suitable routes of administration for the methods of the present disclosure include local administration and systemic administration. Generally, local administration delivers more siRNA complex to a specific site than to the subject's systemic circulation, while systemic administration delivers the siRNA, pharmaceutical composition and / or siRNA complex of the present disclosure to the subject's primary systemic circulation. Considering that the present disclosure is intended to provide a means of preventing and / or treating neurodegenerative diseases, in some embodiments, an administration method capable of delivering the drug to central nervous system tissue is used. In some embodiments, an administration method capable of delivering the drug intrathecally is used. In some embodiments, an administration method capable of injecting the drug into spinal fluid is used.

[0172] The subject may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, intratracheal (aerosol), intracerebroventricular, intrathecal, nasal, rectal, and topical (including buccal and sublingual) administration. The frequency of administration may be once or more times daily, weekly, biweekly, triweekly, monthly, or yearly. The dose of the siRNA, pharmaceutical composition, or siRNA complex described in this disclosure may be a dose that is conventional in the art, and the dose may be determined by various parameters, particularly the age, weight, and sex of the subject. Toxicity and therapeutic efficacy may be measured by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (lethal dose that kills 50% of the colony) and ED 50 (In quantitative response, it refers to the dose that can induce 50% of the maximum response strength, and in numerical response, it refers to the dose at which 50% of experimental subjects have a positive response) may be measured. A range of human doses can be obtained based on data obtained from cell culture assays and animal studies. In some embodiments, the dose during administration of the siRNA, pharmaceutical composition, or formulation made from the siRNA complex is adjusted according to the administration method.

[0173] When administering the siRNA, pharmaceutical composition, and / or siRNA complex described in the present disclosure, for example, the amount of siRNA for male or female, 6-12 week old, C57BL / 6J mouse weighing 18-25 g or ob / ob mouse weighing 30-45 g is as follows: (i) for siRNA complex, the siRNA dose may be 0.001-100 mg / kg body weight, in some embodiments 0.01-50 mg / kg body weight, in further embodiments 0.05-20 mg / kg body weight, in still further embodiments 0.1-15 mg / kg body weight, and in still further embodiments 0.1-10 mg / kg body weight. (ii) for pharmaceutical composition formed by siRNA and a pharma- ceutically acceptable carrier, the siRNA dose may be 0.001-50 mg / kg body weight, in some embodiments 0.01-10 mg / kg body weight, in further embodiments 0.05-5 mg / kg body weight, and in still further embodiments 0.1-3 mg / kg body weight.

[0174] In some embodiments, the present disclosure provides a method for suppressing RPTOR gene expression in a cell, the method comprising contacting the cell with an effective amount of an siRNA and / or pharmaceutical composition and / or siRNA complex of the present disclosure, introducing the siRNA and / or pharmaceutical composition and / or siRNA complex of the present disclosure into the cell, thereby achieving the purpose of suppressing RPTOR gene expression in the cell by an RNA interference mechanism.

[0175] The siRNA dosage in the modified siRNA, pharmaceutical composition and / or siRNA complex provided by the methods provided by the present disclosure for suppressing RPTOR gene expression in cells is generally an amount capable of reducing expression of the target gene and resulting in an extracellular concentration at the surface of the target cell of 1 pM to 1 μM, 0.01 nM to 100 nM, 0.05 nM to 50 nM, or 0.05 nM to about 5 nM. The amount required to achieve this local concentration will vary depending on a variety of factors including the delivery method, the delivery site, the number of cell layers between the delivery site and the target cell or tissue, the delivery route (local or systemic), etc. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

[0176] kit In one aspect, the present disclosure provides a kit, the kit comprising an effective amount of at least one of the siRNA, pharmaceutical composition, and siRNA complex of the present disclosure.

[0177] In some embodiments, the kits described herein may provide the siRNA in a container. In some embodiments, the kits described herein may include a container providing a pharma- ceutically acceptable excipient. In some embodiments, the kits may include other components, such as stabilizers or preservatives. In some embodiments, the kits described herein may include at least one other therapeutic agent in a container separate from the container providing the siRNA described herein. In some embodiments, the kits may include instructions for mixing the siRNA with a pharma- ceutically acceptable carrier and / or additives or other components, if present.

[0178] In the kit of the present disclosure, the siRNA and pharma- ceutically acceptable carriers and / or additives, and the modified siRNA, pharmaceutical composition and / or siRNA complex, and / or pharma- ceutically acceptable carriers and / or additives may be provided in any form, for example, liquid form, dry form, or lyophilized form. In some embodiments, the siRNA and pharma- ceutically acceptable carriers and / or additives, and the pharma- ceutically acceptable carriers and / or additives of the pharmaceutical composition and / or siRNA complex are essentially clean and / or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.

[0179] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited thereto in any way. EXAMPLES

[0180] Unless otherwise specified, all reagents and media used in the following examples are commercially available products, and all procedures such as nucleic acid electrophoresis and real-time PCR are performed with reference to the methods described in Molecular Cloning (Cold Spring Harbor Borough Press (1989)).

[0181] Unless otherwise stated, all reagent percentages provided below are calculated as volume ratios (v / v). Data analysis was performed using Graphpad prism 8.0 statistical analysis software.

[0182] Unless otherwise specified, all reagents and media used in the following examples are commercially available products, and all operations such as nucleic acid electrophoresis and real-time PCR are performed with reference to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0183] Preparation Examples 1 to 5: Synthesis of siRNAs provided by the present disclosure The siRNA sequences listed in Table 2 were synthesized by solid-phase synthesis, and equimolar complementary sense and antisense strands in Table 2 were dissolved in DEPC water, respectively, and then annealed to obtain siRPTORa2-M1X, siRPTORb2-M1X, siRPTORc2-M1X, siRPTORc1-T2S, and siRPTORc1-M1S provided by the present disclosure. Ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ) was added to the siRNA. * The siRNA was diluted to a concentration of 0.2 g / mL (as siRNA) using a 500 sieve (cm (25°C)) and then the molecular weight was detected using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). The measured value was consistent with the theoretical value, indicating that the synthesized siRNA was the double-stranded nucleic acid sequence of the intended design.

[0184] Comparative Preparation Example 1: Synthesis of reference siRNA The sense and antisense strands were synthesized by solid-phase synthesis, each corresponding to the siRNA with siRNA number NC in Table 2. Equimolar sense and antisense strands were dissolved in DEPC water and then annealed to obtain the reference siRNA, numbered NC. NC is a negative control siRNA that has no sequence homology with RPTOR mRNA.

[0185] Table 2 siRNA sequences [Table 4]

[0186] Here, capital letters C, G, U, and A represent the base sequence of nucleotides, lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, lower case letter s represents that the two nucleotides on the left and right sides of the letter s are linked by a thiophosphate group, capital letter P represents that one nucleotide adjacent to the right side of the letter P is a 5'-phosphate nucleotide, and combination letters moe is the combination character moe indicates that the adjacent nucleotide to the left of is a nucleotide having a 2'-O-methoxyethyl modification.

[0187] After preparation of the siRNAs or reference siRNAs of the present disclosure above was completed, they were lyophilized to a solid powder and ready for use.

[0188] Preparations 6 and 7: Synthesis of Complex 1 and Complex 2 Complex 1 of the present disclosure was obtained by the preparation method of "Complex 1" in Preparation Example 1 of WO 2019 / 105437, except that when single strands of the sense and antisense strands were prepared, one nucleoside monomer was bonded to each of the corresponding positions according to the sequences of the sense and antisense strands of siRPTORc1-T2S in Table 2. The molecular weight was detected by liquid chromatography mass spectrometry (LC-MS). As a result, the theoretical value of the sense strand was 7468.3, the actual value of the sense strand was 7467.2, the theoretical value of the antisense strand was 7107.7, and the actual value of the antisense strand was 7106.7. The actual values ​​were consistent with the theoretical values, and it was determined that the synthesized complex 1 was a double-stranded nucleic acid sequence of the intended design, including the group shown in formula (301). The structure of complex 1 is shown by the following formula (301).

[0189] [ka]

[0190] Nu in formula (301) is siRPTORc1-T2S in Table 1 of the present disclosure, the conjugate group is attached to the 3' position of the ribose group at the 3' end of the sense strand of siRPTORc1-T2S, and the conjugate is in the sodium salt form.

[0191] The complex 2 of the present disclosure was prepared by the above method, but the only difference is that when the single strands of the sense and antisense strands were prepared, one nucleoside monomer was bonded to each of the corresponding positions according to the sequences of the sense and antisense strands of siRPTORc1-T2S in Table 2. The molecular weight was detected by liquid chromatography mass spectrometry (LC-MS). As a result, the theoretical value of the sense strand was 7468.3, the actual value of the sense strand was 7467.2, the theoretical value of the antisense strand was 7063.7, and the actual value of the antisense strand was 7062.7. The actual values ​​were consistent with the theoretical values, and it was determined that the synthesized complex 2 was a double-stranded nucleic acid sequence of the intended design, including the group shown in formula (301). The structure of complex 2 is shown by the following formula (301). Nu in formula (301) is siRPTORc1-M1S in Table 1 of the present disclosure, the conjugate group is attached to the 3' position of the ribose group at the 3' end of the sense strand of siRPTORc1-M1S, and the conjugate is in the sodium salt form.

[0192] Experimental Example 1: In vitro suppression activity of siRNA of the present disclosure In this experimental example, the inhibitory activity of siRPTORa2-M1X, siRPTORb2-M1X, and siRPTORc2-M1X of the present disclosure in HepG2 human liver cancer cells in vitro was examined.

[0193] HepG2 human hepatoma cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, RMBIO) at 37°C in 5% CO. 2 The cells were cultured in an incubator containing 95% air.

[0194] HepG2 cells were cultured at 2.0x10 5The cells were seeded at 1 cell / well into a 12-well plate and cultured in 1 mL of cell solution per well for 24 h. After that, all of the medium in the culture well was aspirated, and 500 μL of Opti-MEM medium (GIBCO) was added to each well.

[0195] Using PBS buffer, siRNA dilution standard solutions with a concentration of 20 μM were prepared from each of siRPTORa2-M1X, siRPTORb2-M1X, and siRPTORc2-M1X prepared in Preparation Examples 1 to 3.

[0196] For each siRNA, a 1A1 solution was prepared, with one part of the 1A1 solution containing 48.5 μL of Opti-MEM medium and 1.5 μL of 20 μM siRNA working solution.

[0197] Prepare 1B solution. One part of 1B solution contains 49 µL of Opti-MEM medium and 1 µL of Lipofectamine. TM Includes 2000 (Invitrogen).

[0198] For each siRNA, 1 part of 1B solution and 1 part of 1A1 solution were incubated at room temperature for 20 min to obtain transfection complexes 1Xa, 1Xb, or 1Xc.

[0199] One part of 1B solution was mixed with 50 μL of Opti-MEM medium and incubated at room temperature for 20 min to obtain transfection complex 1×.

[0200] Transfection complex 1Xa was added to two culture wells (both containing the above-mentioned HepG2 cells and 500 μL of Opti-MEM medium, the same below) at an amount of 100 μL / well, and mixed evenly to obtain a transfection mixture with a final concentration of 50 nM, which was designated as test group 1.

[0201] Transfection complex 1Xb was added to two culture wells (both containing the above-mentioned HepG2 cells and 500 μL of Opti-MEM medium, the same below) at an amount of 100 μL / well, and mixed evenly to obtain a transfection mixture with a final concentration of 50 nM, which was designated as test group 2.

[0202] The transfection complex 1Xc was added to two culture wells (each containing the above-mentioned HepG2 cells and 500 μL of Opti-MEM medium, the same below) in an amount of 100 μL / well, and mixed evenly to obtain a transfection mixture with a final concentration of 50 nM, which was designated as test group 3.

[0203] In another two culture wells, transfection complex 1X was added at an addition volume of 100 μL / well, respectively, and mixed evenly to obtain a transfection mixture without siRNA, which was designated as the blank control group.

[0204] After the above test groups 1 to 3 and the blank control group were cultured in the culture wells for 4 hours, the supernatant in each culture well was aspirated, and 1 mL of Opti-MEM medium was added to each well. The 12-well plate was incubated with CO 2 The plate was placed in an incubator and cultured at 37°C for 24 hours.

[0205] Total RNA was extracted from the cells in each well using Seiko Seibutsu's UNIQ-10 column total RNA extraction kit (purchased from Seiko Seibutsu, No. TC13KA4109) according to the method described in the manufacturer's instructions.

[0206] 1 μg of total RNA was taken from each well of cells and reverse-transcribed using the Goldenstar TM The reagents provided by the RT6 cDNA Synthesis Kit (purchased from Beijing Keike Xinye Biotechnology Co., Ltd.) were used. TM Oligo(dT) 17was selected as a primer, and 20 μL of reverse transcription reaction system was prepared according to the reverse transcription operation steps in the kit's manual, and the total RNA of the cells in each well was reverse transcribed. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the reverse transcription reaction system was incubated at 50°C for 50 min, then incubated at 85°C for 5 min, and finally incubated at 4°C for 30 s. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0207] For each reverse transcription reaction system, 5 μL of the solution containing the above cDNA was used as a template, and 20 μL of qPCR reaction system was prepared using the reagents provided by the NovoStart® SYBR qPCR SuperMix Plus kit (purchased from Kinki Protein Technology Co., Ltd., No. E096-01B). The PCR primer sequences for amplifying the target gene RPTOR and the endogenous reference gene GAPDH are as shown in Table 3, and the final concentration of each primer is 0.25 μM. Each qPCR reaction system was placed in an ABI StepOnePlus Real-Time PCR machine and amplified using a three-step method. The amplification process included pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The above denaturation, annealing, and extension steps were repeated a total of 40 times to obtain product W1, which was an amplified target gene RPTOR and endogenous reference gene GAPDH. The product W1 was immediately incubated at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s, and the melting curves of the target gene and the endogenous reference gene GAPDH in the product W1 were collected using a quantitative real-time PCR device, and the Ct values ​​of the target gene RPTOR and the endogenous reference gene GAPDH were obtained.

[0208] Table 3 Primer information [Table 5]

[0209] The comparative Ct (ΔΔCt) method was used to calculate the relative and quantitative expression of the target gene RPTOR in each test group. The calculation method is as follows:

[0210] ΔCt(test group)=Ct(target gene in test group)-Ct(endogenous reference gene in test group) ΔCt(control group)=Ct(gene of interest in control group)-Ct(endogenous reference gene in control group) ΔΔCt(test group)=ΔCt(test group)-ΔCt(average value of control group) ΔΔCt(control group) = ΔCt(control group) - ΔCt(mean value of the control group) ΔCt(mean value of control group) is the arithmetic mean value of the ΔCt(control group) of each of the two culture wells of the control group, so that each culture well of the test group and the control group corresponds to one ΔΔCt value.

[0211] The expression levels of RPTOR mRNA in the test group were normalized based on the mean value of the control group, and the mean value of the expression level of RPTOR mRNA in the blank control group was defined as 100%.

[0212] Relative expression level of RPTOR mRNA in the test group = 2 -ΔΔCt(試験群) ×100% Inhibition rate of RPTOR mRNA in the test group = (1 - relative expression level of RPTOR mRNA in the test group) x 100%

[0213] The experimental results are shown in Figure 1.

[0214] Comparative Experiment 1: In vitro suppression activity of reference siRNA NC The inhibitory activity of the negative reference siRNA NC in human liver cancer cells in vitro was also examined using the method of Experimental Example 1, except that the reference siRNA NC prepared in the Comparative Preparation Example was used instead of siRPTORa2-M1X, siRPTORb2-M1X, or siRPTORc2-M1X to prepare a 20 μM siRNA dilution standard solution for testing. The results are shown in Figure 1.

[0215] 1 is a histogram showing the relative expression level of RPTOR mRNA in in vitro HepG2 human liver cancer cells after transfection with 50 nM of the siRNA of the present disclosure and reference siRNA NC, in which NC represents reference siRNA NC. As can be seen from the results in FIG. 1, in in vitro HepG2 human liver cancer cells, at a concentration of 50 nM, the RPTOR mRNA suppression rate of siRPTORa2-M1X was 76.8%, at a concentration of 50 nM, the RPTOR mRNA suppression rate of siRPTORb2-M1X was 86.8%, and at a concentration of 50 nM, the RPTOR mRNA suppression rate of siRPTORc2-M1X was 78.8%, showing excellent RPTOR mRNA suppression activity and excellent RPTOR gene expression suppression effect.

[0216] Experimental Example 2: In vitro target mRNA suppression activity of the siRNA of the present disclosure In this experimental example, the RPTOR mRNA suppression activity of siRPTORa2-M1X, siRPTORb2-M1X, and siRPTORc2-M1X of the present disclosure at different concentrations in HepG2 human liver cancer cells in vitro was examined.

[0217] HepG2 human hepatoma cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, RMBIO) at 37°C in 5% CO. 2 The cells were cultured in an incubator containing 95% air.

[0218] HepG2 cells were cultured at 2.0x10 5 The cells were seeded at 1 cell / well into a 12-well plate and cultured in 1 mL of cell solution per well for 24 h. After that, all of the medium in the culture well was aspirated, and 500 μL of Opti-MEM medium (GIBCO) was added to each well.

[0219] For each siRNA, PBS buffer was used to prepare siRNA dilution standard solutions of concentrations of 20 μM, 2 μM, and 0.2 μM from siRPTORa2-M1X, siRPTORb2-M1X, and siRPTORc2-M1X prepared in Preparation Examples 1 to 3, respectively.

[0220] For each siRNA, a 2A1 solution was prepared, with one portion of the 2A1 solution containing 48.5 μL of Opti-MEM medium and 1.5 μL of 20 μM siRNA working solution to obtain siRNA working solutions of 2A1a, 2A1b, or 2A1c, respectively.

[0221] For each siRNA, a 2A2 solution was prepared, with one portion of the 2A2 solution containing 48.5 μL of Opti-MEM medium and 1.5 μL of 2 μM siRNA working solution to obtain siRNA working solutions of 2A2a, 2A2b, or 2A2c, respectively.

[0222] For each siRNA, a 2A3 solution was prepared, with one portion of the 2A3 solution containing 48.5 μL of Opti-MEM medium and 1.5 μL of 0.2 μM siRNA working solution to obtain 2A3a, 2A3b, or 2A3c siRNA working solution, respectively.

[0223] Prepare 2B solution: 1 part 2B solution contains 49 µL of Opti-MEM medium and 1 µL of Lipofectamine TM Includes 2000 (Invitrogen).

[0224] A 2X0 solution was prepared, and one part of the 2B solution was mixed with 50 μL of Opti-MEM medium and incubated at room temperature for 20 min to obtain blank transfection complex 2X0.

[0225] A 2X1 solution was prepared by mixing 1 part 2B solution with 1 part 2A1a solution, 1 part 2A2a solution and 1 part 2A3a solution, respectively, and incubating at room temperature for 20 min to obtain transfection complexes 2Xa1, 2Xa2 and 2Xa3, respectively.

[0226] A 2X2 solution was prepared by mixing 1 part 2B solution with 1 part 2A1b solution, 1 part 2A2b solution and 1 part 2A3b solution respectively and incubating at room temperature for 20 min to obtain transfection complexes 2Xb1, 2Xb2 and 2Xb3, respectively.

[0227] 2X3 solutions were prepared by mixing 1 part 2B solution with 1 part 2A1c solution, 1 part 2A2c solution and 1 part 2A3c solution respectively and incubating at room temperature for 20 min to obtain transfection complexes 2Xc1, 2Xc2 and 2Xc3, respectively.

[0228] For each siRNA, transfection complexes 2Xa1, 2Xa2, or 2Xa3 were added at 100 μL / well into two culture wells (each containing the above-mentioned HepG2 cells and 500 μL of Opti-MEM medium, the same below), and mixed evenly to obtain transfection mixtures containing siRPTORa2-M1X at final concentrations of 50 nM, 5 nM, or 0.5 nM, which were designated as test group 1.

[0229] For each siRNA, transfection complexes 2Xb1, 2Xb2, or 2Xb3 were added to two culture wells (each containing the above-mentioned HepG2 cells and 500 μL of Opti-MEM medium, the same below) at an amount of 100 μL / well, and mixed evenly to obtain transfection mixtures containing siRPTORb2-M1X at final concentrations of 50 nM, 5 nM, or 0.5 nM, which were designated as test group 2.

[0230] For each siRNA, transfection complex 2Xc1, 2Xc2 or 2Xc3 was added to two culture wells (each containing the above-mentioned HepG2 cells and 500 μL of Opti-MEM medium, the same below) at an amount of 100 μL / well, and mixed evenly 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.

[0231] Blank transfection complex 2X0 was added to another two culture wells at an addition volume of 100 μL / well, respectively, and mixed evenly to obtain a transfection mixture without siRNA, which was designated as the blank control group.

[0232] After the above test groups 1 to 3 and the blank control group were cultured in the culture wells for 4 hours, the supernatant in each culture well was aspirated, and 1 mL of Opti-MEM medium was added to each well. The 12-well plate was incubated with CO 2 The plate was placed in an incubator and cultured at 37°C for 24 hours.

[0233] The total RNA in the cells of each well was extracted and reverse transcribed using the same method as in Experimental Example 1, and the target gene RPTOR mRNA in each test group was calculated relatively and quantitatively. The results are shown in Figure 2.

[0234] Figure 2 is a histogram showing the relative expression level of RPTOR mRNA in in vitro HepG2 human liver cancer cells after transfection with different concentrations of siRNA of the present disclosure. As can be seen from the results in Figure 2, in in vitro HepG2 human liver cancer cells, siRPTORc2-M1X showed at least 40.5% RPTOR mRNA suppression rate at a low concentration of 0.5 nM, siRPTORa2-M1X showed at least 68.3% RPTOR mRNA suppression rate at a concentration of 5 nM and at least 71.1% RPTOR mRNA suppression rate at a concentration of 50 nM, and siRPTORb2-M1X showed 86.4% RPTOR mRNA suppression rate at a concentration of 50 nM, showing excellent RPTOR gene expression suppression effect.

[0235] Experimental Example 3: Inhibitory activity of siRNA complex in mice The complexes 1 and 2 prepared in Preparation Examples 6 and 7 were dissolved in PBS to prepare 3 mg / ml siRNA complex solutions (as siRNA). C57BL / 6 mice (female, weighing 16-18 g, 6-8 weeks old, purchased from Sigma Co., Ltd.) were randomly divided into 3 groups, with 6 mice per group, and each group was numbered. The siRNA complex 1 solution was administered to each mouse in the first group by subcutaneous injection at the back of the neck, and the body weight was measured and recorded before administration, and the administration volume was 5 mL / kg according to the body weight, and this was designated as test group 1. The siRNA complex 2 solution was administered to each mouse in the second group, and the body weight was measured and recorded before administration, and the administration volume was 5 mL / kg according to the body weight, and this was designated as test group 2. The remaining mice in the remaining groups were administered PBS at a volume of 5 mL / kg, and used as a blank control group.

[0236] The administration time was set as day 1, and on day 8, liver tissues were taken from each mouse in the test group and blank control group and preserved in RNAlater.

[0237] The total RNA in the cells of each well was extracted and reverse transcribed using the same method as in Experimental Example 1, and the target gene RPTOR in each test group was calculated relatively and quantitatively. The primer information used is shown in Table 3 above, and the results of the blank control group were normalized. The results are shown in Table 4.

[0238] Table 4. In vivo suppression activity of siRNA complexes in mice [Table 6]

[0239] As can be seen from the results, compared with the results of the blank control group, at a concentration of 3 mg / kg, the siRNA complexes with different modification schemes disclosed herein still showed an in vivo RPTOR mRNA suppression rate of more than 50% in C57BL / 6j mice within 1 week after administration, demonstrating excellent RPTOR mRNA suppression activity and long-term in vivo stability.

[0240] As can be seen from the above experimental results, the siRNA disclosed herein can effectively suppress RPTOR gene expression in cells, and therefore shows great potential for application in the preparation of drugs for treating and / or preventing diseases associated with abnormal activation of mTORC1 and cellular autophagy, such as NASH or neurodegenerative diseases, or associated symptoms.

[0241] Although the preferred embodiments of the present disclosure have been described in detail above, the present disclosure is not limited to the specific contents of the above embodiments, and several simple modifications can be made to the technical means of the present disclosure within the technical concept of the present disclosure, and all of these simple modifications fall within the scope of protection of the present disclosure.

[0242] It should be noted that the specific technical features described in the above description of the invention may be combined in any suitable manner if not inconsistent, and in order to avoid unnecessary duplication, this disclosure does not separately describe every possible combination manner.

[0243] Furthermore, various different embodiments of the present disclosure can be arbitrarily combined, and should be considered as being similarly disclosed in the present disclosure, unless they deviate from the spirit of the present disclosure.

Claims

1. An siRNA comprising a sense strand and an antisense strand, wherein nucleotides in the siRNA are each independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, wherein the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse-complementary manner at least in part, and the nucleotide sequence I and the nucleotide sequence II are a pair selected from the sequences shown in i) to iii); i) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 are equal in length and have no more than three nucleotide differences; 5'-CUGCCAUGGAGUAUCUGAZ ａ１ -3' (SEQ ID NO: 1), 5'-Z ａ２ UCAGAUACUCCAUGGCAG-3' (SEQ ID NO: 2) However, Z ａ１ is A and Z ａ２ is U, and in said nucleotide sequence I, position Z ａ１ Nucleotide Z corresponding to ａ３ wherein said nucleotide sequence II comprises a sequence ａ２ Nucleotide Z corresponding to ａ４ and Z ａ４ is the first nucleotide at the 5' end of the antisense strand, or ii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 123 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 124 are equal in length and have no more than three nucleotide differences; 5'-ACAACAUCAAGUACUACGZ ｂ１ -3' (SEQ ID NO: 123), 5'-Z ｂ２ CGUAGUACUUGAUGUUGU-3' (SEQ ID NO: 124) However, Z ｂ１ is A and Z ｂ２ is U, and in said nucleotide sequence I, position Z ｂ１ Nucleotide Z corresponding to ｂ３ wherein said nucleotide sequence II comprises a sequence ｂ２ Nucleotide Z corresponding to ｂ４ and Z ｂ４ is the first nucleotide at the 5' end of the antisense strand, or iii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 245 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 246 are equal in length and have no more than three nucleotide differences; 5'-CGACUACUACAUCUCCGUZ ｃ１ -3' (SEQ ID NO: 245), 5'-Z ｃ２ ACGGAGAUGUAGUAGUCG-3' (SEQ ID NO: 246) However, Z ｃ１ is G and Z ｃ２ is C, and in said nucleotide sequence I, position Z ｃ１ Nucleotide Z corresponding to ｃ３ wherein said nucleotide sequence II comprises a sequence ｃ２ Nucleotide Z corresponding to ｃ４ and Z ｃ４ is the first nucleotide at the 5' end of the antisense strand of the siRNA.

2. the nucleotide sequence I differs from the nucleotide sequence shown in SEQ ID NO: 1 by no more than one nucleotide, and / or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 2 by no more than one nucleotide, Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 123 have no more than one nucleotide difference, and / or the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 124 have no more than one nucleotide difference; Alternatively, the nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO: 245 have no more than one nucleotide difference, and / or the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 246 have no more than one nucleotide difference; and / or The nucleotide differences between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 include a difference at position Z a4 , wherein Z a4 is selected from A, C, or G; Alternatively, the nucleotide difference between said nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 124 comprises a difference at position Z b4 , wherein Z b4 is selected from A, C, or G; Alternatively, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 246 comprises a difference at position Z c4 , wherein Z c4 is selected from A, U, or G. The siRNA of claim 1 .

3. Z ａ３ Is Z ａ４ or Z ｂ３ Is Z ｂ４ or Z ｃ３ Is Z ｃ４ The siRNA of claim 1, wherein the nucleotide is complementary to:

4. the sense strand and the antisense strand have the same or different lengths, the sense strand has a length of 19 to 23 nucleotides, and the antisense strand has a length of 19 to 26 nucleotides, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4, 5'-CUGCCAUGGAGUAUCUGAZ ａ３ -3' (SEQ ID NO: 3), 5'-Z ａ４ UCAGAUACUCCAUGGCAG-3' (SEQ ID NO: 4) However, Z ａ３ is selected from A, U, G or C; Z ａ４ Is Z ａ３ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 125, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 126; 5'-ACAACAUCAAGUACUACGZ ｂ３ -3' (SEQ ID NO: 125), 5'-Z ｂ４ CGUAGUACUUGAUGUUGU-3' (SEQ ID NO: 126) However, Z ｂ３ is selected from A, U, G or C; Z ｂ４ Is Z ｂ３ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 247, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 248; 5'-CGACUACUACAUCUCCGUZ ｃ３ -3' (SEQ ID NO: 247), 5'-Z ｃ４ ACGGAGAUGUAGUAGUCG-3' (SEQ ID NO: 248) However, Z ｃ３ is selected from A, U, G or C; Z ｃ４ Is Z ｃ３ The siRNA of claim 1, wherein the nucleotide is complementary to:

5. the sense strand further comprises a nucleotide sequence III, and the antisense strand further comprises a nucleotide sequence IV, wherein the nucleotide sequence III and the nucleotide sequence IV are independently 1 to 4 nucleotides in length, the nucleotide sequence III is linked to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, wherein the term "substantially reverse complementary" refers to the presence of one or less base mismatches between the two nucleotide sequences, and the term "completely reverse complementary" refers to the absence of mismatches between the two nucleotide sequences; Or, the nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO: 1 are equal in length and differ by no more than three nucleotides; the nucleotide sequences III and IV are both one nucleotide in length and the base of nucleotide sequence III is A; or the nucleotide sequences III and IV are both two nucleotides in length and the base sequence of nucleotide sequence III is CA from the 5' end to the 3' end; or the nucleotide sequences III and IV are both three nucleotides in length and the base sequence of nucleotide sequence III is UCA from the 5' end to the 3' end; or the nucleotide sequences III and IV are both four nucleotides in length and the base sequence of nucleotide sequence III is GUCA from the 5' end to the 3' end; Alternatively, the nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO: 123 are equal in length and differ by no more than three nucleotides; the nucleotide sequences III and IV are each one nucleotide in length, and the base of nucleotide sequence III is A; or the nucleotide sequences III and IV are each two nucleotides in length, and the base sequence of nucleotide sequence III is CA from the 5' end to the 3' end; or the nucleotide sequences III and IV are each three nucleotides in length, and the base sequence of nucleotide sequence III is UCA from the 5' end to the 3' end; or the nucleotide sequences III and IV are each four nucleotides in length, and the base sequence of nucleotide sequence III is AUCA from the 5' end to the 3' end; Alternatively, the nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO: 245 are the same length and differ by no more than three nucleotides; the nucleotide sequences III and IV are each one nucleotide long, and the base of nucleotide sequence III is A; or the nucleotide sequences III and IV are each two nucleotides long, and the base sequence of nucleotide sequence III is AA from the 5' end to the 3' end; or the nucleotide sequences III and IV are each three nucleotides long, and the base sequence of nucleotide sequence III is CAA from the 5' end to the 3' end; or the nucleotide sequences III and IV are each four nucleotides long, and the base sequence of nucleotide sequence III is GCAA from the 5' end to the 3' end. The siRNA of claim 1.

6. the antisense strand further comprises a nucleotide sequence V, which is 1 to 3 nucleotides in length and is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand; and / or the sense strand further comprises a nucleotide sequence VI, which is 1 to 3 nucleotides in length and is attached to the 3' end of the sense strand to form a 3' overhang of the sense strand; Or, the nucleotide sequence V and / or the nucleotide sequence VI is 2 nucleotides in length; Or, The nucleotide sequence V and / or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides, or the nucleotide sequence V is complementary to a nucleotide at a corresponding position in a target mRNA, and / or the nucleotide sequence VI is the same as a nucleotide at a corresponding position in a target mRNA. The siRNA of claim 1 .

7. the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 6; 5'-CUGCCAUGGAGUAUCUGAZ ａ３ -3' (SEQ ID NO: 5), 5'-Z ａ４ UCAGAUACUCCAUGGCAGUG-3' (SEQ ID NO: 6) However, the above Z ａ４ is the first nucleotide at the 5' end of the antisense strand, and Z ａ３ is selected from A, U, G or C; Z ａ４ Is Z ａ３ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 8; 5'-CACUGCCAUGGAGUAUCUGAZ ａ３ -3' (SEQ ID NO: 7), 5'-Z ａ４ UCAGAUACUCCAUGGCAGUGAC-3' (SEQ ID NO: 8) However, the above Z ａ４ is the first nucleotide at the 5' end of the antisense strand, and Z ａ３ is selected from A, U, G or C; Z ａ４ Is Z ａ３ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 127, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 128; 5'-ACAACAUCAAGUACUACGZ ｂ３ -3' (SEQ ID NO: 127), 5'-Z ｂ４ CGUAGUACUUGAUGUUGUUG-3' (SEQ ID NO: 128) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 129, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 130; 5'-CAACAACAUCAAGUACUACGZ ｂ３ -3' (SEQ ID NO: 129), 5'-Z ｂ４ CGUAGUACUUGAUGUUGUUGAU-3' (SEQ ID NO: 130) However, the above Z ｂ４ is the first nucleotide at the 5' end of the antisense strand, and Z ｂ３ is selected from A, U, G or C; Z ｂ４ Is Z ｂ３ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 249, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 250; 5'-CGACUACUACAUCUCCGUZ ｃ３ -3' (SEQ ID NO: 249), 5'-Z ｃ４ ACGGAGAUGUAGUAGUCGUU-3' (SEQ ID NO: 250) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 251, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 252; 5'-AACGACUACUACAUCUCCGUZ ｃ３ -3' (SEQ ID NO: 251), 5'-Z ｃ４ ACGGAGAUGUAGUAGUCGUUGC-3 '(SEQ ID NO: 252) However, the above Z ｃ４ is the first nucleotide at the 5' end of the antisense strand, and Z ｃ３ is selected from A, U, G or C; Z ｃ４ Is Z ｃ３ The siRNA of claim 1, wherein the nucleotide is complementary to:

8. The siRNA of claim 1, wherein the siRNA is one of siRPTORa1, siRPTORa2, siRPTORa3, siRPTORb1, siRPTORb2, siRPTORb3, siRPTORc1, siRPTORc2 and siRPTORc3.

9. The siRNA of claim 1, wherein at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide and / or at least one phosphate group is a phosphate group having a modified group.

10. each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide; Or, the fluoro-modified nucleotides are located at nucleotide sequence I and nucleotide sequence II, and from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of nucleotide sequence I are fluoro-modified nucleotides, and from the 5' end to the 3' end, at least the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence II are fluoro-modified nucleotides; Or, the non-fluoro-modified nucleotide is a 2'-O-methoxy-modified or a 2'-O-methoxyethyl-modified nucleotide; Or, at least one of the nucleotides at positions 3 to 6 of nucleotide sequence II from the 5' to 3' end is a 2'-O-methoxyethyl modified nucleotide; Or, the nucleotide at position 3 or 5 of said nucleotide sequence II is a 2'-O-methoxyethyl modified nucleotide; Or, no more than two of the nucleotides at positions 3 to 9 of nucleotide sequence II from the 5' to 3' end are 2'-O-methoxyethyl modified nucleotides; Or, other nucleotides in the sense strand and the antisense strand are 2'-O-methoxy modified nucleotides; Or, The siRNAs include siRPTORa1-M1, siRPTORa1-M2, siRPTORa1-M3, siRPTORa2-M1, siRPTORa2-M2, siRPTORa2-M3, siRPTO Ra3-M1, siRPTORa3-M2, siSRPTORa3-M3, siRPTORb1-M1, siRPTORb1-M2, siRPTORb1-M3, siRPTORb2-M1, siRPTOR b2-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 and siSRPTORc3-M3; Or, the phosphate ester group having a modifying group is a thiophosphate ester group in which at least one oxygen atom of a phosphodiester bond in the phosphate ester group is substituted with a sulfur atom, Or, The siRNAs include 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, and 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, siRPTOR b2-M3S, siRPTORb2-M3X, siRPTORb3-M1S, siRPTORb3-M1X, siRPTORb3-M2S, siRPTORb3-M2X, siRPTORb3-M3S, siRPTORb3-M3X, siRP TORb1-T1S, siRPTORb1-T2S, siRPTORc1-M1S, siRPTORc1-M1X, siRPTORc1-M2S, siRPTORc1-M2X, siRPTORc1-M3S, siRPTORc1-M3X, s 2. The siRNA of claim 1, which is one of iRPTORc2-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, and siRPTORc1-T2S.

11. the 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide; Or, The siRNAs include 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, siRPTORa1-M3SP1, siRPTORa1-M1SP1, siRPTORa1-M2SP1, siRPTORa1-M3 ... a3-M1SP1, siRPTORa3-M2SP1, siRPTORa3-M3SP1, siRPTORa1-M1XP1, siRPT ORa1-M2XP1, siRPTORa1-M3XP1, siRPTORa2-M1XP1, siRPTORa2-M2XP1, siR PTORa2-M3XP1, siRPTORa3-M1XP1, siRPTORa3-M2XP1, siRPTORa3-M3XP1, s iRPTORb1-M1P1, siRPTORb1-M2P1, siRPTORb1-M3P1, siRPTORb2-M1P1, siR PTORb2-M2P1, siRPTORb2-M3P1, siRPTORb3-M1P1, siRPTORb3-M2P1, siRP TORb3-M3P1, siRPTORb1-M1SP1, siRPTORb1-M2SP1, siRPTORb1-M3SP1, siR PTORb2-M1SP1, siRPTORb2-M2SP1, siRPTORb2-M3SP1, siRPTORb3-M1SP1, s iRPTORb3-M2SP1, siRPTORb3-M3SP1, siRPTORb1-M1XP1, siRPTORb1-M2XP1 , siRPTORb1-M3XP1, siRPTORb2-M1XP1, siRPTORb2-M2XP1, siRPTORb2-M3X P1, siRPTORb3-M1XP1, siRPTORb3-M2XP1, siRPTORb3-M3XP1, siRPTORc1-M 1P1, siRPTORc1-M2P1, siRPTORc1-M3P1, siRPTORc2-M1P1, siRPTORc2-M2P 1, siRPTORc2-M3P1, siRPTORc3-M1P1, siRPTORc3-M2P1, siRPTORc3-M3P1,siRPTORc1-M1SP1, siRPTORc1-M2SP1, siRPTORc1-M3SP1, siRPTORc2-M1SP1, siRPTORc2-M2SP 1, siRPTORc2-M3SP1, siRPTORc3-M1SP1, siRPTORc3-M2SP1, siRPTORc3-M3SP1, siRPTORc1-M1 2. The siRNA of claim 1, which is one of siRPTORc1-M2XP1, siRPTORc1-M3XP1, siRPTORc2-M1XP1, siRPTORc2-M2XP1, siRPTORc2-M3XP1, siRPTORc3-M1XP1, siRPTORc3-M2XP1 and siRPTORc3-M3XP1.

12. A composition comprising the siRNA of any one of claims 1 to 11 and a pharmaceutically acceptable carrier, the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1:(1-500); Or, A pharmaceutical composition, wherein the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1:(1-50).

13. 12. An siRNA complex comprising the siRNA according to any one of claims 1 to 11 and a conjugated group conjugated and bound to the siRNA, wherein the conjugated group comprises at least one pharmaceutically acceptable targeting group and / or delivery-assisting group, preferably the conjugated group further comprises a linker, and the linker and / or the targeting group or the delivery-assisting group are bound in that order, preferably each targeting group is selected from a ligand capable of binding to a cell surface receptor, and / or each delivery-assisting group is selected from a group capable of improving the biocompatibility of the siRNA complex in a target organ or tissue.

14. The siRNA conjugate of claim 13, wherein the siRNA conjugate has a structure shown in formula (301), wherein Nu has a sequence corresponding to one of siRPTORa2-M1S, siRPTORb2-M1S, siRPTORc2-M1S, siRPTORc1-T1S, and siRPTORc1-T2S, the conjugated group is attached to the 3' position of the ribose group of the 3'-terminal nucleotide of the siRNA sense strand in Nu, and the siRNA conjugate is in the form of a sodium salt.

15. A composition for treating and / or preventing a disease or symptom associated with modulation of RPTOR function, comprising: The siRNA according to any one of claims 1 to 11, and / or the pharmaceutical composition according to claim 12, and / or the siRNA complex according to claim 13 or 14, Or, The disease or symptom associated with modulation of RPTOR function is a disease caused by activation of mTORC1 and a disease associated with abnormality in cellular autophagy function, Or, The disease or symptom associated with modulation of RPTOR function is a neurodegenerative disease or non-alcoholic steatohepatitis; Or, The neurodegenerative disease is one or more of Alzheimer's disease, amyotrophic lateral sclerosis, frontotemporal dementia, dementia with Lewy bodies, Huntington's disease, Tourette's syndrome, and Parkinson's disease, preferably, the neurodegenerative disease is Alzheimer's disease.

16. A composition for suppressing RPTOR gene expression in a cell, comprising an siRNA described in any one of claims 1 to 11, and / or a pharmaceutical composition described in claim 12, and / or an siRNA complex described in claim 13 or 14.

Images