WO2025040005A1 - Sirna for inhibiting the gene expression of lftslp and composition thereof - Google Patents

Abstract

Provided is a siRNA for inhibiting the gene expression of lfTSLP, said siRNA comprising a sense strand and an antisense strand. The provided siRNA can specifically target the long isoform of TSLP having pro-inflammatory activity, and inhibit the mRNA and protein expression of the long isoform of TSLP, while not affecting the mRNA and protein expression of the short isoform of TSLP having anti-inflammatory activity. Therefore, the siRNA has important clinical value for the treatment of lfTSLP related diseases.

Description

siRNA for inhibiting lfTSLP gene expression and its composition

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application 202311047112.8 filed on August 18, 2023, the entire contents of which are incorporated herein by reference in their entirety.

Technical Field

The present invention provides an siRNA for inhibiting the expression of long subtype thymic stromal lymphopoietin (lfTSLP) and a pharmaceutical composition thereof. The siRNA and the pharmaceutical composition thereof provided by the present invention can treat diseases related to lfTSLP.

Technical Background

Thymic Stromal Lymphopoietin (TSLP) belongs to the interleukin-2 (IL-2) family of cytokines. It is mainly expressed and secreted by lung, skin, and intestinal epithelial cells, and is also expressed in small amounts in airway smooth muscle cells, keratinocytes, stromal cells, mast cells, and other cells. TSLP is an alarm factor that, when stimulated by external stimuli (such as allergens, viral infections, etc.), binds to a heterodimeric receptor composed of its specific receptors, the Thymic Stromal Lymphopoietin Receptor (TSLPR) and the Interleukin-7 receptor alpha chain (IL-7Rα), to establish a ternary complex and activate the occurrence of downstream inflammatory signals.

Human TSLP has two isoforms regulated by independent promoters: long isoform TSLP (lfTSLP) and short isoform TSLP (sfTSLP). The long isoform TSLP (lfTSLP) is upregulated in inflammation and has pro-inflammatory function; the short isoform TSLP (sfTSLP) is expressed in healthy tissues and has anti-inflammatory and antibacterial activities. Under pathological conditions, abnormal activation of lfTSLP signaling is closely related to the occurrence of many diseases, such as asthma, chronic sinusitis, atopic dermatitis, eosinophilic esophagitis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, etc. Research data show that in asthmatic patients, the expression level of lfTSLP is positively correlated with the severity of asthma.

To date, only one antibody drug targeting TSLP has been approved by the FDA for marketing, namely Tezspire (Tezepeluma), which is used as an additional maintenance treatment for severe asthma patients aged 12 years and above without phenotypic restrictions. It requires subcutaneous injection once every four weeks. Its mechanism of action is to bind to TSLP protein and inhibit its binding to TSLPR receptor, thereby inhibiting the occurrence of downstream inflammatory cascade reactions and achieving the purpose of treating asthma.

siRNA targeting the human long isoform-specific TSLP gene can be designed to target the mRNA level with sequence specificity The expression of lfTSLP protein can be inhibited in a targeted manner to treat related diseases caused by abnormal expression of lfTSLP without affecting the expression of sfTSLP. However, to date, no siRNA drug targeting lfTSLP has been used in clinical practice. Therefore, it is urgent to develop a siRNA drug with potential clinical application value, good stability and good biological activity.

Summary of the invention

The present invention provides siRNA that can effectively inhibit the expression of lfTSLP gene, and thus provides medicine and method for preventing and/or treating lfTSLP related diseases.

siRNA

In one aspect, the present invention provides a small interfering RNA (siRNA) for inhibiting the expression of the long subtype TSLP (lfTSLP) gene, wherein the siRNA comprises a sense chain and an antisense chain, wherein the antisense chain comprises at least 14 consecutive nucleotides that are not more than 4 (e.g., 0, 1, 2, 3 or 4) nucleotides different from the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:164, and the sense chain is at least partially complementary to the antisense chain.

The at least partial complementarity means that the two sequences can be completely complementary, or have no more than 6, 5, 4, 3, 2 or 1 mismatched base pairings overall, while retaining the ability to hybridize under relevant conditions. Those skilled in the art can determine the conditions that are most suitable for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides. Such conditions can, for example, be stringent conditions, such as 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, may also be applied.

In some embodiments, the lfTSLP is a long subtype TSLP, such as an mRNA of the sequence shown in Genbank: NM_033035.5. In some embodiments, the siRNA of the present invention targets the sequence shown in SEQ ID NO: 727. In some embodiments, the siRNA of the present invention targets lfTSLP and does not affect the expression of the short subtype TSLP (sfTSLP).

In some embodiments, the antisense strand comprises at least 14 consecutive nucleotides that differ by 0 or 1 nucleotide from the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:164.

In some embodiments, the antisense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that differ from the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:164 by no more than 4 (e.g., 0, 1, 2, 3, or 4) nucleotides. In some embodiments, the antisense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that differ from the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:164 by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides. In some embodiments, the antisense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that differ from the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:164 by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides. 1 to SEQ ID NO: 164 shows a nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 164. In some embodiments, the antisense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that differ by no more than 2 (e.g., 0, 1, or 2) nucleotides from the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 164. In some embodiments, the antisense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that differ by no more than 1 (e.g., 0 or 1) nucleotide from the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 164. In some embodiments, the antisense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) of the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 164.

In some embodiments, the antisense strand is a nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:164.

In some embodiments, the sense strand has no more than 6 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 5 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 4 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 3 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 2 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 1 nucleotide mismatch with the antisense strand. In some embodiments, the sense strand is fully complementary to the antisense strand.

In some embodiments, the sense strand has at least 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides complementary to the antisense strand. In some embodiments, the sense strand has 14 to 19 nucleotides complementary to the antisense strand.

In some embodiments, the sense strand is at least 85% (eg, at least 90%, at least 95%, at least 99%) complementary or fully complementary to the antisense strand over at least 14 consecutive nucleotides.

In some embodiments, the sense strand and the antisense strand form a duplex region of 14 to 30 nucleotide pairs in length, such as 14 to 25 nucleotide pairs, 14 to 23 nucleotide pairs, 14 to 21 nucleotide pairs, 15 to 25 nucleotide pairs, 15 to 23 nucleotide pairs, 15 to 21 nucleotide pairs, 17 to 25 nucleotide pairs, 17 to 23 nucleotide pairs, or 17 to 21 nucleotide pairs, such as 14, 15, 16, 17, 18, 19, 20, or 21 nucleotide pairs. In some embodiments, the sense strand and the antisense strand form a duplex region of 14 to 19 nucleotide pairs in length.

In some embodiments, the antisense strand is 14 to 30 (e.g., 14 to 29, 14 to 28, 14 to 27, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 25, 19 to 23, 21 to 25, 21 to 23) nucleotides in length and the sense strand is 14 to 30 (e.g., 14 to 29, 14 to 28, 14 to 27, 14 to 26, 14 to 25, 14 to 21, 19 to 21) nucleotides in length.

In some embodiments, the antisense strand is 16 to 25 nucleotides in length; and the sense strand is 14 to 23 nucleotides in length.

In some embodiments, the antisense strand is 21 to 23 nucleotides in length; and the sense strand is 19 to 21 nucleotides in length.

In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length.

In some embodiments, the siRNA comprises blunt ends and/or overhanging ends.

In some embodiments, the siRNA comprises one or more single-stranded nucleotide overhangs. For example, an overhang of 1, 2, 3 or 4 nucleotides. In some embodiments, the overhang can be on the sense strand, the antisense strand or any combination thereof. In some embodiments, the overhang is present at the 5' end, the 3' end or both ends of the siRNA antisense strand or the sense strand.

In some embodiments, the 3' end of the antisense strand of the siRNA has an overhang of 2 nucleotides. In some embodiments, the 3' end of the sense strand of the siRNA is blunt-ended.

In some embodiments, the sense strand of the siRNA comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that are not more than 4 (e.g., 0, 1, 2, 3, or 4) nucleotides different from the nucleotide sequence shown in any one of SEQ ID NO: 165 to SEQ ID NO: 328. The at least 14 consecutive nucleotides contained in the sense strand and the antisense strand of the siRNA form a duplex region. In some embodiments, the sense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that are not more than 3 (e.g., 0, 1, 2, or 3) nucleotides different from the nucleotide sequence shown in any one of SEQ ID NO: 165 to SEQ ID NO: 328. In some embodiments, the sense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that differ from the nucleotide sequence shown in any one of SEQ ID NO: 165 to SEQ ID NO: 328 by no more than 2 (e.g., 0, 1, or 2) nucleotides. In some embodiments, the sense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) that differ from the nucleotide sequence shown in any one of SEQ ID NO: 165 to SEQ ID NO: 328 by no more than 1 (e.g., 0 or 1) nucleotide. In some embodiments, the sense strand has a region within the 14 consecutive nucleotides that is at least 85% (e.g., at least 90%, at least 95%, at least 99%) complementary or fully complementary to the antisense strand. In some embodiments, the sense strand comprises at least 14 consecutive nucleotides (e.g., at least 15, at least 16, or at least 17 consecutive nucleotides) of the nucleotide sequence shown in any one of SEQ ID NO: 165 to SEQ ID NO: 328. In some embodiments, the sense strand is the nucleotide sequence shown in any one of SEQ ID NO: 165 to SEQ ID NO: 328.

In some embodiments, the antisense strand of the siRNA comprises GPSZT082UM provided in Table 1. The antisense strand sequence or a portion thereof (eg, at least 14 consecutive nucleotides) of any duplex in GPSZT245UM, and the sense strand of the siRNA comprises the sense strand sequence or a portion thereof (eg, at least 14 consecutive nucleotides) of the duplex.

In some embodiments, the antisense strand of the siRNA comprises the antisense strand sequence of any duplex provided in Table 5 or a portion thereof (e.g., at least 14 consecutive nucleotides thereof). Preferably, the sense strand of the siRNA comprises the sense strand sequence of the duplex or a portion thereof (e.g., at least 14 consecutive nucleotides thereof).

In some embodiments, the antisense strand of the siRNA comprises the antisense strand sequence or a portion thereof (e.g., at least 14 consecutive nucleotides thereof) of any duplex of GPSZT086UM, GPSZT091UM, GPSZT095UM, GPSZT082UM, GPSZT089UM, GPSZT083UM, GPSZT098UM, GPSZT097UM, GPSZT099UM, or GPSZT085UM provided in Table 1. Preferably, the sense strand of the siRNA comprises the sense strand sequence or a portion thereof (e.g., at least 14 consecutive nucleotides thereof) of the duplex.

In some embodiments, the antisense strand of the siRNA comprises GPSZT084UM, GPSZT122UM, GPSZT150UM, GPSZT090UM, GPSZT094UM, GPSZT123UM, GPSZT175UM, GPSZT153UM, GPSZT207UM, GPSZT092UM, GPSZT111UM, GPSZT231UM, GPSZT155UM, GPSZT112UM, GPSZT208UM, GPSZT174UM, GPSZT120 ... Preferably, the sense strand of the siRNA comprises the antisense strand sequence of any duplex of GPSZT177UM, GPSZT121UM, GPSZT220UM, GPSZT176UM, GPSZT223UM, GPSZT213UM, GPSZT148UM, GPSZT233UM, GPSZT191UM, GPSZT232UM, GPSZT160UM, GPSZT230UM, GPSZT188UM, GPSZT128UM, GPSZT221UM, GPSZT130UM or GPSZT149UM or a portion thereof (e.g., at least 14 consecutive nucleotides thereof). Preferably, the sense strand of the siRNA comprises the sense strand sequence of the duplex or a portion thereof (e.g., at least 14 consecutive nucleotides thereof).

In some embodiments, the antisense strand of the siRNA comprises GPSZT084UM, GPSZT122UM, GPSZT150UM, GPSZT090UM, GPSZT094UM, GPSZT123UM, GPSZT175UM, GPSZT153UM, GPSZT092UM, GPSZT111UM, GPSZT231UM, GPSZT112UM, GPSZT174UM, GPSZT12 ... Preferably, the sense strand of the siRNA comprises the antisense strand sequence of any duplex of GPSZT10UM, GPSZT177UM, GPSZT121UM, GPSZT220UM, GPSZT176UM, GPSZT223UM, GPSZT213UM, GPSZT233UM, GPSZT191UM, GPSZT232UM, GPSZT230UM, GPSZT128UM, GPSZT221UM or GPSZT149UM or a portion thereof (e.g., at least 14 consecutive nucleotides thereof). Preferably, the sense strand of the siRNA comprises the sense strand sequence of the duplex or a portion thereof (e.g., at least 14 consecutive nucleotides thereof).

In some embodiments, the antisense strand of the siRNA comprises GPSZT082UM, GPSZT083UM, GPSZT086UM, GPSZT089UM, GPSZT090UM, GPSZT091UM, GPSZT092UM, GPSZT094UM, GPSZT095UM, GPSZT098UM, GPSZT099UM, GPSZT112UM, GPSZT121UM, GPSZT122UM, GPSZT123UM, GPSZT124UM, GPSZT125UM, GPSZT126UM, GPSZT127UM, GPSZT128UM, GPSZT129UM, GPSZT130UM, GPSZT131UM, GPSZT132UM, GPSZT133UM, GPSZT134UM, GPSZT135UM, GPSZT136UM, GPSZT137UM, GPSZT138UM, GPSZT139UM, GPSZT140UM, GPSZT141UM, GPSZT142UM, GPSZT143UM, GPSZT144UM, GPSZT145UM, GPSZT146UM, GPSZT147UM, GPSZT148UM, GPSZT149UM, GPSZT141UM, GPSZT149UM, GPSZT141UM, GPSZZT142UM, GPSZT143UM, GPSZZT144UM, GPSZZT145UM Preferably, the sense strand of the siRNA comprises the antisense strand sequence of any duplex of GPSZT3UM, GPSZT128UM, GPSZT149UM, GPSZT150UM, GPSZT153UM, GPSZT174UM, GPSZT175UM, GPSZT176UM, GPSZT177UM, GPSZT191UM, GPSZT220UM, GPSZT221UM, GPSZT223UM, GPSZT226UM or GPSZT231UM or a portion thereof (e.g., at least 14 consecutive nucleotides thereof). Preferably, the sense strand of the siRNA comprises the sense strand sequence of the duplex or a portion thereof (e.g., at least 14 consecutive nucleotides thereof).

Modification

The siRNA of the present invention can be modified in the nucleoside base structure or in the ribose-phosphate backbone structure to reduce the effect of missing the target, and/or increase the biological stability of the molecule, or increase the physical stability of the duplex formed between the antisense and sense nucleic acids. Therefore, the siRNA sequence comprising any modification is also encompassed within the scope of the present invention. The siRNA molecule comprising ribonucleoside analogs or derivatives must maintain the ability to form duplexes and allow or mediate the specific degradation of the target RNA via the RISC approach.

In some embodiments, the siRNA contains at least one modified nucleotide. The modification need not be the same for each of the plurality of modified ribonucleosides in the siRNA.

In some embodiments, all nucleotides in the sense strand and/or antisense strand of the siRNA are modified nucleotides or nucleotide analogs. In some embodiments, all nucleotides in the sense strand of the siRNA are modified nucleotides or nucleotide analogs, and all nucleotides in the antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

In some embodiments, the siRNA comprises 2'-modified nucleotides.

In some embodiments, the modified nucleotide or nucleotide analog is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxyribonucleotides, 2',3'-split ring nucleotide analogs, 2'-fluoroarabino nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleic acids (LNA), unlocked nucleic acids (UNA) or glycerol nucleotides (GNA), but the present invention is not limited thereto.

In some embodiments, the modified nucleotide or nucleotide analog is selected from 2'-methoxy nucleotides, 2'-fluoro In some embodiments, the sense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the sense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the sense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the sense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the sense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the antisense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the antisense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the sense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the sense strand comprises at least one of 2'-deoxyribonucleotides. In some embodiments, the antisense strand comprises at least one of 2'-methoxy nucleotides, 2'-fluoro nucleotides, or a combination thereof. In some embodiments, the sense strand comprises at least one of 2'-deoxyribonucleotides. In some embodiments, the antisense strand comprises at least one of 2'-deoxyribonucleotides. In some embodiments, the sense strand comprises at least one nucleotide comprising a thiophosphate group. In some embodiments, the antisense strand comprises at least one nucleotide comprising a thiophosphate group. In some embodiments, the antisense strand comprises at least one nucleotide comprising a 5'-phosphate mimic. In some embodiments, the nucleotide comprising a 5'-phosphate mimic is at position 1 of the antisense strand.

In some embodiments, the nucleotides in the sense strand are selected from at least two of 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxyribonucleotides, or locked nucleic acids, and/or the nucleotides in the antisense strand are selected from at least two of 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxyribonucleotides, glycerol nucleotides, nucleotides containing 5'-phosphate esters, or nucleotides containing 5'-phosphate mimetics.

In some embodiments, the siRNA of the present invention comprises a modified internucleoside connection or a modified backbone. The modified internucleoside connection or backbone includes, but is not limited to, phosphorothioate, 2'-O methoxyethyl (MOE), 2'-fluoro, alkyl phosphate, phosphorodithioate, alkyl phosphorothioate, phosphoramidate, carbamate, carbonate, phosphotriester, acetamidate, carboxymethyl ester and combinations thereof.

In some embodiments, the modified nucleotide is a nucleotide in which the phosphate group is modified by a phosphorothioate group, that is, a non-bridging oxygen atom in a phosphodiester bond is substituted with a sulfur atom, thereby replacing the phosphodiester bond with a phosphorothioate diester bond.

In some embodiments, the 5' end and/or 3' end of the sense strand independently comprises 1 or 2 phosphorothioate linkages; and/or the 5' end and/or 3' end of the antisense strand independently comprises 1 or 2 phosphorothioate linkages. In some embodiments, the 5' end of the sense strand comprises 1 or 2 phosphorothioate linkages, and the 5' end and/or 3' end of the antisense strand independently comprises 1 or 2 phosphorothioate linkages.

In some embodiments, the sense strand can include one or more blocking residues or moieties, referred to as "blocking residues." A "capping residue" is a non-nucleotide compound or other moiety that can be incorporated into one or more ends of the nucleotide sequence of an siRNA. In some embodiments, the capping residue is present at the 5' end, the 3' end, or both the 5' end and the 3' end of the sense strand.

In some embodiments, an inverted abasic residue (iab) is added as a capping residue. See F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16. In some embodiments, the 5' end and/or 3' end of the sense strand may contain more than one inverted abasic deoxyribose moiety as a capping residue.

In some embodiments, one or more inverted abasic residues (iab) are added to the 3' end of the sense strand. In some embodiments, one or more inverted abasic residues (iab) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues are included at or near one or more ends of the sense strand of the siRNA.

Inverted abasic residues may be linked via phosphate, phosphorothioate or other internucleoside linkages.

In some embodiments, the antisense strand of the siRNA is 21 nucleotides in length and comprises the following modification pattern:

(1)5’-NmsNfsNmNfNmNfNmNfNmNfNmNmNmNfNmNfNmNfNmsNmsNm-3’ (SEQ ID NO:719);

(2)5’-NmsNfsNmNmNmNfNmNfNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’ (SEQ ID NO:720);

(3)5’-NmsNfsNmNfNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’ (SEQ ID NO:721);

(4)5’-VPNmsNfsNmNfNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’ (SEQ ID NO:728);

(5)5’-VPNmsNfsNmNfNmN(GNA)NmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’ (SEQ ID NO:729);

(6)5’-VPNmsNfsNmNfNmNmN(GNA)NmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’(SEQ ID NO:730);

Wherein, Nm is a methoxy-modified nucleotide, Nf is a fluorinated-modified nucleotide, s is a phosphorothioate linkage, VP represents a 5'-phosphate mimetic, and N(GNA) represents a glycerol nucleotide. The nucleotide may be selected from C, G, U or A.

In some embodiments, the sense strand of the siRNA is 19 nucleotides in length and comprises the following modification pattern:

(1)5’-NmsNmsNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfNmNf-3’ (SEQ ID NO:722);

(2) 5'-NmsNmsNfNmNmNmNfNfNfNmNfNmNfNmNfNmNmNmNm-3' (SEQ ID NO: 723);

(3)5’-NmsNmsNmNmNfNmNfNfN(d)NmNmNmNmNmNmNmNmNmNm-3’(SEQ ID NO:724);

(4)5’-N(LNA)sNmsNfNmNmNmNfNfNfNfNmNfNmNfNmNfNmNmNmNm-3’(SEQ ID NO:725);

(5)5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNfNmNmNmNm-3’ (SEQ ID NO:726);

(6)5’-NmsNmsNmNmNfNmN(d)NfN(d)NmNmNmNmNmNmNmNmNmNmNm-3’(SEQ ID NO:731);

(7)5’-NmsNmNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfNmsNf-3’ (SEQ ID NO:732);

(8)5’-NmsNmNfNmNmNmNfNfNfNmNfNmNfNmNfNmNmNmsNm-3’ (SEQ ID NO:733);

(9)5’-NmsNmNmNmNfNmNfNfN(d)NmNmNmNmNmNmNmNmNmsNm-3’(SEQ ID NO:734);

(10)5’-N(LNA)sNmNfNmNmNmNfNfNfNfNmNfNmNfNmNfNmNmNmsNm-3’(SEQ ID NO:735);

or

(12)5’-NmsNmNmNmNfNmN(d)NfN(d)NmNmNmNmNmNmNmNmNmsNm-3’(SEQ ID NO:737);

Wherein, Nm is a methoxy-modified nucleotide, Nf is a fluorinated-modified nucleotide, N(LNA) is a locked nucleic acid-modified nucleotide, N(d) is a 2'-deoxyribonucleotide, and s is a thiophosphate-linked nucleotide. The nucleotide can be selected from C, G, U or A.

In some embodiments, the siRNA comprises a modification pattern selected from the group consisting of: SEQ ID NO: 719 and SEQ ID NO: 722; SEQ ID NO: 719 and SEQ ID NO: 723; SEQ ID NO: 719 and SEQ ID NO: 724; SEQ ID NO: 719 and SEQ ID NO: 725; SEQ ID NO: 719 and SEQ ID NO: 726; SEQ ID NO: 719 and SEQ ID NO: 731; SEQ ID NO: 719 and SEQ ID NO: 732; SEQ ID NO: 719 and SEQ ID NO: 733; SEQ ID NO: 719 and SEQ ID NO: 734; SEQ ID NO: 719 and SEQ ID NO: 735; SEQ ID NO: 719 and SEQ ID NO: 736; SEQ ID NO: 719 and SEQ ID NO: 737; SEQ ID NO: 720 and SEQ ID NO: 722; SEQ ID NO:720 and SEQ ID NO:723; SEQ ID NO:720 and SEQ ID NO:724; SEQ ID NO:720 and SEQ ID NO:725; SEQ ID NO:720 and SEQ ID NO:726; SEQ ID NO:720 and SEQ ID NO:731; SEQ ID NO:720 and SEQ ID NO:732; SEQ ID NO:720 and SEQ ID NO:733; SEQ ID NO:720 and SEQ ID NO:734; SEQ ID NO:720 and SEQ ID NO:735; SEQ ID NO:720 and SEQ ID NO:736; SEQ ID NO:720 and SEQ ID NO:737; SEQ ID NO:721 and SEQ ID NO:722; SEQ ID NO:721 and SEQ ID NO:723; SEQ ID NO:721 and SEQ ID NO:724; SEQ ID NO:721 and SEQ ID NO:725; SEQ ID NO:726; NO:721 and SEQ ID NO:726; SEQ ID NO:721 and SEQ ID NO:731; SEQ ID NO:721 and SEQ ID NO:732; SEQ ID NO:721 and SEQ ID NO:733; SEQ ID NO:721 and SEQ ID NO:734; SEQ ID NO:721 and SEQ ID NO:735; SEQ ID NO:721 and SEQ ID NO:736; SEQ ID NO:721 and SEQ ID NO:737; SEQ ID NO:728 and SEQ ID NO:722; SEQ ID NO:728 and SEQ ID NO:723; SEQ ID NO:728 and SEQ ID NO:724; SEQ ID NO:728 and SEQ ID NO:725; SEQ ID NO:728 and SEQ ID NO:726; SEQ ID NO:728 and SEQ ID NO:731; SEQ ID NO:728 and SEQ ID NO:732; SEQ ID NO:728 and SEQ ID NO:733; SEQ ID NO:728 and SEQ ID NO:734; SEQ ID NO:728 and SEQ ID NO:735; SEQ ID NO:728 and SEQ ID NO:736; SEQ ID NO:728 and SEQ ID NO:737; SEQ ID NO:729 and SEQ ID NO:722; SEQ ID NO:729 and SEQ ID NO:723; SEQ ID NO:729 and SEQ ID NO:724; SEQ ID NO:729 and SEQ ID NO:725; SEQ ID NO:729 and SEQ ID NO:726; SEQ ID NO:729 and SEQ ID NO:731; SEQ ID NO:729 and SEQ ID NO:732; SEQ ID NO:729 and SEQ ID NO:733; SEQ ID NO:729 and SEQ ID NO:734; SEQ ID NO:729 and SEQ ID NO:735; SEQ ID NO:729 and SEQ ID NO:726; SEQ ID NO:729 and SEQ ID NO:731; SEQ ID NO:729 and SEQ ID NO:732; SEQ ID NO:729 and SEQ ID NO:733; SEQ ID NO:729 and SEQ ID NO:734; SEQ ID NO:729 and SEQ ID NO:735; SEQ ID NO:729 and SEQ ID NO:736; NO:729 and SEQ ID NO:736; SEQ ID NO:729 and SEQ ID NO:737; SEQ ID NO:730 and SEQ ID NO:722; SEQ ID NO:730 and SEQ ID NO:723; SEQ ID NO:730 and SEQ ID NO:724; SEQ ID NO:730 and SEQ ID NO:725; SEQ ID NO:730 and SEQ ID NO:726; SEQ ID NO:730 and SEQ ID NO:731; SEQ ID NO:730 and SEQ ID NO:732; SEQ ID NO:730 and SEQ ID NO:733; SEQ ID NO:730 and SEQ ID NO:734; SEQ ID NO:730 and SEQ ID NO:735; SEQ ID NO:730 and SEQ ID NO:736; SEQ ID NO:730 and SEQ ID NO:737.

In some embodiments, the antisense strand comprises a nucleotide sequence shown in any one of SEQ ID NO:329-523.

In some embodiments, the positive strand comprises a nucleotide sequence shown in any one of SEQ ID NO:524-718.

In some embodiments, the sequences of the sense strand and antisense strand of the siRNA are selected from the sequences provided in Table 3. The sense and antisense strand sequences of any duplex of GPSZT082S1 to GPSZT233S5.

In some embodiments, the antisense strand comprises SEQ ID NOs: 329-331, 333-337, 339, 341, 344-356, 360, 362-363, 365, 367-369, 371-378, 380-381, 383, 385-393, 395-396, 398-404, 407-408, 411-417, 419-420, 421-423 5-426, 428, 430-431, 433-434, 438, 440-441, 446-447, 450-456, 458-459, 463-470, 472-474, 476-477, 479-480, 482, 484-486, 488-498, 500, 502-513, 515-516, 518-521.

In some embodiments, the positive strand comprises SEQ ID NOs: 524-526, 528-532, 534, 536, 539-551, 555, 557-558, 560, 562-564, 566-573, 575-576, 578, 580-588, 590-591, 593-599, 602-603, 606-612, 614-615, 62 0-621, 623, 625-626, 628-629, 633, 635-636, 641-642, 645-651, 653-654, 658-665, 667-669, 671-672, 674-675, 677, 679-681, 683-693, 695, 697-708, 710-711, and the nucleotide sequence shown in any one of 713-716.

In some embodiments, the sequences of the sense strand and antisense strand of the siRNA are selected from the sense strand and antisense strand sequences of any one of the duplexes provided in Table 7.

In some embodiments, the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 330, 333-334, 336, 339, 341, 346-347, 352, 368-369, 372-373, 375, 378, 380, 386, 411-412, 414, 417, 419, 434, 450-451, 453, 456, 458, 463-464, 486, 489-490, 492-493, 495, 497-498, and 503.

In some embodiments, the positive strand comprises the nucleotide sequence shown in any one of SEQ ID NOs:525, 528-529, 531, 534, 536, 541-542, 547, 563-564, 567-568, 570, 573, 575, 581, 606-607, 609, 612, 614, 629, 645-655, 657, 660, 662, 667-668, 681, 684-685, 687-688, 690, 692-693, and 698.

In some embodiments, the sequences of the sense and antisense strands of the siRNA are selected from the group consisting of GPSZT086S4, GPSZT086S5, GPSZT095S1, GPSZT091S5, GPSZT095S2, GPSZT095S4, GPSZT095S5, GPSZT086S3, GPSZT091S2, GPSZT091S4, GPSZT091S3, GPSZT095S3, GPSZT089S5, GPSZT098S5, GPSZT098S3, GPSZT086S1, GPSZT091S5, GPSZT091S2, GPSZT091S4, GPSZT091S3, GPSZT095S3, GPSZT089S5, GPSZT098S5, GPSZT098S3, GPSZT086S1, GPSZT091S5, GPSZT091S2, GPSZT091S4, GPSZT091S3, GPSZT095S3, GPSZT086S1, GPSZT091S5, GPSZT091S2, GPSZT091S4, GPSZT091S3, GPSZT095S3, GPSZT086S1 6S2, GPSZT089S4, GPSZT083S5, GPSZT098S2, GPSZT098S4, GPSZT098S1, GPSZT089S2, GPSZT121S4, GPSZT082S2, GPSZT089S1, GPSZT099S5, GPSZT153S1, GPSZT122S2, GPSZT122S1, GPSZT092S5, GPSZT089S3, GPSZT083S1, GPSZT121S1, GPSZT122S4, GPSZT083S2 or GPSZT177S3.

In some embodiments, the antisense strand comprises SEQ ID NOs: 329, 335, 337, 344-345, 348-351, 353, 355-356, 360, 362, 365, 371, 374, 376-377, 381, 383, 385, 387-393, 395, 399, 401-404, 407-408, 413, 415-416, 420, 425-426, 428, 430-431, 438, 446-447, 452, 454-455, 459, 465-467, 469-470, 473-474, 477, 482, 485, 491, 494, 500, 502, 504-510, 512, 516, 518-519, 521.

In some embodiments, the sense strand comprises SEQ ID NOs: 524, 530, 532, 539-540, 543-546, 548, 550-551, 555, 557, 560, 566, 569, 571-572, 576, 578, 580, 582-588, 590, 594, 596-599, 602-603, 608, 610- The nucleotide sequence shown in any one of 611, 615, 620-621, 623, 625-626, 633, 641-642, 647, 649-650, 654, 660-662, 664-665, 668-669, 672, 677, 680, 686, 689, 695, 697, 699-705, 707, 711, 713-714, and 716.

In some embodiments, the sequences of the sense and antisense strands of the siRNA are selected from the group consisting of GPSZT153S4, GPSZT177S4, GPSZT128S5, GPSZT174S3, GPSZT177S1, GPSZT082S4, GPSZT099S4, GPSZT174S2, GPSZT153S2, GPSZT083S4, GPSZT092S2, GPSZT174S4, GPSZT099S2, GPSZT092S3, GPSZT094S2, GPSZT123S4, GPSZT177S5, GPSZT174S3, GPSZT177S1, GPSZT082S4, GPSZT099S4, GPSZT174S2, GPSZT153S2, GPSZT083S4, GPSZT092S2, GPSZT174S4, GPSZT099S2, GPSZT092S3, GPSZT094S2, GPSZT123S4, GPSZT177S5, GPSZT174S3, GPSZT177S1 S3, GPSZT122S3, GPSZT174S1, GPSZT092S4, GPSZT177S2, GPSZT112S5, GPSZT094S5, GPSZT082S5, GPSZT123S2, GPSZT150S2, GPSZT082S1, GP SZT090S1, GPSZT094S4, GPSZT099S3, GPSZT153S5, GPSZT090S5, GPSZT090S2, GPSZT149S5, GPSZT112S1, GPSZT221S4, GPSZT092S1, GPSZT08 2S3, GPSZT174S5, GPSZT221S1, GPSZT221S3, GPSZT123S5, GPSZT094S3, GPSZT083S3, GPSZT090S3, GPSZT121S2, GPSZT128S4, GPSZT231S2, G PSZT221S5, GPSZT128S2, GPSZT123S1, GPSZT121S5, GPSZT149S4, GPSZT176S1, GPSZT090S4, GPSZT123S3, GPSZT150S5, GPSZT230S2, GPSZT1 The sense and antisense strand sequences of any duplex of GPSZT75S5, GPSZT112S2, GPSZT149S1, GPSZT128S1, GPSZT120S1, GPSZT223S1, GPSZT231S1, GPSZT221S2, GPSZT223S5, GPSZT150S1, GPSZT231S5, GPSZT149S2, GPSZT191S4, GPSZT175S2, GPSZT223S2, GPSZT085S2, GPSZT153S3, GPSZT231S4, GPSZT226S2 or GPSZT226S5.

In some embodiments, the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 334-335, 339, 353, 356, 373-374, 376, 378, 392-393, 395, 408, 411, 417, 428, 431, 434, 446, 450-452, 463, 470, 472-473, 489, 498, 500, 509, 512-513, and 516.

In some embodiments, the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 529-530, 534, 548, 551, 568-569, 571, 573, 587-588, 590, 603, 606, 612, 623, 626, 629, 641, 645-647, 658, 665, 667-668, 684, 693, 695, 704, 707-708, 711.

In some embodiments, the sequences of the sense and antisense strands of the siRNA are selected from GPSZT082S4, GPSZT083S3, GPSZT086S3, GPSZT086S4, GPSZT086S5, GPSZT089S1, GPSZT089S2, GPSZT089S4, GPSZT090S1, GPSZT090S2, GPSZT090S4, GPSZT092S2, GPSZT095S1, GPSZT095S2, GPSZT095S3, GPSZT095S4, GPSZT096S1, GPSZT096S2, GPSZT096S4, GPSZT096S5, GPSZT096S6, GPSZT096S7, GPSZT096S8, GPSZT096S9, GPSZT097S1, GPSZT097S2, GPSZT097S4, GPSZT097S5, GPSZT097S1, GPSZT097S2, GPSZT097S4, GPSZT097S5 The sense and antisense strand sequences of any duplex of GPSZT191S5, GPSZT221S4, GPSZT149S3, GPSZT174S1, GPSZT174S2, GPSZT174S3, GPSZT174S4, GPSZT174S5, GPSZT175S2, GPSZT176S4, GPSZT177S1, GPSZT177S2, GPSZT177S3, GPSZT177S4, GPSZT177S5, GPSZT191S5 or GPSZT221S5.

In some embodiments, the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 335, 339, 353, 356, 378, 392, 395, 411, 417, 431, 434, 450-451, 470, 473, 489, 500, 509, 512, and 516.

In some embodiments, the positive strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 530, 534, 548, 551, 573, 587, 590, 606, 612, 626, 629, 645-646, 665, 668, 684, 695, 704, 707, and 711.

In some embodiments, the sequences of the sense and antisense strands of the siRNA are selected from the sense and antisense strand sequences of any duplex of GPSZT086S3, GPSZT086S4, GPSZT086S5, GPSZT089S4, GPSZT090S1, GPSZT095S1, GPSZT095S2, GPSZT095S3, GPSZT112S5, GPSZT174S1, GPSZT174S2, GPSZT174S3, GPSZT174S4, GPSZT174S5, GPSZT177S1, GPSZT177S2, GPSZT177S3, GPSZT177S4, GPSZT177S5, or GPSZT221S5 provided in Table 10.

In some embodiments, the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 335, 339, 353, 356, 378, 392, 395, 431, 450, 470, 473, 489, 509, and 512.

In some embodiments, the positive strand comprises a nucleotide sequence shown in any one of SEQ ID NOs: 530, 534, 548, 551, 573, 587, 590, 626, 645, 665, 668, 684, 704, and 707.

In some embodiments, the sequences of the sense and antisense strands of the siRNA are selected from the sense and antisense strand sequences of any duplex of GPSZT086S4, GPSZT086S5, GPSZT090S1, GPSZT095S1, GPSZT095S2, GPSZT174S1, GPSZT174S2, GPSZT174S3, GPSZT174S4, GPSZT174S5, GPSZT177S1, GPSZT177S2, GPSZT177S4, or GPSZT177S5 provided in Table 11 or Table 12.

deliver

The siRNA of the invention can be delivered or introduced by any means known in the art (e.g., delivered or introduced into cells in vitro, or delivered or introduced into a patient in vivo). For example, for in vivo delivery, the siRNA can be injected into a tissue site. In vivo delivery can also be performed by a β-glucan delivery system. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection.

In some embodiments, the delivery method includes, but is not limited to, viral delivery (retrovirus, adenovirus, lentivirus, baculovirus, AAV); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC); nanoparticles (cationic polymers, PEI); bacterial delivery (tkRNAi); chemical modification of siRNA (LNA) to increase stability; lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles (low molecular weight polymers or high molecular weight polymers); double-stranded RNA binding motifs (dsRBMs); and other delivery systems known in the art to be suitable for nucleic acid or oligonucleotide delivery.

Conjugate

In one aspect, the invention provides a conjugate comprising at least one siRNA of the invention and a pharmaceutically acceptable targeting molecule. The conjugate of the invention is obtained by coupling the siRNA of the invention with a pharmaceutically acceptable targeting molecule, and the conjugate comprises a pharmaceutically acceptable targeting molecule and an optional linker. The siRNA can be non-covalently coupled to the targeting molecule, or covalently coupled to the targeting molecule.

The pharmaceutically acceptable targeting molecule can be a targeting molecule conventionally used in the field of siRNA administration, which typically enhances the pharmacokinetic or biodistribution properties of the siRNA connected thereto, and improves the cell-specific (or organ-specific) distribution and cell-specific (or organ-specific) uptake of the siRNA. Representative targeting molecules include, but are not limited to, compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies or antibody fragments, antibody mimetics, etc. In some embodiments, the targeting molecules include but are not limited to one or more of the following targeting molecules or their derivatives: integrins; lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); or receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

The linker can be a linker conventionally used in the field of siRNA administration, including but not limited to one or more of the following linkers or their derivatives: amide linker portion, amino linker portion, carbonyl linker portion, carbamate linker portion, urea linker portion, ether linker portion, disulfide linker portion, succinylamino linker portion, etc.

In some embodiments, the targeting molecule can be directly or indirectly linked to the siRNA of the present invention via a linker/linking group. In some embodiments, the targeting molecule is linked to the siRNA via an unstable, cleavable or reversible bond or linker. siRNA connection. In some embodiments, the targeting molecule is connected to at least one end of the sense strand and/or antisense strand of the siRNA. In some embodiments, the targeting molecule is connected to the 5' end and/or 3' end of the sense strand. In some embodiments, the targeting molecule is connected to the 5' end and/or 3' end of the antisense strand.

Pharmaceutical composition

In one aspect, the invention provides a pharmaceutical composition comprising at least one siRNA of the invention.

In some embodiments, the pharmaceutical composition contains 1 siRNA as described above.

In other embodiments, the pharmaceutical composition contains at least two of the siRNAs described above (for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) as active ingredients. Preferably, the at least two siRNAs each target a different target sequence in the lfTSLP gene (for example, the sequence shown in SEQ ID NO: 727), so that it can be expected that they will act simultaneously on different target sequences to bring about a synergistic effect. Here, the so-called "different target sequences" means that there is no overlap between the target sequences, or the number of overlapping continuous nucleotides between the target sequences is less than 5 (for example, the number of overlapping continuous nucleotides is 4, 3, 2, 1, or 0). In this case, the at least two of the above-mentioned siRNAs may be present in any different proportions. Preferably, the at least two siRNAs mentioned above may be present in a molar ratio of 1:100 to 100:1; more preferably, the at least two siRNAs mentioned above may be present in a molar ratio of 1:10 to 10:1, 1:5 to 5:1 or 1:2 to 2:1. In some embodiments, the at least two siRNAs mentioned above are present in the same molar ratio.

In other embodiments, the pharmaceutical composition contains at least one siRNA of the present invention and also contains at least one siRNA targeting other targets (e.g., other genes other than lfTSLP). In this case, the siRNA of the present invention and the siRNA targeting other targets can be present in any different ratios, such as in a molar ratio of 1:100 to 100:1, such as in a molar ratio of 1:10 to 10:1, 1:5 to 5:1 or 1:2 to 2:1, such as in the same molar ratio.

In some embodiments, the pharmaceutical composition comprises an effective amount of siRNA. An "effective amount" means an amount of siRNA that is effective to produce a desired pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or condition, the therapeutically effective amount of a drug for treating the disease or condition is the amount required to achieve at least a 10% reduction in that parameter. For example, a therapeutically effective amount of siRNA targeting lfTSLP can reduce lfTSLP mRNA levels by at least 10%.

In some embodiments, in the pharmaceutical composition described in any of the above embodiments, the siRNA can be linked to a targeting molecule to form a conjugate. Therefore, in some embodiments, the pharmaceutical composition comprises the conjugate of the present invention.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In some embodiments, the pharmaceutically acceptable carrier and/or excipient is a delivery vehicle. The delivery vehicle is A substance that improves the delivery of nucleic acids or oligonucleotides to cells or tissues. Such substances can be any delivery vector known in the art that is suitable for nucleic acid or oligonucleotide delivery, including but not limited to: viruses (retrovirus, adenovirus, lentivirus, baculovirus, AAV); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC); nanoparticles (cationic polymers, PEI); bacteria (tkRNAi); lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles (low molecular weight polymers or high molecular weight polymers); double-stranded RNA binding motifs (dsRBMs), etc.

In some embodiments, the siRNA may be packaged by the delivery vehicle.

In some embodiments, the siRNA can be directly or indirectly connected to the delivery vector via a joint/connecting group. In some embodiments, the delivery vector is connected to the siRNA via an unstable, cleavable or reversible bond or joint. In some embodiments, the delivery vector is connected to at least one end of the sense strand and/or antisense strand of the siRNA. In some embodiments, the delivery vector is connected to the 5' end and/or 3' end of the sense strand. In some embodiments, the delivery vector is connected to the 5' end and/or 3' end of the antisense strand.

In some embodiments, the pharmaceutical composition comprises one siRNA provided by the present invention, and the siRNA is encapsulated by the delivery vector. The one siRNA can be encapsulated in the same delivery vector or in different delivery vectors.

In other embodiments, the pharmaceutical composition comprises at least two siRNAs provided by the present invention (for example, but not limited to, two, three, four, five, six, seven, eight, nine, ten or more), wherein the siRNAs are packaged by the delivery vector. The at least two siRNAs can be packaged in the same delivery vector or in different delivery vectors. Preferably, the at least two siRNAs each target a different target sequence in the lfTSLP gene.

In other embodiments, the pharmaceutical composition comprises at least one siRNA provided by the present invention and siRNA targeting other targets (e.g., other genes other than lfTSLP), wherein the siRNA is packaged by the delivery vector. The siRNA provided by the present invention and siRNA targeting other targets (e.g., other genes other than lfTSLP) can be packaged in the same delivery vector or in different delivery vectors.

In some embodiments, the pharmaceutical composition of the present invention is formulated into a dosage form compatible with its intended route of administration, for example, it can be administered locally (such as direct injection or implantation), systemically, or subcutaneously, intravenously, intraperitoneally, or parenterally, including intracranial (such as intraventricular, intrameningeal, or intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

In some embodiments, the pharmaceutical composition is administered by inhalation, intranasal administration, intratracheal administration, or oropharyngeal inhalation. Formulations suitable for inhalation administration can be prepared by incorporating the required amount of active ingredient into an appropriate solvent, followed by sterile filtration. Typically, formulations for inhalation administration are sterile solutions at physiological pH and have low viscosity. Salts are added to the formulation to balance tonicity. In some cases, surfactants or cosolvents may be added to increase active ingredient solubility and improve aerosol properties. In some cases, excipients may be added to control viscosity in order to ensure the size and distribution of the atomized droplets.

In other embodiments, the pharmaceutical composition can be administered by injection, such as intravenous, intramuscular, subcutaneous, intradermal, intraarticular, intraocular, intraperitoneal or topical administration. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, or phosphate buffered saline (PBS) and the like. The pharmaceutical composition should remain stable under production and storage conditions, and should prevent the contamination of microorganisms such as bacteria and fungi. For example, sterile injection solutions can be prepared by the following method: the active ingredient of the required dose is mixed in an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delayed absorption, preservatives, or any combination thereof) are mixed at the same time, followed by filtration and sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use.

The siRNA of the present invention can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be administered once, multiple times over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

application

Inhibition of lfTSLP expression

The siRNA of the present invention can be used to inhibit the expression of lfTSLP in vitro and/or in vivo. The lfTSLP refers to the long subtype TSLP, such as the mRNA with the sequence shown in Genbank: NM_033035.5.

In one aspect, the present invention provides a method for inhibiting lfTSLP expression in a cell, the method comprising: introducing into the cell an siRNA, a conjugate or a pharmaceutical composition of the present invention. In some embodiments, the method is implemented in vitro. The siRNA of the present invention can be introduced by any nucleic acid delivery method known in the art, such as electroporation or lipofection.

The term "inhibit the expression of lfTSLP" means at least partial suppression of the expression of lfTSLP gene, which can be manifested as a decrease in the amount of detectable lfTSLP mRNA. For example, the degree of inhibition can be expressed as: (mRNA in control cells) - (mRNA in treated cells)/(mRNA in control cells)*100%. Alternatively, the degree of inhibition can be given as a reduction in a parameter functionally associated with lfTSLP gene expression, such as the amount of protein encoded by the lfTSLP gene. In principle, lfTSLP gene silencing can be measured in any cell expressing lfTSLP (constitutively or by genetic engineering), and by any suitable assay. Measurements can be made at multiple time points, before, during and after administration of siRNA, to determine the effect of siRNA. The level or expression of lfTSLP can be measured by evaluation of mRNA (e.g., by Northern blot or PCR) or protein (e.g., Western blot or ELISA). For example, the effect of siRNA on lfTSLP expression can be measured by measuring the transcription rate of the lfTSLP gene (e.g., by RT-PCR). For example, the effect of siRNA on lfTSLP expression can be measured by measuring the expression of a reporter gene (e.g., luciferase) fused to the lfTSLP gene.

In some embodiments, by administering the siRNA of the present invention, the expression of the lfTSLP gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. In some embodiments, by administering the siRNA of the present invention, the expression of the lfTSLP gene is suppressed by at least about 60%, 70% or 80%. In some embodiments, by administering the siRNA of the present invention, the expression of the lfTSLP gene is suppressed by at least about 85%. In some embodiments, by administering the siRNA of the present invention, the expression of the lfTSLP gene is suppressed by at least about 90%. In some embodiments, by administering the siRNA of the present invention, the expression of the lfTSLP gene is suppressed by at least about 95%. In some embodiments, by administering the siRNA of the present invention, the expression of the lfTSLP gene is suppressed by at least about 96%, 97%, 98%, 99% or 100%.

In some embodiments, the siRNA, the conjugate or the pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (eg, siRNA targeting a different target sequence in the lfTSLP gene or siRNA targeting other targets).

In some embodiments, one siRNA provided by the present invention is used. The siRNA is optionally packaged by a delivery vector. In some embodiments, the one siRNA is packaged in the same delivery vector. In other embodiments, the one siRNA is packaged in different delivery vectors.

In other embodiments, at least two siRNAs provided by the present invention (for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) are used, preferably each of the at least two siRNAs targets a different target sequence in the lfTSLP gene. The at least two siRNAs are optionally packaged by a delivery vector. In some embodiments, the at least two siRNAs are packaged in the same delivery vector. In other embodiments, the at least two siRNAs are respectively packaged in different delivery vectors.

In other embodiments, at least one siRNA provided by the present invention and siRNA targeting other targets (e.g., other genes other than lfTSLP) are used. 1fTSLP) is optionally packaged in a delivery vector. In some embodiments, the siRNA provided by the present invention and the siRNA targeting other targets (e.g., other genes other than 1fTSLP) are packaged in the same delivery vector. In other embodiments, the siRNA provided by the present invention and the siRNA targeting other targets (e.g., other genes other than 1fTSLP) are packaged in different delivery vectors.

In some embodiments, the siRNA of the invention inhibits the expression of lfTSLP and does not affect the expression of short isoform TSLP (sfTSLP).

Treatment of lfTSLP-related diseases

The siRNAs of the invention can be used to treat diseases or conditions that would benefit from reduced levels or inhibition of expression of lfTSLP.

In one aspect, the present invention provides a method for preventing and/or treating a disease associated with lfTSLP in a subject, the method comprising administering an effective amount of the siRNA, conjugate or pharmaceutical composition of the present invention to a subject in need thereof. The present invention also relates to the use of the siRNA, conjugate or pharmaceutical composition of the present invention in the preparation of a medicament for treating and/or preventing a disease associated with lfTSLP.

In some embodiments, the disease associated with lfTSLP involves lfTSLP overexpression. lfTSLP overexpression refers to lfTSLP levels (e.g., lfTSLP levels present in plasma or tissues of a subject, and preferably in damaged tissues) that are higher than normal lfTSLP levels (e.g., corresponding levels of healthy controls).

In some embodiments, the disease associated with lfTSLP would benefit from decreased levels or inhibition of expression of lfTSLP.

In some embodiments, the disease associated with lfTSLP is an inflammatory disease, such as an inflammatory respiratory disease, an inflammatory digestive disease, or an inflammatory skin disease.

In some embodiments, the disease associated with lfTSLP is asthma, nasal polyps, allergic rhinitis, chronic sinusitis, atopic dermatitis, eosinophilic esophagitis, chronic obstructive pulmonary disease, or idiopathic pulmonary fibrosis.

In some embodiments, if lfTSLP expression is elevated for a particular disease, treatment with the siRNA of the invention may reduce the level or expression of lfTSLP to a level that is within the normal range considered for individuals without such a disorder.

In some embodiments, the subject is a mammal, such as a human.

In some embodiments, the siRNA, the conjugate or the pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., siRNA targeting a different target sequence in the lfTSLP gene or siRNA targeting other targets), for example, administered simultaneously or sequentially.

In some embodiments, one siRNA provided by the present invention is used. The siRNA is optionally delivered via a delivery vector. In some embodiments, the 1 siRNA is packaged in the same delivery vector. In other embodiments, the 1 siRNA is packaged in different delivery vectors.

In other embodiments, at least two siRNAs provided by the present invention (for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) are used, preferably each of the at least two siRNAs targets a different target sequence in the lfTSLP gene. The at least two siRNAs are optionally packaged by a delivery vector. In some embodiments, the at least two siRNAs are packaged in the same delivery vector. In other embodiments, the at least two siRNAs are respectively packaged in different delivery vectors.

In other embodiments, at least one siRNA provided by the present invention and siRNA targeting other targets (e.g., other genes other than lfTSLP) are used. The siRNA provided by the present invention and siRNA targeting other targets (e.g., other genes other than lfTSLP) are optionally packaged by a delivery vector. In some embodiments, the siRNA provided by the present invention and siRNA targeting other targets (e.g., other genes other than lfTSLP) are packaged in the same delivery vector. In other embodiments, the siRNA provided by the present invention and siRNA targeting other targets (e.g., other genes other than lfTSLP) are respectively packaged in different delivery vectors.

Definition of terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

In this article, lfTSLP mRNA refers to long subtype TSLP (lfTSLP) mRNA. The long subtype TSLP (lfTSLP) mRNA sequence is well known to those skilled in the art, for example, see the mRNA with the sequence shown in Genbank registration number NM_033035.5. Further, unless otherwise specified, the term "target gene" used in this article refers to a gene that can transcribe the above-mentioned lfTSLP mRNA, the term "target mRNA" refers to the above-mentioned lfTSLP mRNA, and the term "inhibiting lfTSLP gene" refers to inhibiting the expression of lfTSLP.

Herein, short-chain TSLP (sfTSLP) mRNA is well known to those skilled in the art, for example, see the mRNA having the sequence shown in Genbank Accession No. NM_138551.5.

In this article, capital letters C, G, U, A, and T represent the base composition of nucleotides, including modified or unmodified nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated modified nucleotide; (LNA) indicates that the nucleotide adjacent to the left of (LNA) is a locked nucleic acid-modified nucleotide; (d) indicates that the nucleotide adjacent to the left of (d) is a 2'-deoxyribonucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are connected by thiophosphate groups; VP indicates that the nucleotide adjacent to the right of VP is a phosphate mimetic at the 5'position; (GNA) indicates that the (GNA) The adjacent nucleotide on the left is a glycerol nucleotide.

As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified ribose moiety, a modified internucleoside bond, or a modified base. Therefore, the term "modified nucleotide" encompasses substitution, addition, or removal (e.g., using a functional group or atom) of an internucleoside bond, a ribose moiety, or a base. Modifications applicable to the present invention include all types of modifications disclosed herein or known in the art. "Methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by a methoxy group. "Fluoro-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by a fluorine group. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids, but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Such as isonucleotides, bridged nucleotides (BNA for short) or acyclic nucleotides.

Among them, the structure of the fluorinated modified nucleotide is as follows:

The structure of a methoxy-modified nucleotide is shown below:

The nucleotide structure of the locked nucleic acid is as follows:

The structure of a 2'-methoxyethyl modified nucleotide is shown below:

The nucleotide structure of the 5'-phosphate analog is shown below:

The structure of glycerol nucleotide (GNA) is shown below:

The structure of Unlocked Nucleic Acid (UNA) is shown below:

As used herein, the term "siRNA" means an RNA molecule that can induce RNAi phenomenon with sequence specificity, is composed of a sense strand and an antisense strand, and has a partially or completely complementary double-stranded structure. In the siRNA involved in the present invention, the length of the complementary double-stranded structure can be 14 to 30 base pairs, for example, 14, 15, 16, 17, 18, 19, 20 or 21 base pairs, for example, 14 to 19 base pairs. In some embodiments of the present invention, the siRNA may also contain modified nucleotides as needed, and the modified nucleotides will not cause the siRNA to significantly weaken or lose the function of inhibiting the expression of the lfTSLP gene. Currently, there are many methods in the art that can be used to modify siRNA, including, for example, backbone modification (such as phosphate group modification), ribose group modification and base modification (Watts, J.K., G.F.Deleavey, and M.J.Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008.13(19-20):p.842-55).

The term "antisense strand" includes a region that is substantially complementary to a target sequence. The term "sense strand" as used herein refers to a strand having a region that is substantially complementary to a region of the antisense strand. The term "complementary region" refers to a region on the antisense strand that is substantially complementary to lfTSLP mRNA or a region on the sense strand that is substantially complementary to the antisense strand. When the complementary region is substantially complementary to the target sequence, the complementary region is substantially complementary to the target sequence. When the sequences are not completely complementary, mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, for example, within 5, 4, 3, 2 or 1 nucleotides of the 5' and/or 3' termini.

In this article, unless otherwise specified, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize with an oligonucleotide of a second sequence under certain conditions and form a double-stranded structure. "At least partially complementary" means that the two sequences can be completely complementary, or generally no more than 6, 5, 4, 3 or 2 or 1 mismatched base pairings, while retaining the ability to hybridize under relevant conditions. In addition, in the case where two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs should not be regarded as mismatches in terms of determining complementarity. In this article, when the above hybridization ability requirements are met, the "complementary" sequence can also include or be completely formed from non-Watson (Watson) -Crick (Crick) base pairs and/or base pairs formed from non-natural and modified nucleotides. Such non-Watson (Watson) -Crick (Crick) base pairs include but are not limited to G: U wobble base pairing or Hoogstein (Hu Gesten) base pairing. Correspondingly, in this article, unless otherwise specified, "mismatch" means that in the siRNA duplex molecule, the bases at corresponding positions are not paired in a complementary form.

A person skilled in the art will be able to determine the conditions most suitable for testing the complementarity of two sequences, depending on the final application of the hybridized nucleotides. Such conditions may, for example, be stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, may also be applied.

In this article, unless otherwise specified, "nucleotide sequence difference" means that the base type of the nucleotide at the same or corresponding position has changed compared to the original nucleotide sequence. For example, when one nucleotide base in the original nucleotide sequence is A, when the nucleotide base at the same or corresponding position is changed to U, C, G or dT, dC, dG, etc., it is considered that there is a difference in the nucleotide sequence at this position. It should be noted here that, compared to the original nucleotide sequence, when the nucleotide at the same or corresponding position differs only in whether there is a modification or the type of modification, it is not considered that there is a difference in the nucleotide sequence at this position.

In this article, unless otherwise specified, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier, delivery vehicle, diluent, excipient and/or salt/ester/hydrate formed thereof, etc., which is usually chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form (such as the siRNA of the present invention) and physiologically compatible with the subject. "Pharmaceutically acceptable carriers and/or excipients" do not play or are not intended to play a therapeutic role at the expected dose. Such ingredients can play the following roles: a) assist in the processing of the drug delivery system during manufacturing, b) protect, support or enhance the stability, bioavailability or patient acceptability of the active ingredient, c) assist in product identification, and/or d) enhance the overall safety, effectiveness, delivery, etc. of the active ingredient during storage and use. Any other properties. For example, the siRNA of the present invention can be packaged by a delivery vehicle. "Pharmaceutically acceptable carriers and/or excipients" include but are not limited to: viruses, lipids Bodies, nanoparticles, bacteria, lipid nanoparticles (LNP), neutral liposomes (NL), polymer nanoparticles, double-stranded RNA binding motifs (dsRBMs), pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delayed absorption, preservatives. For example, viruses include but are not limited to retroviruses, adenoviruses, lentiviruses, baculoviruses, AAV. Liposomes include but are not limited to Lipofectamine, cationic DOTAP, neutral DOPC. Nanoparticles include but are not limited to cationic polymers, PEI. Bacteria include but are not limited to tkRNAi. Polymer nanoparticles include but are not limited to low molecular weight polymers or high molecular weight polymers. For example, pH regulators include but are not limited to phosphate buffers. Surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial agents and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents for maintaining osmotic pressure include but are not limited to sugars, NaCl and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.

In this article, unless otherwise specified, the term "inhibition" refers to the situation where the target gene expression is down-regulated (down-regulation) due to the degradation of the mRNA of the target gene mediated by siRNA. The "down-regulation" refers to the situation where the target gene expression level decreases by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more or even 100% relative to the situation without siRNA treatment. Among them, a 100% decrease in the target gene expression level means that there is no detectable level of target gene expression.

In this article, the term "overhang" or "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the siRNA duplex structure. For example, when the 3' end of one strand of the siRNA extends beyond the 5' end of the other strand (or vice versa), there is an overhang. The siRNA may include an overhang of at least one nucleotide, or the overhang may include at least 2nt, at least 3nt, at least 4nt, at least 5nt or more. The overhang may include nucleotide/nucleoside analogs, or be composed of nucleotide/nucleoside analogs, and the nucleotide/nucleoside analogs include deoxyribonucleotides/nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. The nucleotides of the overhang may be present on the 5' end, 3' end, or both ends of the antisense strand or sense strand of the siRNA. Accordingly, the term "flat end" refers to the absence of nucleotide overhangs.

As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or disorder or symptom in a subject; the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, The effective amount for preventing a disease is an amount sufficient to prevent, prevent, or delay the occurrence of a disease; the effective amount for treating a disease is an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

Advantageous Effects of the Invention

The siRNA of the present invention can effectively inhibit lfTSLP gene expression in vitro and/or in vivo, and can specifically target the long subtype TSLP with pro-inflammatory activity, inhibit the expression of long subtype TSLP mRNA or protein, without affecting the expression of short subtype TSLP with anti-inflammatory activity, and has good stability. Therefore, the siRNA of the present invention can be used to treat diseases or conditions that benefit from the reduction of lfTSLP levels or the inhibition of expression, and has important clinical value for the treatment of lfTSLP-related diseases.

Embodiments of the present invention will be described in detail below in conjunction with examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become implementable for those skilled in the art.

DETAILED DESCRIPTION

The invention is now described in the following non-limiting examples.

Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

It is known to those skilled in the art that the siRNA of the present invention can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis), wherein both solid phase synthesis and liquid phase synthesis have commercial customization services. It is also clear to those skilled in the art that modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleotide monomers with corresponding modifications. Methods for preparing nucleotide monomers with corresponding modifications are well known to those skilled in the art, and commercial monomers are also available on the market.

Preparation Example 1: Unmodified siRNA Synthesis

This preparation example provides a siRNA for inhibiting the expression of lfTSLP. The nucleotide sequence of the siRNA is designed based on the target mRNA, as shown in Table 1.

Table 1: siRNA sequences

Preparation Example 2: Synthesis of modified siRNA

This preparation example provides a modified siRNA for inhibiting the expression of lfTSLP. The modification pattern of the siRNA is shown in Table 2, and the modified siRNA sequence is shown in Table 3.

Modification instructions:

In Table 2-3, Nm is a methoxy-modified nucleotide, Nf is a fluorinated-modified nucleotide, N(LNA) is a locked nucleic acid-modified nucleotide, N(d) is a 2'-deoxyribonucleotide, s is a phosphorothioate linkage, VP represents a 5'-phosphate analog, and N(GNA) represents a glycerol nucleotide.

The sequence name suffixes S1, S2, S3, S4, and S5 in Table 3 represent different modification methods, which are described as follows:

S1 represents the modification pattern of the sense strand SEQ ID NO: 722 + the modification pattern of the antisense strand SEQ ID NO: 719. The sense strand includes the following chemical modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and at positions 2 and 3 are linked by thiophosphate groups, the nucleotides at positions 1, 2, 4, 6, 10, 12, 14, 16, and 18 are methoxy-modified nucleotides, the nucleotides at positions 3, 5, 7, 8, 9, 11, 13, 15, 17, and 19 are fluorinated-modified nucleotides, and the antisense strand includes the following chemical modifications ： From the 5' end to the 3' end, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups, the nucleotides at positions 1, 3, 5, 7, 9, 11, 12, 13, 15, 17, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, and 18 are fluorine-modified nucleotides.

S2 represents the modification pattern of the sense strand SEQ ID NO: 723 + the modification pattern of the antisense strand SEQ ID NO: 720. The sense strand includes the following chemical modifications: from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 2 and 3 are linked by thiophosphate groups, the nucleotides at positions 1, 2, 4, 5, 6, 10, 12, 14, 16, 17, 18, and 19 are methoxy-modified nucleotides, and the nucleotides at positions 3, 7, 8, 9, 11, 13, and 15 are fluorinated modified nucleotides; the antisense strand includes the following chemical modifications ： From the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are linked by thiophosphate groups, the nucleotides at positions 1, 3, 4, 5, 7, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 8, 14, and 16 are fluorine-modified nucleotides.

S3 represents the modification pattern of the sense strand SEQ ID NO: 724 + the modification pattern of the antisense strand SEQ ID NO: 721. The sense strand includes the following chemical modifications: from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and at positions 2 and 3 are linked by thiophosphate groups, the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy-modified nucleotides, the nucleotides at positions 5, 7, and 8 are fluorinated-modified nucleotides, and the nucleotide at position 9 is a deoxyribonucleotide; the antisense strand includes The invention comprises the following chemical modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are linked by thiophosphate groups, the nucleotides at positions 1, 3, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 4, 6, 14, and 16 are fluorinated-modified nucleotides.

S4 represents the positive strand modification pattern SEQ ID NO: 725 + the antisense strand modification pattern SEQ ID NO: 720. The sense strand includes the following chemical modifications: in the direction from the 5' end to the 3' end, the nucleotides at the 1st and 2nd positions, and the 2nd and 3rd positions are linked by thiophosphate groups, the nucleotide at the 1st position is a locked nucleic acid modified nucleotide, the nucleotides at the 2nd, 4th, 5th, 6th, 10th, 12th, 14th, 16th, 17th, 18th, and 19th positions are methoxy modified nucleotides, and the nucleotides at the 3rd, 7th, 8th, 9th, 11th, 13th, and 15th positions are fluorinated modified nucleotides; the antisense strand includes The invention comprises the following chemical modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are linked by thiophosphate groups, the nucleotides at positions 1, 3, 4, 5, 7, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 8, 14, and 16 are fluorinated-modified nucleotides.

S5 indicates the modification pattern of the sense strand SEQ ID NO: 726 + the modification pattern of the antisense strand SEQ ID NO: 720. The sense strand includes the following chemical modifications: from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 2 and 3 are linked by thiophosphate groups, the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 16, 17, 18, and 19 are methoxy-modified nucleotides, and the nucleotides at positions 5, 7, 8, 9, and 15 are fluorinated-modified nucleotides; the antisense strand includes the following chemical modifications ： From the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are linked by thiophosphate groups, the nucleotides at positions 1, 3, 4, 5, 7, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 8, 14, and 16 are fluorine-modified nucleotides.

Antisense strand modification pattern 4 (SEQ ID NO: 728) indicates that: from the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are connected by thiophosphate groups, the nucleotide at position 1 is a methoxy-modified nucleotide containing a 5'-phosphate mimetic, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, and the nucleotides at positions 2, 4, 6, 14, and 16 are fluorine-modified nucleotides.

Antisense strand modification pattern 5 (SEQ ID NO: 729) indicates that: from the 5' end to the 3' end, the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 are connected by thiophosphate groups, the nucleotide at position 1 is a methoxy-modified nucleotide containing a 5'-phosphate mimetic, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, the nucleotides at positions 2, 4, 14, and 16 are fluorine-modified nucleotides, and the nucleotide at position 6 is a glycerol nucleotide.

Antisense strand modification pattern 6 (SEQ ID NO: 730) indicates that, from the 5' end to the 3' end, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are modified by thiolation. Phosphate-linked, the nucleotide at position 1 is a methoxy-modified nucleotide containing a 5'-phosphate mimetic, the nucleotides at positions 3, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy-modified nucleotides, the nucleotides at positions 2, 4, 14, and 16 are fluorine-modified nucleotides, and the nucleotide at position 7 is a glycerol nucleotide.

The positive chain modification pattern 6 (SEQ ID NO:731) indicates that: in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 2 and 3 are linked by thiophosphate groups, the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy-modified nucleotides, the nucleotides at positions 5 and 8 are fluorine-modified nucleotides, and the nucleotides at positions 7 and 9 are 2'-deoxyribonucleotides.

The positive chain modification pattern 7 (SEQ ID NO:732) indicates that: from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 18 and 19 are connected by thiophosphate groups, the nucleotides at positions 1, 2, 4, 6, 10, 12, 14, 16, and 18 are methoxy-modified nucleotides, and the nucleotides at positions 3, 5, 7, 8, 9, 11, 13, 15, 17, and 19 are fluorine-modified nucleotides.

The positive chain modification pattern 8 (SEQ ID NO:733) indicates that: from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 18 and 19 are connected by thiophosphate groups, the nucleotides at positions 1, 2, 4, 5, 6, 10, 12, 14, 16, 17, 18, and 19 are methoxy-modified nucleotides, and the nucleotides at positions 3, 7, 8, 9, 11, 13, and 15 are fluorine-modified nucleotides.

The positive chain modification pattern 9 (SEQ ID NO:734) indicates that: in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 18 and 19 are connected by thiophosphate groups, the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy-modified nucleotides, the nucleotides at positions 5, 7, and 8 are fluorine-modified nucleotides, and the nucleotide at position 9 is a 2'-deoxyribonucleotide.

The positive chain modification pattern 10 (SEQ ID NO:735) indicates that: in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 18 and 19 are connected by thiophosphate groups, the nucleotides at positions 2, 4, 5, 6, 10, 12, 14, 16, 17, 18, and 19 are methoxy-modified nucleotides, the nucleotides at positions 3, 7, 8, 9, 11, 13, and 15 are fluorine-modified nucleotides, and the nucleotide at position 1 is a locked nucleic acid-modified nucleotide.

The positive chain modification pattern 11 (SEQ ID NO:736) indicates that: in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and 18 and 19 are connected by thiophosphate groups, the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 16, 17, 18, and 19 are methoxy-modified nucleotides, and the nucleotides at positions 5, 7, 8, 9, and 15 are fluorine-modified nucleotides.

The positive strand modification pattern 12 (SEQ ID NO: 737) indicates that, from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and positions 18 and 19 are linked by thiophosphate groups, and the nucleotides at positions 1, 2, 3, 4, 6, 10, The nucleotides at positions 11, 12, 13, 14, 15, 16, 17, 18 and 19 are methoxy-modified nucleotides, the nucleotides at positions 5 and 8 are fluorine-modified nucleotides, and the nucleotides at positions 7 and 9 are 2'-deoxyribonucleotides.

Table 2: Modification patterns of siRNA

Table 3: Modified siRNA sequences

The experimental cells involved in the following examples are 293T, which were purchased from the Cell Bank of the Chinese Academy of Sciences.

Example 1: On-target activity detection of unmodified siRNA for inhibition of lfTSLP

This example provides an on-target activity detection experiment of unmodified siRNA for inhibiting lfTSLP. A plasmid vector is constructed using a psiCHECK2 vector for detection. The psiCHECK2 vector is a plasmid vector that can monitor changes in the expression of a target gene fused with a reporter gene. The vector uses Renilla luciferase as the main reporter gene. The target fragment is cloned into the multiple cloning site downstream of the translation stop codon of Renilla luciferase. The RNAi process against the target gene initiated by the synthetic siRNA leads to the shearing and subsequent degradation of the fusion mRNA. By detecting changes in the activity of Renilla luciferase, it can be determined whether the siRNA has a targeting relationship with the target gene fragment. The experimental process is as follows:

(1) Construction of detection plasmid TSLP-psiCHECK2

The detection plasmid was constructed using psiCHECK ^TM -2 (Promega ^TM ) plasmid. The detection plasmid contained the insertion sequence shown below. The insertion sequence was derived from the mRNA sequence shown in the Genbank registration number NM_033035.5. A single copy of the inserted sequence was cloned into the Xho I/Not I site of the psiCHECK ^TM -2 plasmid to obtain the detection plasmid TSLP-psiCHECK2;

Insert sequence: 5’-gtttctttcaggaaaatcttcatcttacaacttgtagggctggtgttaacttacgacttcactaactgtgactttgagaagattaaagcagcctat ctcagtactatttctaaagacctgattacatatatgagtgggaccaaaagtaccgagttcaacaacaccgtctcttgtagcaatcg-3’ (SEQ ID NO: 727).

(2) Cell culture and transfection

In a 96-well plate, 12.5 μl of Opti-MEM containing 20 ng TSLP-psiCHECK2 detection plasmid and 32.5 μl of Opti-MEM plus 0.3 μl Lipofectamine 2000 (purchased from Invitrogen, catalog number 11668-019) were added to each well of 5 μl siRNA and incubated at room temperature for 15 minutes. Then, 50 μl of DMEM complete medium containing 1×10 ⁴ 293T cells (purchased from Transgen Biotech, catalog number FI101-01) was added to each well of the above mixture and cultured at 37°C for 24 hours for subsequent dual luciferase detection experiments. The experimental concentration was carried out at a final siRNA concentration of 1 nM.

(3) Dual luciferase assay

The 5× lysate in the dual luciferase assay kit (purchased from Promega, catalog number E2940) was diluted with water to 1× lysate. The cells obtained by culturing in step (2) were discarded, and each well was diluted and washed twice with PBS buffer (purchased from Hyclone, catalog number SH30256.01), and 50 μL/well of 1× lysate was added to each cell plate, and lysed at room temperature for 20 minutes to obtain a lysed cell plate; 30 μL/well of lysate was drawn from the lysed cell plate and added to an opaque 96-well detection plate, and the dual luciferase assay kit was taken, and two substrates were prepared according to the instructions, and the two substrates were added to new 96-well plates, and 30 μL/well of substrate 1 and substrate 2 were added, respectively, and each time the substrate was added, the multifunctional microplate reader was used for detection, and the numerical results of firefly luciferase and Renilla luciferase were obtained, respectively.

Calculate the luminescence ratio of each well of the ELISA plate = Renilla/Firefly, the luminescence ratio of each test group or control group is the average of the luminescence ratios of three culture wells; take the luminescence ratio of the control group as the benchmark, normalize the luminescence ratio of each test group, and obtain the ratio R of luminescence ratio (test)/luminescence ratio (control), which represents the expression level of Renilla reporter gene, that is, the relative residual activity. The inhibition rate of siRNA is (1-R)×100%, where MOCK is a blank control group with only transfection reagent and plasmid added.

The on-target activity results are shown in Table 4 below, which show that these duplexes have inhibitory activity against lfTSLP expression.

Table 4: On-target activity detection of unmodified siRNA

Example 2: On-target activity detection of unmodified siRNA for inhibition of lfTSLP

The sequences with higher inhibition rates in Example 1 were tested for on-target activity under conditions of siRNA final concentrations of 0.01 nM, 0.1 nM and 1 nM. The test method used was the same as that used in Example 1, except that the final concentrations of siRNA were different, and MOCK was a blank control group in which only transfection reagent and plasmid were added. The test results are shown in Table 5 below, and the results show that these duplexes have good inhibitory activity on lfTSLP expression at different concentrations.

Table 5: On-target activity detection of unmodified siRNA at different concentrations

Example 3: On-target activity detection of modified siRNA for inhibition of lfTSLP

The siRNA with higher inhibition rate in Examples 1 and 2 was chemically modified, and the specific sequence is shown in Table 3. The on-target activity was detected under the condition that the final concentration of siRNA was 1 nM. The test method used was the same as that used in Example 1, except that the siRNA was different, and MOCK was a blank control group with only transfection reagent and plasmid added. The test results are shown in Table 6 below, and the results show that the chemically modified duplex has good inhibitory activity on lfTSLP expression.

Table 6: On-target activity detection of modified siRNA

Example 4: On-target activity detection of modified siRNA for inhibition of lfTSLP

The sequences with higher inhibition rates in Example 3 were tested for on-target activity under conditions of siRNA final concentrations of 0.01 nM, 0.1 nM and 1 nM. The test method used was the same as that used in Example 1, except that the siRNA used and its final concentration were different, and MOCK was a blank control group in which only transfection reagent and plasmid were added. The test results are shown in Table 7 below, and the results show that the chemically modified duplexes have good inhibitory activity on lfTSLP expression at different concentrations.

Table 7: On-target activity detection of modified siRNA at different concentrations

Example 5: Activity detection of modified siRNA in stably transfected cell line hTSLP-A549

Genemax was commissioned to construct a stable cell line hTSLP-A549 that overexpressed human TSLP protein (human TSLP sequence gene number is NM_033035.5) in A549 cell line through lentiviral infection. The stable cell line was cultured using F12K complete medium.

Lipofectamine RNAiMAX (purchased from Invitrogen) was used to transfect siRNA into hTSLP-A549 cells, with final siRNA concentrations of 1nM, 0.1nM, and 0.01nM, respectively. Each siRNA was transfected into 3 replicate wells. The experiment was repeated three times. In addition, MOCK was set as a control. The MOCK group was a group that only added lipofectamine RNAiMAX reagent without any siRNA.

The relative inhibition level of human TSLP mRNA by the siRNA compound of the present invention in the stably transfected cell line hTSLP-A549 was determined by real-time fluorescence quantitative PCR (Quantitative Real-Time PCR, hereinafter referred to as qPCR).

The specific steps are: a total RNA extraction kit (purchased from Suzhou Genema Gene Co., Ltd., catalog number E31008) was used to extract total RNA from the cells in each well according to the method described in the kit manual.

5 μl of the above RNA-containing solution was taken as a template, and real-time fluorescence quantitative PCR was performed using HiScript II U+One Step qRT-PCR Probe Kit (purchased from Nanjing Novozymes Biotech Co., Ltd., catalog number Q222-CN-00). The real-time fluorescence quantitative PCR instrument collected the probe luminescence signals of the target gene TSLP and the internal reference gene GAPDH during the amplification process, and obtained the Ct values of the target gene TSLP and the internal reference gene GAPDH.

Sequence of detection primer

lfTSLP-FO1 5'-3':ATATGAGTGGGACCAAAAGTAC (SEQ ID NO:738);

lfTSLP-RE1 5’-3’：TGCCTGAGTAGCATTTATCTG(SEQ ID NO:739);

HEX-lfTSLP-P1 5’-3’:CGTCTCTTGTAGCAATCGGC(SEQ ID NO:740);

hGAPDH-FO1 5’-3’: CATGAGAAGTATGACAACAGCCT (SEQ ID NO:741);

hGAPDH-RE1 5’-3’: AGTCCTTCCACGATACCAAAGT (SEQ ID NO:742);

FAM-hGAPDH 5’-3’:CAATGCCTCCTGCACCACCAA(SEQ ID NO:743);

The comparative Ct (ΔΔCt) method was used to perform relative quantitative calculation of the expression level of the target gene TSLP in each test group and the control group. The calculation method is as follows:

ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

Wherein, each test group is hTSLP-A549 cells treated with each siRNA, and the control group is hTSLP-A549 cells not treated with siRNA. ΔCt (control group average) is the arithmetic mean of ΔCt (control group) of each of the three culture wells of the control group. Thus, each culture well of the test group and the control group corresponds to a ΔΔCt value.

The expression level of TSLP mRNA in the test group was normalized based on the control group, and the expression level of TSLP mRNA in the control group was defined as 100%.

The relative expression level of TSLP mRNA in the test group = 2 ^{- ΔΔCt (test group)} × 100%.

For the same test group siRNA, the average relative expression level of TSLP mRNA in the test group at each concentration is the arithmetic mean of the relative expression levels of the three culture wells at that concentration.

The inhibition rate of siRNA on TSLP mRNA expression was calculated according to the following formula: inhibition rate = (1-relative expression level of TSLP mRNA in the test group) × 100%.

The results of transfection of the above siRNA at final concentrations of 1 nM, 0.1 nM, and 0.01 nM are shown in Table 8 below. The results show that the chemically modified duplex has good inhibitory activity on TSLP expression in A549 cells at different concentrations.

Table 8: Activity detection of modified siRNA at different concentrations in hTSLP-A549

Example 6: Activity detection of modified siRNA in stably transfected cell line hTSLP-BEAS-2B

Genemax was commissioned to construct a stable cell line hTSLP-BEAS-2B that overexpresses human TSLP protein (human TSLP sequence gene number is NM_033035.5) in the BEAS-2B cell line through lentiviral infection. This example uses the same experimental steps as in Example 5, except that the cells used are hTSLP-BEAS-2B stable cell lines, and the hTSLP-BEAS-2B stable cell lines are cultured using DMEM complete medium.

The results of transfection of the above siRNA at final concentrations of 1 nM, 0.1 nM, and 0.01 nM are shown in Table 9 below. The results show that the chemically modified duplex has good inhibitory activity on TSLP expression in BEAS-2B cells at different concentrations.

Table 9: Activity detection of modified siRNA at different concentrations in hTSLP-BEAS-2B

Example 7: Activity detection of modified siRNA in cell line LN18

This example uses the same experimental steps as in Example 5, except that the cells used are LN18 cells (Jima Gene), and the LN18 cell line is cultured using DMEM complete medium.

The results of transfection of the above siRNA at final concentrations of 1 nM, 0.1 nM, and 0.01 nM are shown in Table 10 below. The results show that the chemically modified duplex has good inhibitory activity on TSLP expression in LN18 cells at different concentrations.

Table 10: Activity detection of modified siRNA at different concentrations in LN18

Example 8: IC50 detection of modified siRNA in stably transfected cell line hTSLP-A549

Seven different concentrations of siRNA were transfected into the stable cell line hTSLP-A549, and the final concentrations of the experiment were 30nM, 10nM, 1nM, 0.1nM, 0.03nM, 0.01nM, and 0.001nM, and the IC50 value was calculated, and the experimental method was the same as Example 5. The experimental results are shown in Table 11 below, and the results show that the chemically modified duplex has good inhibitory activity on TSLP expression in A549 cells.

Table 11: IC50 detection of modified siRNA in hTSLP-A549

Example 9: IC50 detection of modified siRNA in stably transfected cell line hTSLP-BEAS-2B

Seven different concentrations of siRNA were transfected into the stable cell line hTSLP-BEAS-2B, and the final concentrations of the experiment were 30nM, 10nM, 1nM, 0.1nM, 0.03nM, 0.01nM, and 0.001nM, and the IC50 value was calculated. The experimental method was the same as that of Example 6. The experimental results are shown in Table 12 below, and the results show that the chemically modified duplex has good inhibitory activity on TSLP expression in BEAS-2B cells.

Table 12: IC50 detection of modified siRNA in hTSLP-BEAS-2B

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims (26)

A small interfering RNA (siRNA) for inhibiting the expression of the long subtype TSLP (lfTSLP) gene, wherein the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 14 consecutive nucleotides that are not more than 4 (e.g., 0, 1, 2, 3 or 4) nucleotides different from the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:164, and the sense strand is at least partially complementary to the antisense strand. The siRNA according to claim 1, wherein the antisense strand comprises at least 14 consecutive nucleotides that differ by 0 or 1 nucleotide from the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:164. The siRNA according to claim 1 or 2, wherein the positive strand comprises at least 14 consecutive nucleotides that are not more than 4 (e.g., 0, 1, 2, 3 or 4) nucleotides different from the nucleotide sequence shown in any one of SEQ ID NO: 165 to SEQ ID NO: 328; Preferably, the positive strand comprises at least 14 consecutive nucleotides that differ by 0 or 1 nucleotide from the nucleotide sequence shown in any one of SEQ ID NO: 165 to SEQ ID NO: 328; Preferably, the sense strand has a region of at least 85% complementarity with the antisense strand within the 14 consecutive nucleotides. The siRNA according to any one of claims 1 to 3, wherein the siRNA comprises a blunt end and/or an overhang of 1 to 4 nucleotides; Preferably, the siRNA comprises an overhang of 1 or 2 nucleotides; Preferably, the overhang is present at the 5' end and/or the 3' end of the antisense strand and/or the sense strand; Preferably, the 3' end of the antisense strand of the siRNA comprises an overhang of 2 nucleotides. The siRNA according to any one of claims 1 to 4, wherein the lengths of the antisense strand and the sense strand are independently 14 to 30 nucleotides; preferably, the antisense strand is 16 to 25 nucleotides in length; preferably, the sense strand is 14 to 23 nucleotides in length. The siRNA according to any one of claims 1 to 4, wherein the antisense strand is 21 to 23 nucleotides in length, and the sense strand is 19 to 21 nucleotides in length. The siRNA according to any one of claims 1 to 6, wherein the sense strand has no more than 6 (e.g., 0, 1, 2, 3, 4, 5 or 6) nucleotide mismatches with the antisense strand. The siRNA according to any one of claims 1 to 7, wherein the sequences of the sense strand and the antisense strand of the siRNA are selected from the sense strand and the antisense strand sequences of any duplex of GPSZT082UM to GPSZT245UM provided in Table 1; Preferably, the sequences of the sense strand and antisense strand of the siRNA are selected from the sense strand and antisense strand sequences of any duplex provided in Table 5. The siRNA according to any one of claims 1 to 8, wherein the siRNA contains at least one modified nucleotide. The siRNA according to claim 9, wherein all nucleotides in the sense strand and/or antisense strand of the siRNA are modified nucleotides or nucleotide analogs; Preferably, all nucleotides in the sense strand of the siRNA are modified nucleotides or nucleotide analogs, and all nucleotides in the antisense strand of the siRNA are modified nucleotides or nucleotide analogs. The siRNA according to claim 9 or 10, wherein the modified nucleotide or nucleotide analog is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxyribonucleotides, 2',3'-split ring nucleotide analogs, 2'-fluoroarabino nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleic acids, unlocked nucleic acids or glycerol nucleotides. The siRNA according to any one of claims 9 to 11, wherein the nucleotides in the sense strand of the siRNA are selected from at least two of 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxyribonucleotides or locked nucleic acids; and/or the nucleotides in the antisense strand of the siRNA are selected from at least two of 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxyribonucleotides, glycerol nucleotides, nucleotides containing 5'-phosphate esters or nucleotides containing 5'-phosphate mimetics. The siRNA according to any one of claims 9 to 12, wherein the sense strand and/or antisense strand of the siRNA comprises a modified internucleoside linkage; Preferably, the 5' end and/or 3' end of the sense strand independently contain 1 or 2 phosphorothioate linkages; and/or the 5' end and/or 3' end of the antisense strand independently contain 1 or 2 phosphorothioate linkages. The siRNA according to any one of claims 9 to 13, wherein the antisense strand of the siRNA comprises the following modification pattern: (1)5’-NmsNfsNmNfNmNfNmNfNmNfNmNmNmNfNmNfNmNfNmsNmsNm-3’ (SEQ ID NO:719); (2)5’-NmsNfsNmNmNmNfNmNfNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’ (SEQ ID NO:720); (3)5’-NmsNfsNmNfNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’ (SEQ ID NO:721); (4)5’-VPNmsNfsNmNfNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm-3’ (SEQ ID NO:728); or (6)5’-VPNmsNfsNmNfNmNmN(GNA)NmNmNmNmNmNmNfNmNfNmNmNmsNms Nm-3’(SEQ ID NO:730); Among them, Nm is a methoxy-modified nucleotide, Nf is a fluorinated-modified nucleotide, s is a phosphorothioate linkage, VP represents a 5'-phosphate analog, and N(GNA) represents a glycerol nucleotide. The siRNA according to any one of claims 9 to 14, wherein the sense strand of the siRNA comprises the following modification pattern: (1)5’-NmsNmsNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfNmNf-3’ (SEQ ID NO:722); (2)5’-NmsNmsNfNmNmNmNfNfNfNmNfNmNfNmNfNmNmNmNm-3’ (SEQ ID NO:723); (3) 5'-NmsNmsNmNmNfNmNfNfN(d)NmNmNmNmNmNmNmNmNmNm-3' (SEQ ID NO: 724); (4)5’-N(LNA)sNmsNfNmNmNmNfNfNfNfNmNfNmNfNmNfNmNmNmNm-3’(SEQ ID NO:725); (5)5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNfNmNmNmNm-3’ (SEQ ID NO:726); (6)5’-NmsNmsNmNmNfNmN(d)NfN(d)NmNmNmNmNmNmNmNmNmNmNm-3’(SEQ ID NO:731); (7)5’-NmsNmNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfNmsNf-3’ (SEQ ID NO:732); (8)5’-NmsNmNfNmNmNmNfNfNfNmNfNmNfNmNfNmNmNmsNm-3’ (SEQ ID NO:733); (9)5’-NmsNmNmNmNfNmNfNfN(d)NmNmNmNmNmNmNmNmNmsNm-3’(SEQ ID NO:734); (10)5’-N(LNA)sNmNfNmNmNmNfNfNfNfNmNfNmNfNmNfNmNmNmsNm-3’(SEQ ID NO:735); or (12)5’-NmsNmNmNmNfNmN(d)NfN(d)NmNmNmNmNmNmNmNmNmsNm-3’(SEQ ID NO:737); Among them, Nm is a methoxy-modified nucleotide, Nf is a fluorinated-modified nucleotide, N(LNA) is a locked nucleic acid-modified nucleotide, N(d) is a 2’-deoxyribonucleotide, and s is a phosphorothioate linkage. The siRNA according to any one of claims 9 to 15, wherein the antisense strand comprises a nucleotide sequence shown in any one of SEQ ID NOs: 329 to 523; Preferably, the antisense strand comprises SEQ ID NOs: 329-331, 333-337, 339, 341, 344-356, 360, 362-363, 365, 367-369, 371-378, 380-381, 383, 385-393, 395-396, 398-404, 407-408, 411-417, 419-420, 425-4 26, 428, 430-431, 433-434, 438, 440-441, 446-447, 450-456, 458-459, 463-470, 472-474, 476-477, 479-480, 482, 484-486, 488-498, 500, 502-513, 515-516, 518-521. The siRNA according to any one of claims 9 to 16, wherein the sense strand comprises SEQ ID NOs: The nucleotide sequence shown in any one of 524-718; Preferably, the positive strand comprises SEQ ID NOs: 524-526, 528-532, 534, 536, 539-551, 555, 557-558, 560, 562-564, 566-573, 575-576, 578, 580-588, 590-591, 593-599, 602-603, 606-612, 614-615, 620-6 21, 623, 625-626, 628-629, 633, 635-636, 641-642, 645-651, 653-654, 658-665, 667-669, 671-672, 674-675, 677, 679-681, 683-693, 695, 697-708, 710-711, and the nucleotide sequence shown in any one of 713-716. The siRNA according to any one of claims 9 to 17, wherein the sequences of the sense strand and the antisense strand of the siRNA are selected from the sense strand and the antisense strand sequences of any duplex of GPSZT082S1 to GPSZT233S5 provided in Table 3; Preferably, the sequences of the sense strand and antisense strand of the siRNA are selected from the sense strand and antisense strand sequences of any duplex provided in Table 7. A conjugate, wherein the conjugate comprises the siRNA according to any one of claims 1 to 18 and a pharmaceutically acceptable targeting molecule. A pharmaceutical composition, wherein the pharmaceutical composition comprises the siRNA according to any one of claims 1 to 18 or the conjugate according to claim 19, and a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition according to claim 20, wherein the pharmaceutically acceptable carrier is a delivery carrier; preferably, the siRNA is encapsulated by the delivery carrier. Use of the siRNA according to any one of claims 1 to 18, the conjugate according to claim 19 and/or the pharmaceutical composition according to claim 20 or 21 in the preparation of a medicament for treating and/or preventing a disease associated with lfTSLP; Preferably, the siRNA, the conjugate or the pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (eg, siRNA targeting a different target sequence in the lfTSLP gene or siRNA targeting other targets). The use according to claim 22, wherein the disease associated with lfTSLP is an inflammatory disease, such as an inflammatory respiratory disease, an inflammatory digestive disease or an inflammatory skin disease. The use according to claim 22, wherein the disease associated with lfTSLP is selected from asthma, nasal polyps, allergic rhinitis, chronic sinusitis, atopic dermatitis, eosinophilic esophagitis, chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. A method for inhibiting lfTSLP expression in a cell, wherein the method comprises: introducing into the cell the siRNA according to any one of claims 1 to 18, the conjugate according to claim 19 and/or the pharmaceutical composition according to claim 20 or 21; Preferably, the siRNA, the conjugate or the pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (eg, siRNA targeting a different target sequence in the lfTSLP gene or siRNA targeting other targets). A method for preventing and/or treating a disease associated with lfTSLP in a subject, wherein the method comprises administering to a subject in need thereof an effective amount of the siRNA according to any one of claims 1 to 18, the conjugate according to claim 19, and/or the pharmaceutical composition according to claim 20 or 21; Preferably, the disease associated with lfTSLP is an inflammatory disease, such as an inflammatory respiratory disease, an inflammatory digestive disease or an inflammatory skin disease; Preferably, the disease associated with lfTSLP is selected from asthma, nasal polyps, allergic rhinitis, chronic sinusitis, atopic dermatitis, eosinophilic esophagitis, chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis; Preferably, the subject is a mammal, such as a human; Preferably, the siRNA, the conjugate or the pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (eg, siRNA targeting a different target sequence in the lfTSLP gene or siRNA targeting other targets).