Reagent and method for silencing HIF2A

Abstract

The invention belongs to the field of nucleic acid reagents, and particularly relates to a reagent and a method for silencing HIF2A. The dsRNA reagent provided by the invention can efficiently inhibit the expression of the HIF2A, so that the dsRNA reagent can be used for treating or preventing diseases related to the HIF2A, such as cancers, cardiovascular diseases and pulmonary arterial hypertension.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid reagents, specifically relating to reagents and methods for silencing HIF2A. Background Technology

[0002] HIF2A, short for Hypoxia Inducible Factor 2Alpha, is a transcription factor that can be induced to express under hypoxic conditions. HIF2A plays a crucial role in the adaptive response of cells to hypoxia, and it participates in regulating the expression of various genes involved in angiogenesis, cell metabolism, cell proliferation, migration, and invasion.

[0003] HIF2A is a member of the HIF family, which also includes HIF-1α and HIF-3α. Under normoxic conditions, the HIF-α subunit is hydroxylated by proline hydroxylase (PHD) and subsequently binds to Von Hippel-Lindau protein (VHL), leading to rapid degradation via the ubiquitin-proteasome pathway. Under hypoxic or iron-deficient conditions, hydroxylation is inhibited, allowing the HIF-α subunit to stabilize and accumulate within the cell. It then translocates to the nucleus and binds to the HIF-β subunit, forming a HIF heterodimer. This heterodimer binds to the hypoxia response element (HRE), activating the transcription of downstream target genes.

[0004] HIF2A has a wide range of functions. It not only participates in the regulation of iron metabolism, influencing iron absorption and transport, but is also closely related to the development of various diseases, including cancer, cardiovascular disease, and pulmonary hypertension. For example, in cancer development, HIF2A can promote tumor angiogenesis and support tumor cell metabolism and proliferation. In pulmonary hypertension, increased HIF2A activity is associated with disease progression, thus making it a potential therapeutic target. Currently, the function and mechanism of action of HIF2A are still under investigation. With further research, more details about HIF2A and its potential therapeutic applications may be discovered in the future. Summary of the Invention

[0005] The present invention first provides a double-stranded ribonucleic acid (dsRNA) reagent, wherein the dsRNA reagent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising at least 15 consecutive nucleotides having 0, 1, 2 or 3 mismatches with any of the sequences shown in SEQ ID No. 51 to 100, and the sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.

[0006] The present invention also provides a cell containing the dsRNA reagent as described above.

[0007] The present invention also provides a pharmaceutical composition comprising the dsRNA reagent as described above and a pharmaceutically acceptable carrier.

[0008] The present invention also provides a method for inhibiting the expression of HIF2A in cells, comprising: contacting the cells with the dsRNA reagent as described above or the pharmaceutical composition as described above, to inhibit the expression of HIF2A in the cells.

[0009] The present invention also provides the use of the dsRNA reagent or the pharmaceutical composition thereof in any of the following aspects: 1) treating and / or preventing HIF2A-related diseases; 2) preparing a medicament for treating and / or preventing HIF2A-related diseases.

[0010] The dsRNA reagent provided by this invention can efficiently inhibit the expression of HIF2A, and therefore can be used to treat or prevent HIF2A-related diseases, such as cancer, cardiovascular disease and pulmonary hypertension. Attached Figure Description

[0011] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0012] Figure 1 IC50 detection of candidate sequences in human renal clear cell adenocarcinoma 786-O cells in this embodiment of the invention - 1.

[0013] Figure 2 IC50 detection of candidate sequences in human renal clear cell adenocarcinoma 786-O cells in this embodiment of the invention - 2.

[0014] Figure 3 The IC50 detection results of the 5'-(E)-VP modified sequence in human hepatocellular carcinoma HepG2 cells in the embodiments of the present invention are shown.

[0015] Figure 4 This study aims to detect the off-target effects of the candidate sequence DN01087b in human clear cell adenocarcinoma 786-O cells in this embodiment of the invention.

[0016] Figure 5 This invention relates to the detection of off-target effects of the candidate sequence DN01090a in human clear cell adenocarcinoma 786-O cells.

[0017] Figure 6This invention illustrates the inhibitory effect of candidate sequences on different renal cell carcinoma cells in various embodiments.

[0018] In each of the attached figures, the two numbers ending with the siRNA number and the letters indicating the single-stranded DNA molecular linker are used to refer to the results of different siRNAs; as an example, "DN01071a" is referred to as "71a" in the attached figure. Detailed Implementation

[0019] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0020] Terminology Explanation

[0021] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0022] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0023] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0024] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0025] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0026] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0027] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0028] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0029] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0030] In this invention, the terms "double-stranded RNA," "dsRNA," or "siRNA" refer to iRNA comprising an RNA molecule or molecular complex having a hybrid double-stranded region comprising two antiparallel and substantially complementary nucleic acid strands, which will be referred to as having a "sense" and "antisense" orientation relative to the target RNA. The double-stranded region can have any length required to allow specific degradation of the target RNA, for example, via a RISC pathway, but typically the length will range from 9 to 36 base pairs, for example, 15 to 30 base pairs. Considering double-stranded regions between 9 and 36 base pairs, the double-stranded region can have any length within this range, for example 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 base pairs. 1, 33, 34, 35, or 36 base pairs and any subrange thereof, including but not limited to 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 1 to 30 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 dsRNA lengths range from 19 to 22 base pairs. The lengths of dsRNA produced in cells by treatment with Dicer and similar enzymes are typically in the range of 19 to 22 base pairs. One strand of the dsDNA duplex region includes a sequence substantially complementary to the region of the target RNA. The two strands forming the duplex structure can originate from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. When the double-stranded region is formed by the two strands of a single molecule, the molecule may have a double-stranded region separated by a single-stranded nucleotide chain (referred to herein as a “hairpin loop”) between the 3’ end of one strand and the 5’ end of the corresponding other strand forming the double-stranded structure.A hairpin loop may include at least one unpaired nucleotide; in some embodiments, a hairpin loop may include at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides. In cases where the two substantially complementary strands of the dsRNA comprise separate RNA molecules, those molecules do not necessarily but may be covalently linked. In some embodiments, when the two strands are covalently linked by means other than a hairpin loop, and the linking structure is a connector.

[0031] In this invention, when referring to the expression of a given gene, the terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” mean, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from mRNA in a cell, cell population, tissue, organ, or subject in which the gene is transcribed, when the cell, cell population, tissue, organ, or subject is treated with the nucleic acid described herein, the expression of said gene is reduced compared to a second cell, cell population, tissue, organ, or subject who has not been treated in this way.

[0032] In this invention, "completely complementary" means that in a hybridization pair of nucleobase or nucleotide sequences, all (100%) bases in the adjacent sequence of the first oligonucleotide hybridize with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0033] In this invention, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all of the bases in the adjacent sequence of the first oligonucleotide hybridizes with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0034] In this invention, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all of the bases in the adjacent sequence of the first oligonucleotide hybridizes with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0035] In this invention, the term "at least partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, the first oligonucleotide and the second oligonucleotide are partially complementary, substantially complementary, or completely complementary.

[0036] In this invention, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of iRNA (e.g., dsRNA). For example, a nucleotide overhang exists when the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa. dsRNA may include an overhang of at least one nucleotide; alternatively, the overhang may include at least two, three, four, or five or more nucleotides. The nucleotide overhang may include or consist of nucleotide / nucleoside analogs comprising deoxynucleotides / nucleosides. The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleotide overhang may be present at the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0037] In this invention, the term "treatment" refers to a method or procedure taken to provide relief or reduction of the number, severity, and / or frequency of one or more disease symptoms in a subject. The treatment may include prevention, management, preventative treatment, and / or suppression or reduction of the number, severity, and / or frequency of one or more disease symptoms in a subject.

[0038] In this invention, the term "link" means the combination of two compounds or molecules through a covalent bond. Unless otherwise stated, as used herein, the term "link" may refer to a link between a first compound and a second compound, with or without any intermediate atoms or groups of atoms.

[0039] In this invention, the pharmaceutical composition comprises a pharmacologically effective amount of a therapeutic agent (e.g., iRNA) and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeuticly effective amount,” or simply “effective amount” means the amount of a pharmaceutical agent (e.g., iRNA) that effectively produces the desired pharmacological, therapeutic, or preventative outcome. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 10%, then a therapeutically effective amount of a drug used to treat that disease or condition is the amount necessary to reduce that parameter by at least 10%. For example, a therapeutically effective amount of HIF2A-targeting iRNA can reduce the level of HIF2A mRNA or the level of HIF2A protein by any measurable amount, such as at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0040] In this invention, when referring to the sense strand or antisense strand, it means the two strands that make up a double-stranded repressive ribonucleic acid (RNA) molecule, and does not include the single-stranded deoxyribonucleic acid (DNA) molecules involved in some sequences.

[0041] dsRNA reagent

[0042] This invention relates to a double-stranded ribonucleic acid (dsRNA) reagent, wherein the dsRNA reagent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with any of the sequences shown in SEQ ID Nos. 51 to 100, and the sense strand comprising a nucleotide sequence at least partially complementary to the antisense strand. This invention has found that when the antisense strand has the above-described sequence, the dsRNA reagent exhibits a significantly better inhibitory effect on HIF2A.

[0043] In some embodiments, the antisense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

[0044] In some embodiments, the positive strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

[0045] In this invention, the justice chain and the antisense chain can have the same length or different lengths.

[0046] All nucleotide groups in the above dsRNA reagent can be unmodified or contain at least one modified nucleotide group, and the modification can be on nucleotides at any position.

[0047] In some implementations, the justice chain and the antisense chain may be partially complementary, substantially complementary, or completely complementary to each other.

[0048] In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides having 0, 1, 2, or 3 mismatches with any of the sequences shown in SEQ ID No. 64, 67, 69, 72, 74, 84, or 92.

[0049] In some embodiments, the positive strand comprises at least 15 consecutive nucleotides having 0, 1, 2 or 3 mismatches with the nucleotides at positions 1 to 19 of any of the sequences shown in SEQ ID No. 1 to 50.

[0050] In some embodiments, the positive chain comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the nucleotides 1 to 19 of any of the sequences shown in SEQ ID No. 14, 17, 19, 22, 24, 34, 42.

[0051] In practical implementation, those skilled in the art can combine the sequences provided in this invention by taking into account the complementarity of the sense and antisense strands, thereby obtaining the combined dsRNA reagent (siRNA).

[0052] In a preferred embodiment of the present invention, as shown in Table 1, the dsRNA reagent is selected from at least one of the following: siRNA-1, whose sense strand sequence is nucleotides 1-19 of SEQ ID No. 1 and antisense strand sequence is SEQ ID No. 51; siRNA-2, whose sense strand sequence is nucleotides 1-19 of SEQ ID No. 2 and antisense strand sequence is SEQ ID No. 52; siRNA-3, whose sense strand sequence is nucleotides 1-19 of SEQ ID No. 3 and antisense strand sequence is SEQ ID No. 53; siRNA-4, whose sense strand sequence is nucleotides 1-19 of SEQ ID No. 4 and antisense strand sequence is SEQ ID No. 54; siRNA-5, ..., siRNA-48, siRNA-49, and siRNA-50, whose sense strand sequence is nucleotides 1-19 of SEQ ID No. 5 and antisense strand sequence is SEQ ID No. 55.

[0053] In some specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sequence shown in SEQ ID No. 64, and the sense strand comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the nucleotides at positions 1 to 19 of the sequence shown in SEQ ID No. 14. In some preferred embodiments, the antisense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the sequence shown in SEQ ID No. 64, and the sense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the nucleotides at positions 1 to 19 of the sequence shown in SEQ ID No. 14.

[0054] In some specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sequence shown in SEQ ID No. 67, and the sense strand comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the nucleotides from positions 1 to 19 of the sequence shown in SEQ ID No. 17. In some preferred embodiments, the antisense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the sequence shown in SEQ ID No. 67, and the sense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the nucleotides from positions 1 to 19 of the sequence shown in SEQ ID No. 17.

[0055] In some specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sequence shown in SEQ ID No. 69, and the sense strand comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the nucleotides at positions 1 to 19 of the sequence shown in SEQ ID No. 19. In some preferred embodiments, the antisense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the sequence shown in SEQ ID No. 69, and the sense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the nucleotides at positions 1 to 19 of the sequence shown in SEQ ID No. 19.

[0056] In some specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sequence shown in SEQ ID No. 72, and the sense strand comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the nucleotides from positions 1 to 19 of the sequence shown in SEQ ID No. 22. In some preferred embodiments, the antisense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the sequence shown in SEQ ID No. 72, and the sense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the nucleotides from positions 1 to 19 of the sequence shown in SEQ ID No. 22.

[0057] In some specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sequence shown in SEQ ID No. 74, and the sense strand comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the nucleotides from positions 1 to 19 of the sequence shown in SEQ ID No. 24. In some preferred embodiments, the antisense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the sequence shown in SEQ ID No. 74, and the sense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the nucleotides from positions 1 to 19 of the sequence shown in SEQ ID No. 24.

[0058] In some specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sequence shown in SEQ ID No. 84, and the sense strand comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the nucleotides at positions 1 to 19 of the sequence shown in SEQ ID No. 34. In some preferred embodiments, the antisense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the sequence shown in SEQ ID No. 84, and the sense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the nucleotides at positions 1 to 19 of the sequence shown in SEQ ID No. 34.

[0059] In some specific embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the sequence shown in SEQ ID No. 92, and the sense strand comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with the nucleotides at positions 1 to 19 of the sequence shown in SEQ ID No. 42. In some preferred embodiments, the antisense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the sequence shown in SEQ ID No. 92, and the sense strand comprises 0, 1, 2, or 3 nucleotides having mismatches with the nucleotides at positions 1 to 19 of the sequence shown in SEQ ID No. 42.

[0060] In some specific implementations, the sequences of the justice chain and the antisense chain are shown in Table 1 below:

[0061] Table 1

[0062]

[0063]

[0064]

[0065] Note: Column 2 indicates the position of the first base of the target gene in the human HIF2A mRNA sequence. In this article, the reference sequence of the target gene is the coding sequence of human HIF2A mRNA, NM_001430.5.

[0066] In some specific embodiments, when the sequence identity of the sense or antisense strand of the nucleic acid with the corresponding sequence mentioned in this invention is less than 100% or differs by more than one nucleotide, it still has an inhibitory effect on HIF2A that is similar to (e.g., still has an efficacy equivalent to 80-120%, 85-115%, or 90-110% of the corresponding sequence) or equivalent to (e.g., still has an efficacy equivalent to 95-105% of the corresponding sequence). Such nucleic acid sequences are also within the protection scope of this invention.

[0067] The advantages of the technical solutions regarding bare sequences (i.e., unmodified sequences) mentioned in this invention do not depend on the choice of modification method or target vector. The following details the applicable modification schemes and further preferred modification schemes:

[0068] According to the nucleic acid of the present invention, the nucleic acid contains a nucleotide group as a basic structural unit, the nucleotide group containing a phosphate group, a ribose group and a base, and preferably, the nucleic acid contains at least one modified nucleotide group. The modified nucleic acid has an inhibition efficiency of no less than 50% against HIF2A (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0069] According to the nucleic acid of the present invention, the modified nucleotide group is a nucleotide group whose phosphate group and / or ribose group are modified. The modified site can be at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 of the sense and / or antisense strands. At least positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 of the nucleotides at positions 44 and 45.

[0070] In some embodiments, the dsRNA reagent comprises at least one modified nucleotide.

[0071] In some embodiments, the modified nucleotide is selected from one or more of the following: deoxynucleotides, 5'-terminal vinyl phosphate (5'-(E)-VP) modified nucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides. Modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholinonucleotides, aminophosphates, non-natural bases including nucleotides, tetrahydropyran modified nucleotides, 1,5-dehydrohexyl modified nucleotides, cyclohexenyl modified nucleotides, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimics, ethylene glycol modified nucleotides, and 2-O-(N-methylacetamide) modified nucleotides.

[0072] In some preferred embodiments, the modified nucleotide is one or more of the following: 5'-terminal vinyl phosphate modified nucleotide, 2'-O-methyl modified nucleotide, and 2'-fluorine modified nucleotide;

[0073] In some embodiments, the nucleotides of the sense strand and the antisense strand each include 3 to 5 2'-fluorine modified nucleotides, and the other nucleotides are 2'-O-methyl modified nucleotides.

[0074] In some embodiments, the antisense strand has 2'-fluoronucleotides at positions 3 to 5 (e.g., 3, 4, or 5) of the nucleotides at positions 2, 6, 8, 14, and 16 counting from the 5' end, and the other nucleotides are 2'-O-methyl modified nucleotides.

[0075] In some embodiments, the positive strand has 3 to 4 (e.g., 3 or 4) nucleotides at positions 5, 7, 8 and 9 counting from the 5' end as 2'-fluoronucleotides, and the other nucleotides are 2'-O-methyl modified nucleotides.

[0076] The present invention has found that, after the above modifications, the off-target effect can be further reduced.

[0077] In some preferred embodiments, the antisense strand has 2'-fluoronucleotides at positions 2, 6, 8, 14, and 16 counting from the 5' end, and the remaining nucleotides are 2'-O-methyl modified nucleotides; and the sense strand has 2'-fluoronucleotides at positions 5, 7, 8, and 9 counting from the 5' end, and the remaining nucleotides are 2'-O-methyl modified nucleotides. This modification scheme provides superior modification effects in the dsRNA reagent of the present invention.

[0078] In some embodiments, the 5' end of the antisense strand is a vinyl phosphate-modified nucleotide.

[0079] In some embodiments, the last 2 to 4 (e.g., 2, 3, or 4) nucleotides at the 5' and 3' ends of the antisense strand contain phosphate thioester nucleotide bonds, and the last 2 to 4 (e.g., 2, 3, or 4) nucleotides at the 5' end of the sense strand contain phosphate thioester nucleotide bonds.

[0080] In some specific embodiments, the last three nucleotides at the 5' and 3' ends of the antisense strand contain two phosphate thioester nucleotide bonds, and the last three nucleotides at the 5' end of the sense strand contain two phosphate thioester nucleotide bonds.

[0081] In some embodiments, the dsRNA reagent further includes a single-stranded deoxyribonucleic acid molecule; the 5' end of the single-stranded deoxyribonucleic acid molecule is covalently linked to the 3' end of the sense strand, or the 5' end of the single-stranded deoxyribonucleic acid molecule is covalently linked to the 3' end of the antisense strand.

[0082] In some specific embodiments, the single-stranded deoxyribonucleic acid molecule is covalently linked to the sense or antisense strand via disulfide bonds.

[0083] In some specific embodiments, the single-stranded deoxyribonucleic acid molecule includes nucleotides having 0, 1, 2, or 3 mismatches with the sequence shown in TTTTACGTGAGCACGGAAC (SEQ ID No. 101).

[0084] It should be specifically noted that, in this invention, the dsRNA reagent linked to the single-stranded deoxyribonucleic acid molecule is only one embodiment of the invention and does not constitute a limitation on the dsRNA reagent used in this invention. Related research data of this invention show that, even without the presence of this single-stranded deoxyribonucleic acid molecule, the dsRNA reagent described in this invention still exhibits superior inhibitory effects against HIF2A.

[0085] In some embodiments, the antisense strand has an antisense strand sequence as shown in any of Tables 4-5, having at least 15 consecutive nucleotides with 0, 1, 2, or 3 mismatches, and the sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand.

[0086] In some embodiments, the positive strand has a positive strand sequence as shown in any of Tables 4-5, having at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches.

[0087] In some embodiments, the antisense strand sequence has at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the antisense strand sequence shown in DN01084, and the sense strand sequence has at least 15, 16, 17, 18, or 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense strand sequence shown in DN01084.

[0088] In some specific embodiments, the antisense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the antisense strand sequence shown in DN01084, and the sense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the sense strand sequence shown in DN01084.

[0089] In some embodiments, the antisense strand sequence has at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the antisense strand sequence shown in DN01086, and the sense strand sequence has at least 15, 16, 17, 18, or 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense strand sequence shown in DN01086.

[0090] In some specific embodiments, the antisense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the antisense strand sequence shown in DN01086, and the sense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the sense strand sequence shown in DN01086.

[0091] In some embodiments, the antisense strand sequence has at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the antisense strand sequence shown in DN01087, and the sense strand sequence has at least 15, 16, 17, 18, or 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense strand sequence shown in DN01087.

[0092] In some specific embodiments, the antisense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the antisense strand sequence shown in DN01087, and the sense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the sense strand sequence shown in DN01087.

[0093] In some embodiments, the antisense strand sequence has at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the antisense strand sequence shown in DN01088, and the sense strand sequence has at least 15, 16, 17, 18, or 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense strand sequence shown in DN01088.

[0094] In some specific embodiments, the antisense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the antisense strand sequence shown in DN01088, and the sense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the sense strand sequence shown in DN01088.

[0095] In some embodiments, the antisense strand sequence has at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the antisense strand sequence shown in DN01089, and the sense strand sequence has at least 15, 16, 17, 18, or 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense strand sequence shown in DN01089.

[0096] In some specific embodiments, the antisense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the antisense strand sequence shown in DN01089, and the sense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the sense strand sequence shown in DN01089.

[0097] In some embodiments, the antisense strand sequence has at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the antisense strand sequence shown in DN01090, and the sense strand sequence has at least 15, 16, 17, 18, or 19 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense strand sequence shown in DN01090.

[0098] In some specific embodiments, the antisense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the antisense strand sequence shown in DN01090, and the sense strand sequence has 0, 1, 2 or 3 mismatched nucleotides with the sense strand sequence shown in DN01090.

[0099] In some specific embodiments, the 5' end of the antisense strand may be a vinyl phosphate-modified nucleotide, based on any of the above specific sequences. For example, in some specific embodiments, the antisense strand sequence has 0, 1, 2, or 3 mismatched nucleotides with the antisense strand sequence shown in DN01098, and the sense strand sequence has 0, 1, 2, or 3 mismatched nucleotides with the sense strand sequence shown in DN01098. In some specific embodiments, the antisense strand sequence has 0, 1, 2, or 3 mismatched nucleotides with the antisense strand sequence shown in DN01099, and the sense strand sequence has 0, 1, 2, or 3 mismatched nucleotides with the sense strand sequence shown in DN01099. In some specific embodiments, the antisense strand sequence has 0, 1, 2, or 3 mismatched nucleotides with the antisense strand sequence shown in DN01100, and the sense strand sequence has 0, 1, 2, or 3 mismatched nucleotides with the sense strand sequence shown in DN01100.

[0100] In some specific embodiments, based on any of the above specific sequences, the 5' end of a single-stranded deoxyribonucleic acid molecule can be covalently linked to the 3' end of the sense strand or the 3' end of the antisense strand via a disulfide bond, and the single-stranded deoxyribonucleic acid molecule includes nucleotides having 0, 1, 2 or 3 mismatches with the sequence shown in SEQ ID No. 101.

[0101] In some embodiments, the length of the double-stranded region is 15 to 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotide pairs.

[0102] In some embodiments, the length of the double-stranded region is 17 to 23 nucleotides (e.g., about 17, 18, 19, 20, 21, 22 or 23).

[0103] In some embodiments, the length of the double-stranded region is 19 to 30 nucleotides (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30).

[0104] In some embodiments, at least one of the sense strand and the antisense strand includes a 3' overhang of at least two nucleotides.

[0105] In some specific embodiments, at least one of the sense strand and the antisense strand includes a 3' overhang of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0106] In some embodiments, at least one of the sense and antisense strands is attached to one or more ligands selected from the following: lectins, glycoproteins, lipids, aptamers, polymers, polypeptides, proteins, lipophilic molecules, polysaccharides, vitamins, antibodies or antibody fragments, light-sensitive oligonucleotides (OliP), lipid nanoparticles (LNPs), exosomes, metal nanoparticles, polymer nanoparticles, viral vectors, and haptens.

[0107] In some specific embodiments, the ligand is selected from one or more of the following: thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimicry.

[0108] In some specific embodiments, the ligand includes one or more N-acetylgalactosamine (GalNAc) and / or one or more N-acetylgalactosamine (GalNAc) derivatives.

[0109] In some embodiments, the ligand is attached to the 3' or 5' end of the sense or antisense strand of the nucleic acid.

[0110] Based on common knowledge in the art, the dsRNA reagent (siRNA) of this invention exhibits superior inhibitory effects when applied to different targeted drug delivery systems. In other words, the effectiveness of the naked sequence and the modified sequence in this invention does not depend on the choice of targeting vector.

[0111] Related products

[0112] The present invention also provides a cell containing the dsRNA reagent as described above.

[0113] The cells can be used for gene function research, disease model research, or drug screening.

[0114] In some embodiments, the cells do not develop into an animal individual. In some specific embodiments, the cells may be microbial cells or animal cells, but the animal cells are not animal embryonic stem cells or cells at various stages of formation and development (e.g., germ cells, fertilized egg cells, etc.).

[0115] The present invention also provides a pharmaceutical composition comprising the dsRNA reagent as described above and a pharmaceutically acceptable carrier.

[0116] The pharmaceutical composition can be prepared using conventional methods from the dsRNA reagent and the pharmaceutically acceptable carrier. For example, the pharmaceutical composition can be an injection solution. The injection solution can be used for subcutaneous, intramuscular, or intravenous injection.

[0117] According to the pharmaceutical composition of the present invention, there are no particular requirements for the amount of nucleic acid or targeted drug delivery system and pharmaceutically acceptable carrier. Generally, the content of the pharmaceutically acceptable carrier can be 1-100,000 parts by weight relative to 1 part by weight of the dsRNA reagent (e.g., 1 part by weight, 5 parts by weight, 10 parts by weight, 50 parts by weight, 100 parts by weight, 500 parts by weight, 1000 parts by weight, 5000 parts by weight, 100000 parts by weight, or any value between any two of the above).

[0118] Methods and Applications

[0119] The present invention also provides a method for inhibiting the expression of HIF2A in cells, comprising: contacting the cells with the dsRNA reagent as described above or the pharmaceutical composition as described above, to inhibit the expression of HIF2A in the cells.

[0120] In some implementations, the cells are in a subject, for example, a human subject, such as a subject with HIF2A-related disease.

[0121] In some embodiments, the cells are located in vitro. The method is based on research purposes or is used to construct animal models.

[0122] In some embodiments, contacting the cells with the nucleic acid inhibits HIF2A expression by at least 50%, 60%, 70%, 80%, 90%, or 95% (e.g., compared to HIF2A expression levels prior to the first contact of the cells with the nucleic acid; e.g., before administering a first dose of the nucleic acid to the subject). In some embodiments, inhibiting HIF2A expression reduces HIF2A protein levels in a subject's serum sample by at least 50%, 60%, 70%, 80%, 90%, or 95%, e.g., compared to HIF2A expression levels prior to the first contact of the cells with the nucleic acid.

[0123] The present invention also provides the use of the dsRNA reagent or the pharmaceutical composition thereof in any of the following aspects: 1) treating and / or preventing HIF2A-related diseases; 2) preparing a medicament for treating and / or preventing HIF2A-related diseases.

[0124] In some implementations, the disease is: (i) a disease associated with increased or elevated HIF2A levels; or (ii) a disease that would benefit from reduced HIF2A expression.

[0125] In some embodiments, the disease is selected from one or more of the following: tumor, tumor metastasis, cardiovascular disease, pulmonary hypertension (PAH), polycystic kidney disease, hereditary polycythemia (Chuvash disease), liver fibrosis, dyslipidemia, diabetes, non-alcoholic fatty liver disease (NAFLD), anemia, inflammation, chronic inflammation, rheumatoid arthritis, and inflammatory bowel disease.

[0126] In some embodiments, the tumor includes one or more of the following: brain tumor, renal cell carcinoma (RCC), pancreatic neuroendocrine tumor, hepatocellular carcinoma, lung cancer, neurogenic tumor, multiple myeloma, and multiple endocrine adenoma type 2.

[0127] In this invention, the subject can be a mammal, including primates (such as humans, non-human primates such as monkeys and chimpanzees), non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds. In some embodiments, the subject is preferably a primate, more preferably a human. Administration can be administered via various routes, depending on whether local or systemic treatment is required. Administration methods include, but are not limited to, intravenous administration, intra-arterial administration, subcutaneous administration, intraperitoneal administration, percutaneous administration (e.g., via implanted devices), and intrasoft tissue administration. Dosage can be referred to the foregoing and will not be repeated here.

[0128] Example

[0129] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0130] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0131] The abbreviations used in the following embodiments refer to the following terms as shown in Table 2:

[0132] Table 2

[0133]

[0134]

[0135] I. Research Objectives

[0136] In human clear cell adenocarcinoma cells 786-O, naked sequences and modified siRNAs of the DN01 project were screened using qRT-PCR. Candidate sequences with good inhibitory effects on HIF2A mRNA expression were selected for IC50 detection and off-target effect detection.

[0137] 1. In the first and second screening experiments, the inhibition rates of naked sequences (see Table 1, with naked sequence PC in Table 3 added as a positive control) and modified sequences (see Table 4) in human renal clear cell adenocarcinoma 786-O cells were detected. The data from the two experiments were analyzed to select the preferred sequences for the third screening experiment. The candidate sequences for IC50 detection were finally determined by combining the data from the three experiments.

[0138] In the modified sequences in Table 4, 7 pairs of naked sequences, namely DN01014, DN01017, DN01019, DN01022, DN01024, DN01034, and DN01042, were selected from the first round of screening. Three different modification schemes and two different single-stranded DNA molecular adapters were designed. In addition, 6 pairs of modified sequences (DN01092 to DN01097) were designed for modification sequence screening.

[0139] In the reference rules of this invention, a and b differ only in their single-stranded DNA molecular adapters. When a or b is removed from the siRNA number, it means that the dsRNA reagent does not contain the single-stranded DNA molecular adapter and disulfide bond. For example, DN01086 means that DN01086b does not contain the disulfide bond connected to the 3' end of the antisense strand and the single-stranded DNA molecular adapter.

[0140] 2. Six concentration gradients were designed to detect the IC50 of candidate sequences DN01074b, DN01075b, DN01077a, DN01078b, DN01086b, DN01087b, DN01088b, DN01089a, DN01090a, and DN01090b selected from screening in human clear cell adenocarcinoma cells 786-O. PC3, as shown in Table 3, was added as a positive control. DN01084, DN01087, and DN01090 were 5'-(E)-VP modified (specific sequences are shown in Table 5), and their IC50 was detected in human hepatocellular carcinoma cells HepG2. The experimental method was the same as that used in human clear cell adenocarcinoma cells 786-O.

[0141] 3. In addition to the 10 genes selected in the first off-target detection, other potential off-target genes were added, and some candidate sequences were detected.

[0142] 4. Inhibitory effects of DN01086b, DN01087b, DN01088b, DN01089a, and DN01090a on human clear cell carcinoma Caki-1, human renal cell adenocarcinoma 769-P, human renal cell adenocarcinoma ACHN, and human renal cell carcinoma A498.

[0143] II. Experimental Design

[0144] This experiment used human clear cell adenocarcinoma 786-O cells to verify the inhibition rate of siRNA. A 12-well plate was used with 40,000 cells per well, and Lipofectamine was employed. TM Transfection was performed using Lipo3000 transfection reagent. For the screening experiment, the working concentration of siRNA was 5 nM, and the transfection time was 24 hours. For the IC50 detection experiment, the working concentrations of siRNA were 0.0391 nM, 0.15625 nM, 0.625 nM, 2.5 nM, 10 nM, and 40 nM, with a transfection time of 24 hours. In the off-target effect detection experiment, the detection primers are shown in Table 6, the working concentration of siRNA was 10 nM, and the transfection time was 24 hours.

[0145] Table 3 shows the sequences used as positive controls (PCs).

[0146]

[0147] Table 4 Modified sequence information

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155] Table 5. Sequences modified with 5'-(E)-VP

[0156]

[0157] Table 6. Primers for detecting off-target effects

[0158]

[0159]

[0160] III. Experimental Materials

[0161] 3.1 Experimental Apparatus

[0162] As shown in Table 7.

[0163] Table 7

[0164] name model factory Biosafety cabinet AC2-6S1-TC ESCO Micro-volume refrigerated high-speed centrifuge Fresco21 ThermoFisher Biological inverted microscope Primovert Zeiss 37℃ CO2 incubator CelMate ESCO low-speed centrifuge Mini-6K Hangzhou Aosheng Miniature Vortex MTV-1 Hangzhou Aosheng Real-time quantitative PCR instrument Roche LightCycler 480 instrument II Luo Real-time quantitative PCR instrument Q900 Coolbo Microplate centrifuge Mini-P25 Hangzhou Aosheng

[0165] 3.2 Experimental Reagents

[0166] As shown in Table 8.

[0167] Table 8

[0168] name brand Item number OPTI-MEM I Gibco 31985-070 <![CDATA[Lipofectamine TM 3000 transfection reagent]]> Invitrogen L3000015 RNase-Free Water (Sterile) LABLEAD D0055WJ-500ml RNA-easy Isolation Reagent Vazyme R701-01 Anhydrous ethanol Titan G73537B Isopropanol Titan G75885B HiScript IIOne Step qRT-PCR SYBR Green Kit Vazyme Q221

[0169] IV. Experimental Methods

[0170] 4.1 Cell Culture

[0171] 786-O cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. Cells in the exponential growth phase were collected, resuspended to a concentration of 40,000 cells / mL, and plated into 12-well plates with 1 mL of medium and 40,000 cells per well.

[0172] 4.2 Transfection

[0173] Observe the cells under a microscope. Transfection begins when the cell confluence reaches 50-70%. Prepare EP tubes according to the number of siRNAs to be transfected and label them. Add 50 μL of OPTI-MEM and the corresponding volume of siRNA to each tube (the working concentration of siRNA for screening was determined in preliminary experiments to be 5 nM, with a transfection time of 24 hours; for IC50 detection experiments, the concentration gradients were 0.0391 nM, 0.15625 nM, 0.625 nM, 2.5 nM, 10 nM, and 40 nM, with a transfection time of 24 hours; for off-target effect detection experiments, the working concentration of siRNA was 10 nM, with a transfection time of 24 hours) to prepare mixing solution A. Vortex mix thoroughly and set aside. Add 3 μL of Lipofectamine to each well. TM Calculate the amounts of 3000 transfection reagent and 50 μL OPTI-MEM and prepare mixed solution B. Vortex the mixture and set aside. Add 50 μL of solution B to each tube of OPTI-MEM and siRNA mixed solution A, vortex the mixture, and incubate at room temperature for 15 min. Add 100 μL of siRNA-lipid complex (solution A and B mixture) to each well of cells, mix using the cross-hatching method, and incubate at 37°C in a 5% CO2 cell culture incubator.

[0174] NC: 100 μL of lipid complex added, without siRNA; blank: blank control without lipid complex and siRNA.

[0175] 4.3 Preparation of the test sample

[0176] The siRNA solution was prepared in a biosafety cabinet.

[0177] 4.4 RNA Extraction and Concentration Determination

[0178] Remove the culture medium from the 12-well plate. Add 400 μL of RNA-easy Isolation Reagent to each well, ensuring it fully covers the cell surface. Then, use a pipette to thoroughly lyse the cells. Transfer the lysis buffer to a centrifuge tube, add 160 μL of RNase-free ddH2O, invert to mix, and incubate at room temperature for 5 min. Centrifuge at 12,500 rpm for 15 min at room temperature.

[0179] Remove the centrifuge tube and carefully aspirate 500 μL of the upper aqueous phase into a new centrifuge tube. Add an equal volume of isopropanol, invert to mix, and incubate at room temperature for 10 min. Centrifuge at 12,500 rpm for 10 min at room temperature, and discard the supernatant. Add 0.5 mL of 75% ethanol and gently wash the RNA precipitate. Centrifuge at 9,100 rpm for 3 min at room temperature, and discard the supernatant. Centrifuge at 9,100 rpm for 1 min at room temperature, and remove any remaining ethanol using a micropipette tip.

[0180] After air-drying the residual ethanol at room temperature for 2 minutes, add 20 μL of RNase-free ddH2O to dissolve it.

[0181] 4.5 Quantitative Real-Time PCR (qRT-PCR) Experiment

[0182] The reaction system was prepared according to the following Table 9:

[0183] Table 9

[0184]

[0185] In a 15 mL centrifuge tube, add all components except primers and template, and label it A. Label 1.5 mL EP tubes, and add 56 μL of A and 350 ng of total RNA to each of the seven tubes, and mix well. Mix the upstream and downstream primers for the internal control gene hsACTB and the target gene hsHIF2A and set aside.

[0186] Add 8 μL of B and 2 μL of C to each well of the PCR plate according to the arrangement. Cover with sealing film, centrifuge at 2,500 rpm for 1 min, and then transfer to the PCR machine.

[0187] Place the plate in a qRT-PCR instrument and run the following procedure:

[0188] Reverse transcription: 55℃, 15 min;

[0189] Pre-denaturation: 95℃, 30 seconds;

[0190] Cyclic reaction: 95℃, 10 sec; 60℃, 35 sec; 40 cycles;

[0191] Melting curves: 95℃, 15sec; 60℃, 60sec; 95℃, 15sec.

[0192] The running time is approximately 1.5 hours. Analyze the experimental results and calculate 2. -ΔΔCt .

[0193] V. Experimental Results

[0194] 5.1 Results of naked sequence screening

[0195] The inhibition rate of the naked sequence in human renal clear cell adenocarcinoma 786-O cells is shown in Table 10.

[0196] Table 10

[0197]

[0198]

[0199] Based on the analysis of the results, DN01014, DN01017, DN01019, DN01022, DN01024, DN01034, and DN01042 were selected to proceed to the next stage of screening.

[0200] 5.2 Screening results of inhibition rate of modified sequences

[0201] The inhibition rate of the modified sequence in human renal clear cell adenocarcinoma 786-O cells is shown in Table 11 below.

[0202] Table 11

[0203]

[0204]

[0205] Based on the analysis of the results, a third screening experiment was conducted to select the preferred sequences.

[0206] 5.3 Screening results of suppression rate of dominant sequences

[0207] The inhibition rate of the dominant sequences from the previous screening in human renal clear cell adenocarcinoma 786-O cells is shown in Table 12 below.

[0208] Table 12

[0209] siRNA numbering Inhibition rate (%) DN01074b 65.85％ DN01075a 51.03％ DN01075b 58.25％ DN01076a 63.14％ DN01077a 67.47％ DN01078a 60.77％ DN01078b 69.43％ DN01084b 59.67％ DN01085b 22.08％ DN01086b 67.91％ DN01087a 59.95％ DN01087b 70.06％ DN01088b 65.37％ DN01089a 64.89％ DN01089b 47.51％ DN01090a 82.32％ DN01090b 73.02％ DN01091a 68.14％ DN01091b 67.47％ DN01097b 64.40％

[0210] Based on the analysis of the results, DN01074b, DN01075b, DN01077a, DN01078b, DN01086b, DN01087b, DN01088b, DN01089a, DN01090a, and DN01090b were selected as candidate sequences for further screening.

[0211] 5.4 Analysis of IC50 Detection Results of Candidate Sequences

[0212] The IC50 experiment results of the 10 candidate sequences obtained from the third round of screening were analyzed. Figures 1-2The results of the summary analysis are shown in Table 13. According to the curve and IC50 results, the sequences with better inhibition effects are DN01087b, DN01088b, DN01086b, DN01090a, and DN01089a.

[0213] Table 13.10 shows the IC50 of candidate sequences.

[0214] siRNA IC50(nM) DN01077a 0.4782 DN01089a 0.1513 DN01090a 0.3221 DN01074b 0.1232 DN01075b 0.5228 DN01078b 0.2842 DN01086b 0.2088 DN01087b 0.01392 DN01088b 0.01223 DN01090b 0.7613 PC3 0.087

[0215] 5.5 IC50 results of 5'-(E)-VP modified sequences in human hepatocellular carcinoma HepG2 cells

[0216] The IC50 results of 5'-(E)-VP modified DN01084, DN01087, and DN01090 in human hepatocellular carcinoma HepG2 cells are shown in Table 14 below. Figure 3 .

[0217] Table 14

[0218] siRNA IC50(nM) DN01098 (based on DN01090) 0.08038 DN01099 (based on DN01087) 0.02752 DN01100 (based on DN01084) 0.06830

[0219] The results show that the modified sequence of the present invention still has a better inhibitory effect even without single-stranded DNA molecular adapters.

[0220] 5.6 Off-target effect analysis of DN01087b and DN01090a

[0221] DN01086b, DN01087b, and DN01088b are three modified sequences of the bare sequence numbered DN01034, and DN01090a and DN01089a are two modified sequences of the bare sequence numbered DN01042. Off-target effects of DN01087b and DN01090a were detected using primers for potential off-target genes corresponding to the bare sequences. Except for some genes with low basal expression in 786-O cells (corresponding primer numbers 1, 5, 6, 7, 8, 9, 208, 209, 151, 152, 158, 159, 191, 192, 195, 198, 201, 203, 206, and 212), no significant reduction was detected in the expression of the remaining genes (fold change less than 0.5).

[0222] Conclusion: No off-target effects were detected in DN01087b (based on DN01034) and DN01090a (based on DN01042) sequences in 786-O cells. The detection results corresponded to the following: Figure 4 and Figure 5 DN01086b, DN01087b, DN01088b, DN01090a, and DN01089a can be used preferentially for subsequent experiments.

[0223] 5.7 Inhibitory effects of candidate siRNAs in different renal cell carcinomas

[0224] The results of the inhibitory effects of DN01086b, DN01087b, DN01088b, DN01089a, and DN01090a on human clear cell renal cancer cells Caki-1, human renal cell adenocarcinoma cells 769-P, human renal cell adenocarcinoma cells ACHN, and human renal cancer cells A498 are shown in [the table below]. Figure 6 The results showed that the above siRNA sequence significantly inhibited the expression of HIF2A in all four types of renal cell carcinoma.

[0225] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A dsRNA reagent, wherein, The dsRNA reagent comprises a sense strand and an antisense strand forming a double-stranded region. The antisense strand comprises at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with any of the sequences shown in SEQ ID No. 51 to 100, and the sense strand comprises a nucleotide sequence that is at least partially complementary to the antisense strand.

2. The dsRNA reagent according to claim 1, wherein, The positive strand comprises at least 15 consecutive nucleotides having 0, 1, 2 or 3 mismatches with the nucleotides at positions 1 to 19 of any of the sequences shown in SEQ ID No. 1 to 50.

3. The dsRNA reagent according to claim 1, wherein, The antisense strand includes 0, 1, 2 or 3 mismatched nucleotides with the sequence shown in SEQ ID No. 64, and the sense strand includes 0, 1, 2 or 3 mismatched nucleotides with positions 1 to 19 of the sequence shown in SEQ ID No. 14; Alternatively, the antisense strand may include 0, 1, 2, or 3 mismatched nucleotides with the sequence shown in SEQ ID No. 67, and the sense strand may include 0, 1, 2, or 3 mismatched nucleotides with positions 1 to 19 of the sequence shown in SEQ ID No. 17; Alternatively, the antisense strand may include 0, 1, 2, or 3 mismatched nucleotides with the sequence shown in SEQ ID No. 69, and the sense strand may include 0, 1, 2, or 3 mismatched nucleotides with positions 1 to 19 of the sequence shown in SEQ ID No. 19; Alternatively, the antisense strand may include 0, 1, 2, or 3 mismatched nucleotides with the sequence shown in SEQ ID No. 72, and the sense strand may include 0, 1, 2, or 3 mismatched nucleotides with positions 1 to 19 of the sequence shown in SEQ ID No. 22; Alternatively, the antisense strand may include 0, 1, 2, or 3 mismatched nucleotides with the sequence shown in SEQ ID No. 74, and the sense strand may include 0, 1, 2, or 3 mismatched nucleotides with positions 1 to 19 of the sequence shown in SEQ ID No. 24; Alternatively, the antisense strand may include 0, 1, 2, or 3 mismatched nucleotides with the sequence shown in SEQ ID No. 84, and the sense strand may include 0, 1, 2, or 3 mismatched nucleotides with positions 1 to 19 of the sequence shown in SEQ ID No. 34; Alternatively, the antisense strand may include 0, 1, 2, or 3 mismatched nucleotides with the sequence shown in SEQ ID No. 92, and the sense strand may include 0, 1, 2, or 3 mismatched nucleotides with positions 1 to 19 of the sequence shown in SEQ ID No.

42.

4. The dsRNA reagent according to any one of claims 1 to 3, wherein, The dsRNA reagent comprises at least one modified nucleotide; The modified nucleotide is selected from one or more of the following: deoxynucleotides, 5'-terminal vinyl phosphate modified nucleotides, 3'-terminal deoxythymidine nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, and morpholinonucleotides. Acids, aminophosphates, non-natural bases including nucleotides, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimics, ethylene glycol-modified nucleotides, and 2-O-(N-methylacetamide)-modified nucleotides; preferably one or more of the following: 5'-terminal vinyl phosphate-modified, 2'-O-methyl-modified, 2'-fluorine-modified, and thiophosphate-modified nucleotides.

5. The dsRNA reagent according to claim 4, wherein, The nucleotides of the sense strand and the antisense strand each include 3 to 5 nucleotides modified with 2'-fluorine, and the other nucleotides are nucleotides modified with 2'-O-methyl. Preferably, in the antisense strand, positions 3 to 5 of the nucleotides at positions 2, 6, 8, 14, and 16 counting from the 5' end are 2'-fluoronucleotides, and the other nucleotides are 2'-O-methyl modified nucleotides; in the sense strand, positions 3 to 4 of the nucleotides at positions 5, 7, 8, and 9 counting from the 5' end are 2'-fluoronucleotides, and the other nucleotides are 2'-O-methyl modified nucleotides.

6. The dsRNA reagent according to claim 5, wherein, The 5' end of the antisense strand is a vinyl phosphate-modified nucleotide.

7. The dsRNA reagent according to any one of claims 4 to 6, wherein, The last 2-4 nucleotides at the 5' and 3' ends of the antisense strand contain thiophosphate nucleotide bonds, and the last 2-4 nucleotides at the 5' end of the sense strand contain thiophosphate nucleotide bonds.

8. The dsRNA reagent according to any one of claims 4 to 7, wherein, The dsRNA reagent further includes a single-stranded deoxyribonucleic acid molecule; the 5' end of the single-stranded deoxyribonucleic acid molecule is covalently linked to the 3' end of the sense strand, or the 5' end of the single-stranded deoxyribonucleic acid molecule is covalently linked to the 3' end of the antisense strand; Preferably, the single-stranded deoxyribonucleic acid molecule is covalently linked to the sense or antisense strand via disulfide bonds; Preferably, the single-stranded deoxyribonucleic acid molecule includes nucleotides having 0, 1, 2 or 3 mismatches with the sequence shown in SEQ ID No.

101.

9. The dsRNA reagent according to claim 1, wherein, The antisense strand has at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches in the antisense strand sequence as shown in any one of Tables 4 to 5, and the sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; preferably, the sense strand has at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches in the sense strand sequence as shown in any one of Tables 4 to 5.

10. The dsRNA reagent according to claim 9, wherein, The sequence of the antisense strand has 0, 1, 2 or 3 mismatched nucleotides with the antisense strand sequence shown in DN01084, and the sequence of the sense strand has 0, 1, 2 or 3 mismatched nucleotides with the sense strand sequence shown in DN01084. Alternatively, the sequence of the antisense strand has 0, 1, 2, or 3 mismatched nucleotides with the antisense strand sequence shown in DN01086, and the sequence of the sense strand has 0, 1, 2, or 3 mismatched nucleotides with the sense strand sequence shown in DN01086. Alternatively, the sequence of the antisense strand has 0, 1, 2, or 3 mismatched nucleotides with the antisense strand sequence shown in DN01087, and the sequence of the sense strand has 0, 1, 2, or 3 mismatched nucleotides with the sense strand sequence shown in DN01087. Alternatively, the sequence of the antisense strand has 0, 1, 2, or 3 mismatched nucleotides with the antisense strand sequence shown in DN01088, and the sequence of the sense strand has 0, 1, 2, or 3 mismatched nucleotides with the sense strand sequence shown in DN01088. Alternatively, the sequence of the antisense strand has 0, 1, 2, or 3 mismatched nucleotides with the antisense strand sequence shown in DN01089, and the sequence of the sense strand has 0, 1, 2, or 3 mismatched nucleotides with the sense strand sequence shown in DN01089. Alternatively, the sequence of the antisense strand has 0, 1, 2, or 3 mismatched nucleotides with the antisense strand sequence shown in DN01090, and the sequence of the sense strand has 0, 1, 2, or 3 mismatched nucleotides with the sense strand sequence shown in DN01090.

11. The dsRNA reagent according to any one of claims 1 to 10, wherein, The length of the double-stranded region is 15 to 30 nucleotide pairs; preferably 17 to 23 nucleotides, or 19 to 30 nucleotides.

12. The dsRNA agent according to any one of claims 1 to 11, wherein, At least one of the sense strand and the antisense strand includes a 3' overhang of at least two nucleotides.

13. The dsRNA reagent according to any one of claims 1 to 12, wherein, At least one of the sense and antisense strands is attached to one or more ligands selected from the following: lectins, glycoproteins, lipids, nucleic acid aptamers, polymers, polypeptides, proteins, lipophilic molecules, polysaccharide molecules, vitamins, antibodies or antibody fragments, photosensitive oligonucleotides, lipid nanoparticles, exosomes, metal nanoparticles, polymer nanoparticles, viral vectors, and haptens.

14. A cell containing the dsRNA reagent according to any one of claims 1 to 13.

15. A pharmaceutical composition comprising the dsRNA reagent of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

16. A method for inhibiting the expression of HIF2A in cells, comprising: The cells are contacted with the dsRNA reagent of any one of claims 1 to 13 or the pharmaceutical composition of claim 15 to inhibit the expression of HIF2A in the cells.

17. The use of the dsRNA reagent according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 15 in any of the following aspects: 1) Treatment and / or prevention of diseases associated with HIF2A; 2) To prepare medicines for the treatment and / or prevention of diseases associated with HIF2A; The preferred disease is: (i) a disease associated with increased or elevated HIF2A levels; or (ii) Diseases that will benefit from reduced HIF2A expression.

18. The application according to claim 17, wherein, The diseases mentioned are selected from one or more of the following: tumors, tumor metastases, cardiovascular diseases, pulmonary hypertension, polycystic kidney disease, hereditary polycythemia, liver fibrosis, dyslipidemia, diabetes, non-alcoholic fatty liver disease, anemia, inflammation, chronic inflammation, rheumatoid arthritis, and inflammatory bowel disease. Preferably, the tumor includes one or more of the following: brain tumor, renal cell carcinoma, pancreatic neuroendocrine tumor, hepatocellular carcinoma, lung cancer, neurogenic tumor, multiple myeloma, and multiple endocrine adenoma type 2.

Images