CN120202298A

CN120202298A - A dsRNA molecule for inhibiting sod1 gene expression and its application

Info

Publication number: CN120202298A
Application number: CN202480001710.8A
Authority: CN
Inventors: 石延君; 王凌宇; 苏晓晔; 张雪艳; 邵昱; 陈慧瑜
Original assignee: CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Current assignee: CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Priority date: 2023-08-29
Filing date: 2024-08-29
Publication date: 2025-06-24
Also published as: WO2025045130A1

Abstract

A modified double stranded RNAi molecule and uses thereof are provided. In particular, it relates to a double stranded RNAi molecule inhibiting expression of the sod1 gene, comprising a sense strand and an antisense strand that are complementary to form a double stranded region, said sense strand or said antisense strand comprising or consisting of 15-25 nucleotides, at least one nucleotide of said double stranded RNAi agent being modified. Also provided is the use of the modified double stranded RNAi molecules with high inhibitory activity and stability, and also with high permeability of the nervous system, for the treatment and/or prevention of sod1 gene mediated diseases.

Description

DsRNA molecule for inhibiting sod1 gene expression and application thereof

Technical Field

The application belongs to the field of molecular biology, relates to a modified dsRNA molecule and application thereof, and in particular relates to a dsRNA molecule for inhibiting the expression of a sod1 gene, a pharmaceutical composition thereof and a method for reducing the expression level of the sod1 gene by using the dsRNA molecule or the pharmaceutical composition thereof.

Background

RNA interference (RNAi) is widely found in species in nature, and RNAi was first found in nematodes since ANDREW FIRE and Craig Mello et al 1998, and after Tuschl and PHIL SHARP et al 2001 confirmed that RNAi was also present in mammals, a series of advances have been made in the study of the mechanism principle, gene function, and clinical application of RNAi. RNAi plays a key role in various organism protection mechanisms such as virus infection prevention, transposon jump prevention and the like. Products developed based on RNAi mechanisms are very promising drug candidates. The small interfering RNA (SMALL INTERFERING RNA, SIRNA) can play the role of RNA interference, and is a main tool for realizing RNAi.

Soluble SOD1 enzymes (also known as Cu/Zn superoxide dismutase) are one of the superoxide dismutases that can protect against oxidative damage by biomolecules by catalyzing the disproportionation of superoxide to hydrogen peroxide (H ₂O₂). Superoxide anions (O ^2-) are potentially harmful cellular byproducts, mainly produced by errors in oxidative phosphorylation in mitochondria. Mutations in the SOD1 gene are associated with dominant inherited forms of ALS, a condition characterized by selective degeneration of upper and lower motor neurons. There is a tight genetic linkage between familial ALS and missense mutations in SOD1 genes. Toxicity of mutant SOD1 is thought to be due to the reduced protective effect of the active enzyme on the nucleus (loss of function in the nucleus) caused by initial misfolding (gain of function), a process that may be associated with ALS pathogenesis.

ALS is of two different types, sporadic and familial. Sporadic ALS is the most common form of disease, accounting for 90% to 95% of all cases, which can affect anyone anywhere. ALS patients have a relatively high proportion of rare patients and have a high mortality rate. Familial ALS means that the disease is inherited. In these families, each offspring has 30% -60% of the chance of inherited genetic mutations and may suffer from the disease. There are also some therapeutic agents currently available to slow down the loss of physiological function or to prolong survival of ALS, including riluzole, edaravone, tofersen, and the like. However, the currently known therapeutic agents do not alleviate the symptoms of all ALS patients, and no method is currently known to cure ALS.

Based on the intractability of the related diseases, the treatment means are extremely lacking, and there is a great unmet treatment need, so other inhibitors aiming at the target point still need to be developed, so that the inhibitors have better curative effect, specificity, stability, targeting or tolerance and the like.

Summary of The Invention

The present application provides modified double stranded RNAi molecules, pharmaceutical compositions containing the modified double stranded RNAi molecules and uses thereof.

Specifically, in one aspect, the application provides a double stranded RNAi agent that inhibits expression of the sod1 gene, comprising a sense strand and an antisense strand that are complementary to form a double-stranded region, said sense strand and/or said antisense strand comprising or consisting of 15-25 nucleotides, said antisense strand being complementary to at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of SEQ ID No.1 or SEQ ID No. 3, said double-stranded region being 15-25bp in length, at least one nucleotide of said double stranded RNAi agent being modified;

The modification is selected from any one or more of locked nucleic acid modification, open or unlocked nucleic acid modification, 2 '-methoxyethyl modification, 2' -O-methyl modification, 2 '-O-allyl modification, 2' -C-allyl modification, 2 '-fluoro modification, 2' -deoxy modification, phosphorothioate backbone modification, DNA modification, and lipophilic modification.

In some embodiments, the sense strand of the double stranded RNAi agent comprises the nucleotide sequence of UAGCUGUAGAAAUGUAUCCUG (SEQ ID NO: 1) and/or the antisense strand comprises the nucleotide sequence of CAGGAUACAUUUCUACAGCUAGC (SEQ ID NO: 2), or the sense strand of the double stranded RNAi agent comprises the nucleotide sequence of AGCUGUAGAAAUGUAUCCUGA (SEQ ID NO: 3) and/or the antisense strand comprises the nucleotide sequence of UCAGGAUACAUUUCUACAGCUAG (SEQ ID NO: 4).

In some embodiments, the sense strand of the double stranded RNAi agent consists of the nucleotide sequence of UAGCUGUAGAAAUGUAUCCUG (SEQ ID NO: 1) and/or the antisense strand of the double stranded RNAi agent consists of the nucleotide sequence of CAGGAUACAUUUCUACAGCUAGC (SEQ ID NO: 2), or the sense strand of the double stranded RNAi agent consists of the nucleotide sequence of AGCUGUAGAAAUGUAUCCUGA (SEQ ID NO: 3) and/or the antisense strand of the double stranded RNAi agent consists of the nucleotide sequence of UCAGGAUACAUUUCUACAGCUAG (SEQ ID NO: 4).

In some embodiments, the double stranded RNAi agent is modified in a manner comprising (1) a sense strand of 17-21nt in length, such as 17, 18, 19, 20, or 21nt, alternating with 2 '-O-methyl modified regions and 2' -fluoro modified regions, each modified region having a consecutive number of nucleotides of any of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides, the first modified region from the 5 'end and the 3' end being modified in the same manner, and (2) an antisense strand of 19-23nt in length, such as 19, 20, 21, 22, or 23nt, alternating with 2 '-O-methyl modified regions, 2' -fluoro modified regions, unmodified regions, or DNA regions, each modified region having consecutive nucleotide lengths of any of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides, the first modified region from the 5 'end and the 3' end being modified in the same manner;

And the continuous nucleotide regions at positions 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7 from the 5 'terminus, and the continuous nucleotide regions at positions 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7 from the 3' terminus, are each joined by a phosphorothioate backbone, and in some embodiments, the continuous nucleotide regions at positions 1 to 3 from the 5 'terminus, and the continuous nucleotide regions at positions 1 to 3 from the 3' terminus are each joined by a phosphorothioate backbone.

In some embodiments, the lipophilic modification is one or more lipophilic moieties conjugated to one or more internal positions (i.e., nucleotides within the strand) on at least one strand of the double stranded RNAi agent, selected from the group consisting of saturated or unsaturated linear or branched C ₄-C₃₀ hydrocarbon chain, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyl hexanol, glycerol, borneol, menthol, 1, 3-propanediol, palmitic acid, myristic acid, dimethoxytrityl, or phenoxazine;

In some embodiments, the one or more lipophilic moieties are conjugated to one or more of the following internal positions, counted from the 5 'end of each strand, positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, the conjugation being on a base or sugar ring, in some embodiments the lipophilic moiety is attached to the 2' position of the sugar ring.

In some embodiments, the double stranded RNAi agent comprises:

(1) A sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%,96%,97%,98%, 99%, or 100% identity to nucleotide sequence UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and a corresponding modification and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%,96%,97%,98%, 99%, or 100% identity to nucleotide sequence CmsAfsGmGmAmUfAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 6) and a corresponding modification;

(2) A sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%,96%,97%,98%, 99%, or 100% identity to nucleotide sequence UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and a corresponding modification and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%,96%,97%,98%, 99%, or 100% identity to nucleotide sequence CmsAfsGmGmAmUmAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 7) and a corresponding modification;

(3) A sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%,96%,97%,98%, 99%, or 100% identity to nucleotide sequence AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and a corresponding modification and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%,96%,97%,98%, 99%, or 100% identity to nucleotide sequence UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 9) and a corresponding modification;

(4) A sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%,96%,97%,98%, 99%, or 100% identity to nucleotide sequence AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and a corresponding modification and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%,96%,97%,98%, 99%, or 100% identity to nucleotide sequence UmsCfsAmGmGmAmUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 10) and a corresponding modification;

Wherein Am, um, cm and Gm respectively represent ribonucleotides A, U, C and G modified by 2 '-O-methyl, af, uf, cf and Gf respectively represent ribonucleotides A, U, C and G modified by 2' -fluoro, and s respectively represent that the front and rear nucleotides are linked by phosphorothioate backbones.

In some embodiments, the double stranded RNAi agent comprises an antisense strand complementary to the target gene described above, a sense strand complementary to the antisense strand, and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand (i.e., nucleotides within the strand), optionally via a linker or carrier;

The one or more lipophilic moieties are conjugated to one or more of the positional nucleotides selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5 'end of each strand, the lipophilic moiety being attached to a base or to a sugar ring, in some embodiments the lipophilic moiety being attached to the 2' position of the sugar ring;

in some embodiments, the lipophilic moiety is selected from a saturated or unsaturated linear or branched C4-C30 hydrocarbon chain, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyl hexanol, glycerol, borneol, menthol, 1, 3-propanediol, palmitic acid, myristic acid, dimethoxytrityl, or phenoxazine.

In some embodiments, the lipophilic moiety is selected from the group consisting of a saturated or unsaturated linear or branched C ₁₀-C₂₅ hydrocarbon chain, a further preferred saturated or unsaturated linear or branched C ₁₂-C₂₂ hydrocarbon chain, a further preferred saturated or unsaturated linear or branched C ₁₄-C₂₀ hydrocarbon chain, a further preferred lipophilic moiety is selected from the group consisting of a saturated or unsaturated linear or branched C ₁₆ hydrocarbon chain, a C ₁₇ hydrocarbon chain, a C ₁₈ hydrocarbon chain, and in some embodiments, a saturated or unsaturated linear or branched C ₁₆ hydrocarbon chain;

the lipophilic moiety is attached to the base or to the sugar ring, in some embodiments the lipophilic moiety is attached to the 2' position of the sugar ring.

In some embodiments, the double stranded RNAi agent has a sense strand structure shown in SEQ ID NO. 12 and the double stranded RNAi agent has an antisense strand structure shown in SEQ ID NO. 10 or SEQ ID NO. 13.

In some embodiments, the lipophilic portion of the double stranded RNAi agent is conjugated to the double stranded iRNA agent via a linker comprising an ether, urea, carbonate, amine, amide, phosphodiester, sulfonamide bond, or carbamate.

In another aspect, the present application provides a biomaterial selected from any one of the following groups:

(A) A DNA molecule capable of producing any of the double stranded RNAi agents described above;

(B) A vector capable of expressing any of the double stranded RNAi agents described above;

(C) A reagent or kit comprising any one of the double stranded RNAi agents described above or the DNA molecule described in (a) or (B) described above or the vector;

(D) A pharmaceutical composition comprising any of the double stranded RNAi agents described above and a pharmaceutically acceptable additional component.

In another aspect, the application provides the use of a double stranded RNAi agent selected from any one of the following groups:

(I) Use of any of the aforementioned double stranded RNAi agents or any of the aforementioned biomaterials for inhibiting the expression of the sod1 gene or for preparing a product for inhibiting the expression of the sod1 gene;

(II) use of any of the double stranded RNAi agents described above or any of the biological materials described above for reducing SOD1 mRNA expression or SOD1 protein concentration in different brain region tissues and spinal cords, or for preparing a product for reducing SOD1 mRNA expression or SOD1 protein concentration in different brain region tissues and spinal cords;

(III) use of any of the double stranded RNAi agents described above or any of the biological materials described above for reducing SOD1 protein concentration and/or neurofilament protein concentration in serum and CSF, or in the manufacture of a product for reducing SOD1 protein concentration and/or neurofilament protein concentration in serum and CSF;

(IV) use of any of the double stranded RNAi agents or any of the biological materials described above for the prevention and/or treatment of a disease mediated by a mutated sod1 gene, or for the preparation of a product for the prevention and/or treatment of a disease mediated by a mutated sod1 gene;

(V) use of any of the double stranded RNAi agents or any of the biological materials described above for alleviating symptoms of a disease mediated by a mutated sod1 gene, or for the preparation of a product for alleviating symptoms of a disease mediated by a mutated sod1 gene;

The aforementioned diseases mediated by the mutated sod1 gene include Amyotrophic Lateral Sclerosis (ALS) or neurodegenerative diseases, etc.

It is to be understood that the aspects and embodiments of the application described herein include aspects and embodiments that "comprise," consist of, "and" consist essentially of. The preferred embodiments of the present application have been described in detail above, but the present application is not limited thereto. Within the scope of the technical idea of the application, a number of simple variants of the technical solution of the application are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the application, all falling within the scope of protection of the application.

The technical effects are as follows:

The application has the unexpected technical effects that 1, the modified double-stranded RNAi agent has high stability and high inhibition activity in vivo and in vitro, which is obviously superior to the existing like products on the market, 2, the modified double-stranded RNAi agent has better capacity of being endocytosed by cells while maintaining higher inhibition activity and stability, the purpose of reducing the using amount of the double-stranded RNAi agent and reducing the toxicity and the cost can be achieved, and 3, the modified double-stranded RNAi agent can enter target cells and target tissues without a transfection reagent, and the negative influence such as the cell or tissue toxicity of the transfection reagent is reduced. 4. The modified double-stranded RNAi agent not only can well delay the disease state, but also can prolong the survival time to a great extent, thereby providing possibility for ALS targeted treatment.

It should be noted that while many modifications can be made to improve the performance of double stranded RNAi agents, such attempts have generally been difficult to elucidate both the mediating RNA interference and the increased stability in serum (e.g., increased resistance to nucleases and/or prolonged duration). The modified double-stranded RNAi agent has high stability and high inhibition activity, and unexpected technical effects are achieved.

Description of the drawings:

FIG. 1 Gene expression data in U-251MG cells for candidate modification sequences. The test results for each concentration are S952, S954 and NC, respectively, from left to right.

FIG. 2 Gene expression data in U-251MG cells for candidate sequence modification strategies. The test results for each concentration are S592.11, S592.21, S594.11, S594.21 and NC, respectively, from left to right.

FIGS. 3A-3E, gene expression data for candidate modification sequence S594.21 and aCSF control in mice. FIG. 3A shows prefrontal cortex region gene expression data, FIG. 3B shows cerebellum region gene expression data, FIG. 3C shows cervical vertebra region gene expression data, FIG. 3D shows lumbar vertebra region gene expression data, and FIG. 3E shows thoracic vertebra region gene expression data.

FIG. 4A-FIG. 4B show in vivo rod-rotating behavioural efficacy test of candidate modified sequences in mice, FIG. 4A shows in vivo rod-rotating behavioural efficacy test data of ICV administration mice in late onset of disease, and FIG. 4B shows in vivo rod-rotating behavioural efficacy test data of IT administration mice in early onset of disease.

FIG. 5A-FIG. 5B show the in vivo reverse hanging behavioural efficacy test of candidate modified sequences in mice, FIG. 5A shows the in vivo reverse hanging behavioural efficacy test data of ICV administration mice in late onset of disease, and FIG. 5B shows the in vivo reverse hanging behavioural efficacy test data of IT administration mice in early onset of disease.

FIG. 6 is a drug efficacy test of the effect of candidate modified sequence onset late ICV administration on mouse pole climbing behaviours.

FIG. 7 test of efficacy of candidate modified sequence onset late ICV administration on weight saving in mice.

FIG. 8 is a graph showing efficacy test of candidate modified sequence onset late ICV administration on the effect of survival in mice.

FIG. 9 is a drug efficacy test of the effect of IT administration in early onset of candidate modified sequences on survival of mice.

Detailed Description

The application provides a dsRNA molecule, a reagent, a kit and a pharmaceutical composition thereof for inhibiting the expression of a sod1 gene, and a method and application of the dsRNA molecule, the reagent, the kit or the pharmaceutical composition in inhibiting or reducing the expression of the sod1 gene or treating a disease or a symptom mediated by the sod1 gene.

In particular, in one aspect, the application provides a double stranded RNAi agent that inhibits the expression of the sod1 gene, comprising a sense strand and an antisense strand that are complementary to form a double stranded region, wherein the sense strand or the antisense strand comprises 15-25 nucleotides, wherein the antisense strand is complementary to at least 15, 16, 17, 18,19, 20, or 21 consecutive nucleotides of SEQ ID NO:1 or SEQ ID NO:3, wherein the double stranded region is 15-25bp, preferably 19-21bp in length, and wherein at least one nucleotide in the double stranded RNAi agent is modified, wherein the modification is selected from any one or more of a Locked Nucleic Acid (LNA) modification, a open loop or non-locked (UNA) modification, a 2 '-methoxyethyl modification, a 2' -O-methyl modification, a 2 '-O-allyl modification, a 2' -C-allyl modification, a 2 '-fluoro modification, a 2' -deoxy modification, a phosphorothioate backbone modification, a DNA modification, a fluorescent probe modification, and a lipophilic modification.

In some embodiments, the application provides a double stranded RNAi agent that inhibits the expression of the sod1 gene, comprising a sense strand and an antisense strand that are complementary to form a double stranded region, the sense strand or the antisense strand consisting of 15-25 nucleotides, the antisense strand being complementary to at least 15, 16, 17, 18,19, 20, or 21 consecutive nucleotides of SEQ ID NO:1 or SEQ ID NO:3, the double stranded region being 15-25bp in length, preferably 19-21bp, at least one nucleotide of the double stranded RNAi agent being modified, the modification selected from any one or more of a Locked Nucleic Acid (LNA) modification, a open loop or non-locked (UNA) modification, a 2 '-methoxyethyl modification, a 2' -O-methyl modification, a 2 '-O-allyl modification, a 2' -C-allyl modification, a 2 '-fluoro modification, a 2' -deoxy modification, a phosphorothioate backbone modification, a DNA modification, a fluorescent probe modification, a lipophilic modification.

The double stranded RNAi agents of the application consist of two strands, wherein the strand that binds to the target mRNA is referred to as the antisense or guide strand and the other strand is referred to as the sense or passenger strand. The term "antisense strand" refers to a strand of a double stranded RNAi agent that includes a region that is fully or substantially complementary to a target sequence. The term "sense strand" refers to a strand of a double stranded RNAi agent that includes a region that is substantially complementary to a region that is the term antisense strand as defined herein. The term "complementary region" refers to a region on the antisense strand that is fully or substantially complementary to a target mRNA sequence. In cases where the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in an internal or terminal region of the molecule. As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions.

The double-stranded RNAi agent promotes sequence-specific degradation of the sod1 mRNA through RNAi effect, and achieves suppression of sod1 gene expression or reduction of the level of sod1 gene expression.

In some embodiments, the application provides a double stranded RNAi agent having a sense strand with a nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3 and an antisense strand with a nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4.

In some embodiments, the sense strand of the double stranded RNAi agent comprises the nucleotide sequence of UAGCUGUAGAAAUGUAUCCUG (SEQ ID NO: 1) and/or the antisense strand of the double stranded RNAi agent comprises the nucleotide sequence of CAGGAUACAUUUCUACAGCUAGC (SEQ ID NO: 2), in some embodiments, the sense strand of the double stranded RNAi agent comprises the nucleotide sequence of AGCUGUAGAAAUGUAUCCUGA (SEQ ID NO: 3) and/or the antisense strand of the double stranded RNAi agent comprises the nucleotide sequence of UCAGGAUACAUUUCUACAGCUAG (SEQ ID NO: 4).

In some embodiments, the sense strand of the double stranded RNAi agent consists of the nucleotide sequence shown in UAGCUGUAGAAAUGUAUCCUG (SEQ ID NO: 1) and/or the antisense strand of the double stranded RNAi agent consists of the nucleotide sequence shown in CAGGAUACAUUUCUACAGCUAGC (SEQ ID NO: 2), in some embodiments, the sense strand of the double stranded RNAi agent consists of the nucleotide sequence shown in AGCUGUAGAAAUGUAUCCUGA (SEQ ID NO: 3) and/or the antisense strand of the double stranded RNAi agent consists of the nucleotide sequence shown in UCAGGAUACAUUUCUACAGCUAG (SEQ ID NO: 4).

In some embodiments, the double stranded RNAi agents provided by the present application are modified in a manner comprising (1) a sense strand of 17-23nt, such as 17, 18, 19, 20, 21nt, 22nt, or 23nt; preferably 19-23nt, such as 19, 20, 21nt, 22nt, or 23nt, more preferably 19-21nt, such as 19, 20, or 21nt, the sense strand consists of alternating 2' -O-methyl modified regions and 2' -fluoro modified regions, each modified region having a contiguous number of nucleotides of any one of 1, 2,3, 4,5, 6, 7, 8, 9, 10, 11, or 12 nucleotides, the first modified region from the 5' end and the 3' end being modified in the same manner, and (2) an antisense strand having a length of 19-25nt, such as 19, 20, 21, 22, 23nt, 24n, or 25nt, preferably 19-23nt, such as 19, 20, 21nt, 22nt, or 23nt, consisting of alternating 2' -O-methyl modified regions, 2' -fluoro modified regions, unmodified regions, or DNA regions, each modified region having a contiguous nucleotide length of any one of 1, 2,3, 4,5, 6, 7, 8, 9, 10, 11, or 12 nucleotides, and the 3' end being modified in the same manner;

And in the sense strand and the antisense strand, the 1 st to 2 nd, 1 st to 3 rd, 1 st to 4 th, 1 st to 5 th, 1 st to 6 th, or 1 st to 7 th consecutive nucleotide regions from the 5 'end and the 1 st to 2 nd, 1 st to 3 rd, 1 st to 4 th, 1 st to 5 th, 1 st to 6 th, or 1 st to 7 th consecutive nucleotide regions from the 3' end are each linked by a phosphorothioate backbone, preferably, the 1 st to 3 rd consecutive nucleotide regions from the 5 'end and the 1 st to 3 rd consecutive nucleotide regions from the 3' end are each linked by a phosphorothioate backbone.

In some embodiments, the double stranded RNAi agent comprises (1) an antisense strand having an overhang of the 5 '(s) mN(s) mN3' structure at the 3 'end, (2) the antisense strand is modified with fluoro at least positions 2, 14, 16 from the 5' end, with methoxy as other positions as possible, (3) the antisense strand is modified with at least two thio modifications from the 3 'and 5' ends, (4) the sense strand is modified with consecutive fluoro at position 7 from the 5 'end, and positions 9-11 as other positions as possible for methoxy modification, and (5) the sense strand is modified with at least two thio modifications from the 5' end.

In some embodiments, the double stranded RNAi agent comprises (1) a sense strand comprising 21 nucleotides, alternating between a 2 '-fluoro modified region and a 2' -O-methyl modified region, each modified region being 1 to 7 nucleotides in length, the first modified region being modified in the same manner from the 5 'end to the 3' end, and (2) an antisense strand comprising 23 nucleotides, alternating between a 2 '-O-methyl modified region and a 2' -fluoro modified region, each modified region being 1 to 5 nucleotides in length, and the continuous nucleotide region at positions 1 to 7 from the 5 'end, and the continuous nucleotide region at positions 1 to 7 from the 3' end, each connected by a phosphorothioate backbone, preferably the continuous nucleotide region at positions 1 to 3 from the 5 'end, and/or the continuous nucleotide region at positions 1 to 3 from the 3' end, each connected by a phosphorothioate backbone.

In some embodiments, the double stranded RNAi agent comprises an antisense strand complementary to the target gene, a sense strand complementary to the antisense strand, and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand (i.e., nucleotides within the strand), optionally via a linker or carrier. Wherein the lipophilic moiety is selected from a saturated or unsaturated linear or branched C ₄-C₃₀ hydrocarbon chain, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxy hexanol, glycerol, borneol, menthol, 1, 3-propanediol, palmitic acid, myristic acid, dimethoxytrityl or phenoxazine.

In some embodiments, one or more lipophilic moieties are conjugated to one or more selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counted from the 5 'end of each strand, preferably one or more of the lipophilic moieties are conjugated to one or more selected from the group consisting of positions 5,6, 7, 15 and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counted from the 5' end of each strand, more preferably position 6 on the sense strand.

In some embodiments, the ligand (lipophilic moiety) is attached to the 3 'end, 5' end, and/or sequence middle of the double stranded RNAi agent.

In some embodiments, the lipophilic moiety comprises a saturated or unsaturated C ₄-C₃₀ hydrocarbon chain, more specifically the lipophilic moiety comprises a saturated or unsaturated C ₆-C₁₈ hydrocarbon chain, most preferably a saturated or unsaturated C ₁₆ hydrocarbon chain.

In some embodiments, the lipophilic moiety is conjugated to the double stranded RNAi agent via a linker containing an ether, urea, carbonate, amine, amide, phosphodiester, sulfonamide bond, or carbamate.

In some embodiments, the lipophilic moiety comprises a saturated or unsaturated linear or branched C ₁₀-C₂₅ hydrocarbon chain, further preferably a saturated or unsaturated linear or branched C ₁₂-C₂₂ hydrocarbon chain, further preferably a saturated or unsaturated linear or branched C ₁₄-C₂₀ hydrocarbon chain, further preferably the lipophilic moiety comprises a saturated or unsaturated linear or branched C ₁₆ hydrocarbon chain, a C ₁₇ hydrocarbon chain, a C ₁₈ hydrocarbon chain, the lipophilic moiety is attached to a base or to a sugar ring, preferably the lipophilic moiety is attached to a sugar ring, further preferably the lipophilic moiety is attached to the 2' position of the sugar ring, for the 3' end, the 5' end and/or the sequence middle of the double stranded RNAi agent. In some embodiments, the nucleotide at position 6 from the 5 'end of the sense strand is modified with 2' -O-hexadecyl.

In another embodiment of the present application, in the double stranded RNAi agent, the nucleoside comprising the lipophilic moiety has the structure shown in formula I:

wherein B is a natural or modified base, n=15;

preferably, the lipophilic moiety is attached to the middle of the sense strand of the double stranded RNAi agent;

in one example of the application, in the double stranded RNAi agent, the lipophilic moiety is C ₁₆;

the 5 '-position of the 5' -terminal nucleotide of the antisense strand is either linked or not linked to a Vinyl Phosphate (VP).

In some embodiments of the application, the double stranded RNAi agent comprises a modification motif selected from any one of:

(1) The sense strand is NMSNMSNMNMNMNMNFNMNFNFNFNMNMNMNMNMNMNMNMSNMSNM of the sequence,

The antisense strand NMSNFSNMNMNMNFNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMSNMSNM;

(2) The sense strand is NMSNMSNMNMNMNMNFNMNFNFNFNMNMNMNMNMNMNMNMSNMSNM of the sequence,

The antisense strand NMSNFSNMNMNMNMNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMSNMSNM;

(3) The sense strand is NMSNMSNMNMNMN (HD) NFNMNFNFNFNMNMNMNMNMNMNMNMSNMSNM of the sequence,

Antisense strand NMSNFSNMNMNMNFNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMSNMSNM

(4) The sense strand is NMSNMSNMNMNMN (HD) NFNMNFNFNFNMNMNMNMNMNMNMNMSNMSNM of the sequence,

The antisense strand NMSNFSNMNMNMNMNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMSNMSNM;

Wherein Nm represents a ribonucleotide modified by a 2' -O-methyl group, nf represents a ribonucleotide modified by a 2' -fluoro group, s represents that the two nucleotides are linked by a phosphorothioate backbone, and N (hd) represents a ribonucleotide modified by a 2' -O-C16 group.

In one example of the application, the double stranded RNAi agent comprises any one or more selected from the group consisting of:

(1) A sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to nucleotide sequence UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and the corresponding modification and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to nucleotide sequence CmsAfsGmGmAmUfAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 6) and the corresponding modification;

(2) A sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to nucleotide sequence UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and the corresponding modification and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to nucleotide sequence CmsAfsGmGmAmUmAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 7) and the corresponding modification;

(3) A sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to nucleotide sequence AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and the corresponding modification and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to nucleotide sequence UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 9) and the corresponding modification, or

(4) A sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to nucleotide sequence AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and the corresponding modification and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to nucleotide sequence UmsCfsAmGmGmAmUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 10) and the corresponding modification;

(1) The sense strand nucleic acid sequence consists of UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and a further 0-5 (e.g., 0, 1,2, 3, 4 or 5) nucleotides at the 5 'and/or 3' end,

The antisense strand nucleotide sequence consists of CmsAfsGmGmAmUfAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 6) and another 0-5 (e.g., 0, 1,2,3,4, or 5) nucleotides at the 5 'and/or 3' ends;

(2) The sense strand nucleic acid sequence consists of UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and a further 0-5 (e.g., 0, 1,2, 3, 4 or 5) nucleotides at the 5 'and/or 3' end,

The antisense strand nucleotide sequence consists of CmsAfsGmGmAmUmAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 7) and another 0-5 (e.g., 0, 1,2,3,4, or 5) nucleotides at the 5 'and/or 3' ends;

(3) The sense strand nucleotide sequence consists of AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and a further 0-5 (e.g., 0, 1,2, 3, 4 or 5) nucleotides at the 5 'and/or 3' end,

The antisense strand nucleotide sequence consists of UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 9) and another 0-5 (e.g., 0, 1,2,3,4, or 5) nucleotides at the 5 'and/or 3' ends;

(4) The sense strand nucleotide sequence consists of AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and a further 0-5 (e.g., 0, 1,2, 3, 4 or 5) nucleotides at the 5 'and/or 3' end,

The antisense strand nucleotide sequence consists of UmsCfsAmGmGmAmUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 10) and another 0-5 (e.g., 0, 1,2,3,4, or 5) nucleotides at the 5 'and/or 3' ends;

(5) The sense strand nucleotide sequence consists of UmsAmsGmCmUmG (hd) UfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 11) and a further 0-5 (e.g., 0, 1,2,3,4 or 5) nucleotides at the 5 'and/or 3' end,

(6) The sense strand nucleotide sequence consists of UmsAmsGmCmUmG (hd) UfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 11) and 0-5 (e.g., 0, 1,2,3,4 or 5) nucleotides at the other 5 'and/or 3' ends,

(7) The sense strand nucleotide sequence consists of AmsGmsCmUmGmU (hd) AfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 12) and a further 0-5 (e.g., 0, 1,2,3,4 or 5) nucleotides at the 5 'and/or 3' end,

The antisense strand nucleotide sequence consists of UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 9) and a further 0-5 (e.g., 0, 1,2,3,4 or 5) nucleotides at the 5 'and/or 3' end, and

(8) The sense strand nucleotide sequence consists of AmsGmsCmUmGmU (hd) AfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 12) and a further 0-5 (e.g., 0, 1,2,3,4 or 5) nucleotides at the 5 'and/or 3' end,

wherein Am, um, cm and Gm represent 2' -O-methyl modified ribonucleotides A, U, C and G, af, uf, cf and Gf represent 2' -fluoro modified ribonucleotides A, U, C and G, respectively,(s) represent that the front and back two nucleotides are linked by phosphorothioate backbone, and G (hd) and U (hd) represent 2' -O-C16 modified G and U, respectively.

In some embodiments, the nucleic acid sequence of the double stranded RNAi agent is formed by ligating a vinyl phosphate at the 5 'position of the 5' nucleotide of the antisense strand in a double stranded RNAi agent as previously described.

(1) The sense strand nucleic acid sequence consists of UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5),

The antisense strand nucleotide sequence consists of VP-CmsAfsGmGmAmUfAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 18);

(2) The sense strand nucleic acid sequence consists of UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5),

The antisense strand nucleotide sequence consists of VP-CmsAfsGmGmAmUmAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 19);

(3) The sense strand nucleotide sequence consists of AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8),

The antisense strand nucleotide sequence consists of VP-UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 20);

(4) The sense strand nucleotide sequence consists of AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8),

The antisense strand nucleotide sequence consists of VP-UmsCfsAmGmGmAmUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 13);

(5) The sense strand nucleotide sequence consists of UmsAmsGmCmUmG (hd) UfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 11),

(6) The sense strand nucleotide sequence consists of UmsAmsGmCmUmG (hd) UfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 11) and 0-5 nucleotides at the other 5 'and/or 3' ends,

(7) The sense strand nucleotide sequence consists of AmsGmsCmUmGmU (hd) AfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 12),

The antisense strand nucleotide sequence consists of VP-UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 20), and

(8) The sense strand nucleotide sequence consists of AmsGmsCmUmGmU (hd) AfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 12),

Wherein Am, um, cm and Gm represent 2 '-O-methyl modified ribonucleotides A, U, C and G respectively, af, uf, cf and Gf represent 2' -fluoro modified ribonucleotides A, U, C and G respectively,(s) represent that the front and back two nucleotides are linked by phosphorothioate backbone, G (hd) and U (hd) represent 2'-O-C16 modified G and U respectively, and VP-represents that the 5' -position of the nucleotide linked thereto is linked with vinyl phosphate.

In some embodiments, the double stranded RNAi agent has a sense strand structure shown in SEQ ID NO. 12 and the double stranded RNAi agent has an antisense strand structure shown in SEQ ID NO. 10. In some embodiments, the double stranded RNAi agent has a sense strand structure shown in SEQ ID NO. 12 and the double stranded RNAi agent has an antisense strand structure shown in SEQ ID NO. 13.

In another aspect, the application relates to a method of reducing expression of a target gene in a cell, the method comprising contacting the cell with the double stranded RNAi agent comprising an antisense strand complementary to the target gene, a sense strand complementary to the antisense strand, and one or more lipophilic moieties conjugated to one or more internal positions (i.e., nucleotides within the strand) on at least one strand, optionally via a linker or carrier. Wherein the lipophilic moiety is a saturated or unsaturated, linear or branched C ₄-C₃₀ hydrocarbon chain, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxy hexanol, glycerol, borneol, menthol, 1, 3-propanediol, palmitic acid, myristic acid, dimethoxytrityl or phenoxazine.

In some embodiments, the lipophilic moiety is selected from the group consisting of a saturated or unsaturated linear or branched C ₄-C₃₀ hydrocarbon chain, preferably the lipophilic moiety is selected from the group consisting of a saturated or unsaturated linear or branched C ₁₀-C₂₅ hydrocarbon chain, further preferably a saturated or unsaturated linear or branched C ₁₂-C₂₂ hydrocarbon chain, further preferably a saturated or unsaturated linear or branched C ₁₄-C₂₀ hydrocarbon chain, further preferably the lipophilic moiety is selected from the group consisting of a saturated or unsaturated linear or branched C ₁₆ hydrocarbon chain, a C ₁₇ hydrocarbon chain, a C ₁₈ hydrocarbon chain, further preferably the nucleotide at positions 6-8 from the 5 'end of the sense strand is modified with a 2' -O-hexadecyl group. In some embodiments, the nucleotide at position 6 from the 5 'end of the sense strand is modified with 2' -O-hexadecyl.

More particularly the lipophilic moiety comprises a saturated or unsaturated C ₁₆ hydrocarbon chain, the individual nucleoside structure comprising said C16 is shown in formula I:

wherein B is a natural or modified base (e.g., A, T, G, C, U), n=15;

The 5 'position of the 5' nucleotide of the antisense strand may or may not be attached to a Vinyl Phosphate (VP).

The present application provides illustrative examples of double stranded RNAi agents S592.1, S592.2, S594.1, S594.2.

Still further, exemplary double stranded RNAi agents are as in any one of table 1:

TABLE 1

In some embodiments, the sense and antisense strand structures of the double stranded RNAi agent are as shown in table 2:

TABLE 2

Wherein Am, um, cm and Gm respectively represent ribonucleotides A, U, C and G modified by 2 '-O-methyl, af, uf, cf and Gf respectively represent ribonucleotides A, U, C and G modified by 2' -fluoro, and s respectively represent that the front and rear nucleotides are linked by phosphorothioate backbones. U (hd) and G (hd) represent ribonucleotides U and G, respectively, which are 2 '-O-hexadecyl groups, and VP-represents the linkage of VP (vinyl phosphate) to the 5' -position of the nucleotide to which it is linked.

The double stranded RNAi agent is capable of inhibiting sod1 gene expression in a human, monkey, rat, or mouse.

Biological materials associated with the double stranded RNAi agents are also within the scope of the application.

The biological material associated with the double stranded RNAi agent can be any of the following:

(A) A DNA molecule capable of producing the double stranded RNAi agent;

(B) A vector capable of expressing the double stranded RNAi agent;

(C) A reagent or kit comprising said double stranded RNAi agent or said DNA molecule of (a) or said vector of (B) above;

(D) A pharmaceutical composition consisting of the double stranded RNAi agent and a pharmaceutically acceptable additional component.

The pharmaceutical compositions include a pharmacologically effective amount of the double stranded RNAi agents of the application and other pharmaceutically acceptable components. An "effective amount" refers to an amount of double stranded RNAi agent effective to produce the desired pharmacological therapeutic effect. "other components" include water, saline, dextrose, buffers (e.g., PBS), excipients, diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, preservatives or combinations thereof.

The pharmaceutical composition can be used for preventing and/or treating a disease mediated by the sod1 gene, or for alleviating symptoms of a disease mediated by the sod1 gene.

The diseases mediated by the sod1 gene include Amyotrophic Lateral Sclerosis (ALS) and neurodegenerative diseases.

The application also provides an application as shown in any one of the following:

(I) Use of said double stranded RNAi agent or said biological material for inhibiting the expression of the sod1 gene or for the preparation of a product for inhibiting the expression of the sod1 gene.

Wherein the inhibition of sod1 gene expression is inhibition or reduction of the level of sod1 gene expression in human, monkey, rat or mouse in cells in vivo or in vitro. The inhibition of sod1 gene expression is at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% inhibition or reduction of the level of sod1 gene expression. Detection of target gene, target RNA, or target protein levels can be used to predict or assess activity, efficacy, or therapeutic outcome.

The cell is a mammalian cell, such as a primate cell, or a human cell, that expresses sod 1. Preferably, the sod1 gene is expressed at high levels in the target cells. More preferably, the cells are derived from brain, salivary gland, heart, spleen, lung, liver, kidney, intestine, or tumor. Even more preferably, the cell is an SK-N-SH, SH-SY5Y, U87MG, or U-251MG cell in the CNS system. Still more preferably, the cell is U-251MG.

In the in vivo application, the pharmaceutical composition may be administered by any suitable means, such as parenteral administration, including intramuscular, intravenous, arterial, intraperitoneal, or subcutaneous injection, ventricular administration, intrathecal administration. Modes of administration include, but are not limited to, single administration or multiple administrations.

In some embodiments, a single dose of the pharmaceutical composition may last for a long period of time, with a decrease in sod1 expression lasting at least 3, 7, 28 days or longer.

(II) use of the double stranded RNAi agent or the biological material for reducing SOD1 mRNA expression or SOD1 protein concentration in or for the preparation of a product for reducing SOD1 mRNA expression or SOD1 protein concentration in different brain region tissues and spinal cords (vertebrae, lumbar vertebrae, thoracic vertebrae);

Wherein, the reduction of the concentration of the SOD1 protein in the tissues and spinal cord (vertebra, lumbar vertebra, thoracic vertebra) of different brain areas of human, monkey, rat or mouse is to reduce the concentration of the SOD1 protein in the tissues and spinal cord (vertebra, lumbar vertebra, thoracic vertebra) of different brain areas of human, monkey, rat or mouse.

The SOD1 protein concentration or content in the tissue and spinal cord (spine, lumbar, thoracic) of different brain regions is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%.

(III) use of the double stranded RNAi agent or the biological material in the prevention and/or treatment of a disease mediated by the sod1 gene, or in the manufacture of a product for the prevention and/or treatment of a disease mediated by the sod1 gene;

(IV) use of the double stranded RNAi agent or the biological material for alleviating symptoms of a disease mediated by the sod1 gene, or for preparing a product for alleviating symptoms of a disease mediated by the sod1 gene;

the disease mediated by the sod1 gene includes Amyotrophic Lateral Sclerosis (ALS) or other neurodegenerative disease.

(V) use of the double stranded RNAi agent or the biological material for reducing SOD1 protein concentration and/or neurofilament protein concentration in serum and CSF or for preparing a product for reducing SOD1 protein concentration and/or neurofilament protein concentration in serum and CSF.

Terminology:

REL (Relative expression level) relative mRNA expression levels.

ALS amyotrophic lateral sclerosis.

N refers to ribonucleotides, including ribonucleotides divided into adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide and uracil ribonucleotide.

DN refers to deoxyribonucleotide.

Nm 2' OMe modified ribonucleotides.

Nf 2' F modified ribonucleotides.

The nucleotide sequence(s) is PS skeleton, i.e. 5' -thio modified phosphate skeleton.

Nhd 2' -O-hexadecyl modified ribonucleotides.

VP-denotes the linkage of VP (vinyl phosphate) to the 5' -position of the nucleotide to which it is linked.

DNA modification in the present application, the DNA modification of double stranded RNAi agent refers to the replacement of ribonucleotides in the double stranded RNAi agent with deoxyribonucleotides, wherein the nucleotides are identical, except for the difference in ribose species.

ICV, ventricular administration by injection.

Late administration in the present application is generally carried out in disease model mice 14-15 weeks of age.

Early administration refers generally to administration of disease model mice in the present application at 5-8 weeks of age.

IT injection, intrathecal injection administration.

DCA: dichloroacetic acid.

CAPA capping reagent A (20% acetic anhydride in acetonitrile, v/v).

CAPB capping reagent B (N-methylimidazole: pyridine: acetonitrile=2:3:5).

ACN, acetonitrile.

TEAA, triethylamine acetic acid.

Trityl-off synthesis of detrityl

ESI-MS, electrospray mass spectrometry;

IEX HPLC, ion exchange high performance liquid chromatography;

GAPDH glyceraldehyde-3-phosphate dehydrogenase (GLYCERALDEHYDE-3-phosphate dehydrogenase);

NC group is negative control group, namely transfection reagent control group;

aCSF is artificial cerebrospinal fluid (sterile), also known as cerebrospinal fluid mimicking fluid. In scientific research, artificial cerebrospinal fluid is often used for maintaining pH balance and tissue oxygen transport, rat brain microdialysis experiments, culture of hippocampal tissue sections, etc.

As used herein, a percentage of "identity," e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to a degree of similarity between amino acid sequences or between nucleotide sequences, as determined by sequence alignment, of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, by introducing gaps or the like, it is possible to determine the ratio of the number of positions having the same base or amino acid residue to the total number of positions after two sequences have the same residue at as many positions as possible. The percentage of "identity" may be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. One preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found on the corresponding pages of the NCBI website. It is particularly noted that when describing a nucleotide sequence that has at least a certain percentage identity to a certain nucleotide sequence and corresponding modification (e.g. "nucleotide sequence that has at least 90% identity to nucleotide sequence UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and corresponding modification"), the sequence alignment takes into account the modification of each nucleotide monomer of the certain nucleotide sequence, i.e. requires that both nucleotides be identical throughout the monomer (including the artificial modification).

As used herein, "complementary" of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid by conventional Watson-Crick base pairing. Percent complementarity means that the nucleotides in a shorter one of the two nucleic acid molecules that can form hydrogen bonds with the other nucleic acid molecule (i.e., watson-Crick base pairing) are a percentage of the total nucleotides in the shorter one of the nucleotides (e.g., about 5, 6, 7, 8, 9, 10 out of 10 are about 50%,60%,70%,80%,90% and 100% complementary, respectively). "fully complementary" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of any one of at least about 70%,75%,80%,85%,90%,95%,96%,97%,98%,99% or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions. For a single base or a single nucleotide, pairing of A with T or U, C with G or I is referred to as complementary, pairing or matching, and vice versa, according to Watson-Crick base pairing rules, while other base pairing is referred to as non-complementary.

As used herein, "hybridization" of a nucleic acid refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between nucleotide residues. Hydrogen bonding may occur through watson crick base pairing, hoogstein binding, or any other sequence-specific manner. The complex may include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or a combination thereof.

The term "nucleotide", in addition to referring to naturally occurring ribonucleotides or deoxyribonucleotide monomers, is also understood herein to refer to structural variants thereof, including derivatives and analogs, which are functionally equivalent with respect to the particular context in which the nucleotide is used, unless the context clearly indicates otherwise. For example, "nucleotide" refers to deoxyribonucleotide or ribonucleotide. The nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having a modified purine or pyrimidine base or modified ribose moiety. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications on the sugar or base portion of a nucleotide include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, and substitution of carbon and nitrogen atoms of the base with other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2' -O-methyl nucleotides, locked Nucleic Acids (LNA), peptide Nucleic Acids (PNA), and morpholino oligonucleotides (morpholino). In some embodiments, a "nucleotide" of the application does not comprise a non-natural nucleotide with a modified base. In some embodiments, a "nucleotide" of the application does not comprise a base modified nucleotide. In the present application, "G", "C", "a", "T" and "U" generally represent nucleotides based on guanine, cytosine, adenine, thymine and uracil, respectively. However, in the context of RNA and in RNA sequences, as is not specifically stated, "T" refers to uridine or uracil. It should be understood that the terms "nucleotide", "nucleotide residue" and "base" are used interchangeably herein in reference to the context of a nucleotide sequence. The number of base pairs is in bp, and one bp is one base pair. The number of nucleotides is in nt, one nt being one nucleotide.

As used herein, "3 'end" refers specifically to the position of the first nucleotide or first base pair of the 3' end of a single nucleotide sequence or double-stranded polynucleotide, and thus "3 'end" is used interchangeably with "3' end nucleotide". "5 'end" refers specifically to the position of the first nucleotide or first base pair of the 5' end of a single nucleotide sequence or double stranded polynucleotide, and thus "5 'end" is used interchangeably with "5' end nucleotide".

As used herein, the term "nucleic acid molecule" may be used to refer to any molecule having a nucleotide sequence of more than 2 nucleotides linked by a phosphoester linkage, or a modified phosphoester linkage (e.g., phosphorothioate linkage).

Herein, siRNA is synonymous with double stranded RNAi agent and is interchangeable.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is to be understood that the present application encompasses the various aspects, embodiments, and combinations of the aspects and/or embodiments described herein. The above description and the examples which follow are intended to illustrate and not limit the scope of the application. Within the scope of the technical idea of the application, a number of simple variants of the technical solution of the application are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the application, all falling within the scope of protection of the application.

The practice of the application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology.

It is to be understood that the present application encompasses the various aspects, embodiments, and combinations of the aspects and/or embodiments described herein. The above description and the examples which follow are intended to illustrate and not limit the scope of the application. Other aspects, improvements and modifications within the scope of the application will be apparent to those skilled in the art to which the application pertains. Accordingly, those of ordinary skill in the art will recognize that the scope of the application also includes such improvements and modifications to the aspects and embodiments.

Examples:

EXAMPLE 1 Sod1-siRNA Activity screening

1.1, SiRNA design

According to the sequence of the human sod1 mRNA, the sod1 siRNAs are designed at different positions, all single siRNAs can target all transcripts of target genes (shown in table 3), and the siRNA sequences (shown in table 4) have the lowest homology with all other non-target gene sequences through sequence similarity software comparison.

TABLE 3 target genes

TABLE 4 high throughput screening sequences

1.2 SiRNA Synthesis

All modified or unmodified nucleic acid compounds used in the present application (e.g., as shown in tables 4 and 7) were synthesized in a theoretical yield of 1. Mu. Mol based on the specification, using 1. Mu. Mol of the universal Frit solid support [. Sup.Comma organism) all oligonucleotides were prepared on LK-192X synthesizer using phosphoramidite natural nucleotide monomers or modified phosphoramidite nucleotide monomers (e.g., 2'-O-C16 modified phosphoramidite nucleotide monomers, 5' -VP modified phosphoramidite nucleotide monomers, 2'-OMe modified phosphoramidite nucleotide monomers, 2' -F modified phosphoramidite nucleotide monomers, etc.) from commercial sources. After completion, the kit solid support was transferred to a 2mL centrifuge tube, 1.2mL of ammonia was added and placed in a 36 ℃ oven for 16h to deprotect the protecting group. Then cooling to room temperature, concentrating in vacuum for 30min, filtering the solution into a sample injection bottle through a 0.22um filter membrane, performing single-chain purification by adopting a semi-preparative reverse phase purification instrument, wherein the elution gradient is 7% -30% (ACN: 100mM TEAA), the time is 12min, the flow rate is 5mL/min, concentrating in vacuum after purification preparation, and spin-drying at room temperature. Finally, the sample is dissolved with water, desalted and the final oligonucleotide product is eluted. All properties and purities were confirmed using ESI-MS and IEX HPLC, respectively. Ultraviolet concentration by using an enzyme-labeled instrument, mixing and combining the sense strand and the antisense strand with equal molar weight into a new 2ml EP (eppendorf) tube, heating for 5min at 95 ℃, slowly annealing to room temperature, and finally spin-drying at room temperature by using a vacuum concentrator to obtain a final product.

1.3, Sod1-siRNA transfected cells

The cells used in the embodiment of the application are all from the classical collection of the Chinese academy, and can also be from other sources available to the public, and other reagents are all commercially available. Wherein the cells used were human astroma cell line U-251MG (model: CBP 60300).

Cells were plated in MEM medium (Gibco) containing 10% fetal bovine serum in 5% co ₂, 37 ℃ incubator and transfected when the cells were in log phase and in good condition (70% confluency). The cell concentration was adjusted to 2X 10 ⁵/ml, 1ml of cell solution was added to each well of the 24-well plate, and cultured overnight in a 5% CO ₂, 37℃incubator. The transfection complex was prepared by mixing 250. Mu.L of Opti-MEM (Gibco) and 5. Mu.L of 10nM siRNA, mixing 250. Mu.L of Opti-MEM and 2.5. Mu.L of Lipofectamine 2000reagent transfection reagent (Thermo), standing for 5min, and then mixing the two mixtures, and standing for 20min. MEM medium in 24 well plates was pipetted off, the transfection complex described above was added to each well and incubated for 6h in a 5% CO ₂, 37℃incubator. The transfection complex in 24 well plates was then aspirated, 1mL of complete medium (MEM+10% FBS+1% nonessential amino acids (NEAA) +1mM sodium pyruvate) was added to each well, and cultured in a 5% CO2, 37℃incubator for 24 hours, followed by harvest.

Each cell plating set a transfection reagent control set in addition to the test set. There were 3 replicates for both the test and control groups.

1.4 Real-time quantitative PCR analysis of target mRNA levels

1) Cells were lysed 24h after siRNA transfection, and total RNA was extracted from the cells using column extraction kit (Nuo Wei, fastpure Cell/Tissue Total RNA isolation Kit V2, cat. RC 112-01).

2) Fluorescent quantitative PCR (i.e. qPCR) in one step:

The GAPDH gene is used as an internal reference gene, a Taqman one step Real-time PCR kit is used for carrying out real-time fluorescence quantitative PCR reaction, and a CFX96 fluorescence quantitative PCR instrument of Bio-Rad company in America is used for carrying out PCR reaction. The primers used are shown in Table 5:

TABLE 5 primer sequence information

3) Analysis of data

After the end of the PCR reaction, the Ct error of 6 replicates (3 transfection replicates per sample, 3 qPCR replicates) of one sample was ± 0.5, and relative quantification was performed using CFX 2.1 software. Table 6 shows the average value of the expression level of the target gene of the selected siRNA relative to that of NC group (transfection reagent control group) (the relative expression level of mRNA of NC group is 1).

1.5, Naked sequence screening

U-251MG cells were transfected with siRNA in Table 4 using three concentrations of 100nM, 1nM and 0.1 nM. Cells were lysed after 24h and the amount of SOD1 mRNA therein was measured using one-step fluorescent quantitative PCR, the average results are shown in table 6, and the specific statistical results are shown in figure 1.

TABLE 6 average value of real-time quantitative PCR detection results

Wherein the 2 siRNA molecules are homologous to human and cynomolgus monkey, i.e. are highly homologous to human and cynomolgus monkey mRNA in table 3, and can target human and cynomolgus monkey SOD1 mRNA simultaneously.

Example 2 optimization of sod1-siRNA inhibition Activity assay

To further confirm these 2 highly active double stranded RNAi agents (also referred to as sirnas in the present application), we performed sequence modification optimization (table 7) on their synthesis, transfection, quantitative PCR detection steps and PCR primers, respectively, as in example 1. The transfected cells were U-251MG cells, and Table 8 shows the average value of the expression levels of the target genes relative to the NC group (the relative expression level of mRNA in the NC group was 1). Wherein NC is a transfection reagent (Lipofectamine 2000; thermo) control group.

TABLE 75 highly active double stranded RNAi agent modification sequences

Wherein Am, um, cm and Gm respectively represent ribonucleotides A, U, C and G modified by 2 '-O-methyl, af, uf, cf and Gf respectively represent ribonucleotides A, U, C and G modified by 2' -fluoro,(s) represent that the front and back two nucleotides are linked by phosphorothioate backbone, U (hd) and G (hd) respectively represent ribonucleotides U and G modified by 2 '-O-hexadecyl, and VP-represents that the 5' -position of the nucleotide linked thereto is linked with VP (vinyl phosphate).

TABLE 8 average value of real-time quantitative PCR detection results

The results in Table 8 and FIG. 2 show that 100nM of chemically modified S594.21 has higher inhibitory activity in U-251MG cells relative to S592.11, S592.21 and S594.11.

Example 3 in vivo efficacy detection-sod 1-mRNA detection

The test used male, 14 Zhou Zhouling SPF grade hSOD1 ^G93A mice (Jiangsu and Co., ltd.) with transgenic expression of the G93A mutant form of human SOD 1. Mutant SOD1 is toxic, can cause motor neuron degeneration, heterozygotes exhibit a phenotype similar to Amyotrophic Lateral Sclerosis (ALS) in humans, and can be used to study neuromuscular diseases including amyotrophic lateral sclerosis. Screening of hSOD1 ^G93A positive mice based on transgene copy number following group entry, the group entry mice were randomly grouped, single ventricle Injection (ICV) was administered (test group injection S594.21, sequences and modifications are shown in table 7; control group injection of artificial cerebrospinal fluid (aCSF); group of administration is shown in table 9), and different brain regions (prefrontal cortex, cerebellum) and spinal cord (cervical, lumbar, thoracic) of the mice were collected on days 3, 7, 28 post-administration, followed by euthanasia of the mice. The different tissues collected were used for sod1-mRNA level detection.

TABLE 9 test protocol for mice dosed with siRNA drug hSOD1 ^G93A by ICV

Sod1-mRNA detection

3.1 Methods

And selecting different brain areas and spinal cord tissues of the frozen mice, and grinding by a full-automatic grinder. Trizol method is used for extracting total RNA in different brain areas and spinal cord tissues of mice, and determining the purity and concentration of the RNA. mRNA expression levels of the sod1 gene were detected using qRT-PCR STARTER KIT (Northenan Fastpure Cell/Tissue Total RNA isolation Kit V2; cat. RC 112-01) and compared between groups. Specific real-time quantitative PCR method and PCR primers used in the same manner as in example 1.

3.2 Results

The test results showed that the relative expression level of the sod1 gene mRNA was significantly lower in the ICV-dosed, S594.21-dosed group than in the control group (FIGS. 3A-3E), and that the difference was statistically significant (P < 0.01). Referring to table 10, relative to the control group, the prefrontal cortex, cerebellum, cervical, lumbar, thoracic vertebrae of the S594.21 dosing group were down-regulated by 29.48%, 38.39%, 67.67%, 31.66%, 7.7% mRNA levels relative to the aCSF group, respectively, 3 days after dosing, the prefrontal cortex, cerebellum, cervical, lumbar, thoracic vertebrae of the S594.21 dosing group were down-regulated by 36.81%, 51.95%, 56.15%, 68.41%, 59.46% mRNA levels relative to the aCSF group, respectively, after dosing for 7 days, and the prefrontal cortex, cerebellum, cervical, lumbar, thoracic vertebrae of the S594.21 dosing group were down-regulated by 65.32%, 50.72%, 80.49%, 79.69%, 84.34% mRNA levels relative to the aCSF group, respectively, suggesting that the drug may allow for low frequency dosing, thereby facilitating the patient. Meanwhile, the siRNA of the application can obviously reduce the mRNA expression level of the sod1 gene of brain and spinal cord tissues of mice.

TABLE 10 percentage of reduction of sod1 mRNA levels by S594.21 RNAi agent relative to aCSF group

Example 4 in vivo efficacy detection-behavioural rescue detection

Experiments were performed using SPF grade hSOD1 ^G93A mice (Jiangsu Ji and Co., ltd.) of 5 or 14 weeks old, which strain is commonly used for studies of neuromuscular diseases such as ALS,50% of mice have a lifespan of about 157.1.+ -. 9.3 days), and single administration was performed by intrathecal Injection (IT) or ventricular Injection (ICV) (see Table 11 for groups). Environmental adaptation (handle) treatment and baseline (baseline) test 3 days before administration were performed one week before administration, followed by stick-turning, reverse hanging, pole-climbing behavioural test, which was performed once every other week after administration.

TABLE 11 ICV/IT dosing WT, hSOD1 ^G93A mouse protocol

4.1 Environment adaptive treatment

All mice are subjected to 7-day mouse environment adaptation (handle) by test operators before test operation and administration, so that the effect of the stimulus of the test operators on the behavior of the mice is eliminated, namely, a transparent cylinder with an opening at two ends, a diameter of 10cm and a length of 20cm is placed in a mouse cage, and the mice are lifted to a position 60cm away from the ground after the mice automatically climb into the cylinder, so that the mice can freely move for 5min. During this time, the condition of the mice was observed in real time through the transparent cylinder.

4.2 Rotating rod test

Mice were allowed to acclimate for 30 minutes in the rotameter test room before the start of the experiment. The rotating rod was rubbed with 75% alcohol. The first day mice were placed on a 4rpm rotating bar for 60s of acclimation, each mouse being acclimatized 3 times, 30min apart. The following day, mice were subjected to adaptation training on 8rpm rotor bars for 60s, each mouse was adapted 3 times, 30min apart. On the third day, the mice were tested on an accelerated rotating rod, the speed of which was slowly accelerated from 4rpm to 40rpm, the acceleration time was 360s, and the total maximum time period was kept at 40rpm for 240s, i.e., 600s, and the residence time of the mice on the rotating rod was recorded. After the test is finished, the bar rotating instrument is cleaned and wiped by 75% alcohol. After the first week is completed, only 8rpm of adaptation training is needed for the following time.

4.3 Reverse hanging test

Mice were allowed to acclimate for 30 minutes in the inverted test room before the start of the experiment. And (3) horizontally placing the mice on the fine iron grids, after the mice are gripped, turning the fine iron grids to be buckled on the transparent box top with a proper height, starting timing after turning, stopping timing when the mice drop from the fine iron grids, and recording the grasping time of the mice. Each mouse was tested twice, two hours apart.

4.4 Pole climbing test

Mice were acclimatized in advance in the pole-climbing test room for 30 minutes. Before the test starts, the mouse is allowed to continuously complete three pole climbing adaptability exercises, and the purpose is to enable the mouse to complete smooth pole climbing. After the test is started, the mice are placed on the rod top, timing is started when the heads of the mice face downwards and the body is vertical, the forelimbs are landed, timing is stopped, and the climbing time of the mice along the rod is recorded. Each mouse was tested 3 times at 1 hour intervals.

4.5 Results

The test results show that ICV/IT administration can significantly improve the exercise capacity and muscle capacity of the mice with disease, and the difference has statistical significance (P < 0.01) (the stick turning behavior results are shown in figures 4A and 4B, the reverse hanging behavior results are shown in figures 5A and 5B, and the stick climbing behavior results are shown in figure 6). Wherein:

Mice 15W, ICV were dosed, compared with control group aCSF, the dosing group S594.21 had significantly longer time to turn the stick at the experimental end (normalized treatment of FIG. 4A, mean: 0.17vs 1.18), significantly longer time to reverse the experimental end (normalized treatment mean:0.04vs 0.74 of FIG. 5A), significantly shorter time to climb the stick at the experimental end (normalized treatment mean:1.43vs 0.72 of FIG. 6), and it could be concluded that compound S594.21 significantly improved the locomotor and muscular capabilities of the ill mice, and that the differences were statistically significant (P < 0.01);

Mice 7W, IT were dosed with significantly longer stick time at the end of the experiment for dosing group S594.3 (FIG. 4B normalized treatment mean:0.52;0.54vs 1.03), significantly longer reverse hanging time (FIG. 5B normalized treatment mean:0.24;0.35vs 1.13) compared to control groups aCSF and Tofersen, and it was concluded that compound S594.3 significantly improved motor and muscle capacity in the ill mice, and that the differences were statistically significant (P < 0.01).

The foregoing test results are all based on a single administration suggesting that the drug may allow for less frequent administration, thereby improving the patient's morbidity and improving the quality of life.

Example 5 in vivo drug detection-survival assessment and weight detection

5.1 Method

After entering the animal feeding room from the mice, the mice were subjected to environmental adaptation treatment for 7 days by the experimental operators, and then single administration of intrathecal Injection (IT) or ventricular Injection (ICV) (see table 11 for group). Before and after administration, a special feeder should pay attention to observe the gait of the mice and weakness of the forelimbs and hindlimbs, and check the mental appearance and survival of the mice. The mice were placed aside and were unable to turn over for 30s, and the death of the animals was judged.

Weight measurements were taken weekly for behavioural testing, and the weight of each mouse was taken 3 times with the mice relatively stationary, taking into account that the mice' movements caused fluctuations in weight, and averaged.

5.2 Results

The test results of the S594.21 molecule show (figures 7 and 8) that ICV administration (20 mice/group) can improve the weight of the mice with diseases at late onset (namely SPF grade hSOD1 ^G93A mice with 15 weeks of age), and the current data of the administration group show that compared with the aCSF control group, the S594.21 injection group can remarkably prolong the survival time of the mice with diseases by 23 weeks, and the difference compared with the vehicle group has statistical significance (P < 0.01). Suggesting that the drug may allow for less frequent administration, thereby improving patient weight and prolonging survival, allowing patients to survive with high quality. The longest survival time of the current mice is at least 41 weeks, and the S594.21 group prolongs the survival time of the mice by nearly 6 months relative to the alpha CSF control group, which is obviously superior to other small nucleic acid medicines with the same target in the grinding process in the prior art, for example, the survival time of the mice is prolonged by only 40 days by the SOD1 ASO product (tofersen) with the same target (Alex McCampbell et al, 2018) in the current literature.

The test results of the S594.3 molecule (12/group) show (fig. 9) that single administration of300 μg/at early onset IT significantly prolonged the survival of the diseased mice by 9 weeks compared to the aCSF (artificial cerebrospinal fluid) panel, and that the difference was statistically significant (P < 0.01) using T-test detection compared to Tofersen, with significant advantages in the inhibition of SOD1 mRNA levels. Suggesting that the drug may allow for less frequent administration, thereby improving patient weight and prolonging survival, allowing patients to survive with high quality.

Claims

A double-stranded RNAi agent for inhibiting the expression of the sod1 gene, comprising a sense strand and an antisense strand that complement each other to form a double-stranded region, wherein the sense strand and/or the antisense strand comprises or consists of 15-25 nucleotides, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3, the length of the double-stranded region is 15-25 bp, and at least one nucleotide in the double-stranded RNAi agent is modified;

The modification is selected from any one or more of the following: locked nucleic acid modification, open ring or non-locked nucleic acid modification, 2'-methoxyethyl modification, 2'-O-methyl modification, 2'-O-allyl modification, 2'-C-allyl modification, 2'-fluoro modification, 2'-deoxy modification, phosphorothioate backbone modification, DNA modification, and lipophilic modification.

The double-stranded RNAi agent according to claim 1, wherein the sense strand of the double-stranded RNAi agent comprises the nucleotide sequence of UAGCUGUAGAAAUGUAUCCUG (SEQ ID NO: 1) or consists of the sequence shown in SEQ ID NO: 1, and the antisense strand comprises the nucleotide sequence of CAGGAUACAUUUCUACAGCUAGC (SEQ ID NO: 2) or consists of the sequence shown in SEQ ID NO: 2; or

The sense strand of the double-stranded RNAi agent includes the nucleotide sequence of AGCUGUAGAAAUGUAUCCUGA (SEQ ID NO: 3) or consists of the sequence shown in SEQ ID NO: 3, and the antisense strand includes the nucleotide sequence of UCAGGAUACAUUUCUACAGCUAG (SEQ ID NO: 4) or consists of the sequence shown in SEQ ID NO: 4.

The double-stranded RNAi agent according to claim 2, wherein: the modification method of the double-stranded RNAi agent includes:

(1) sense strand: 19-23 nt in length, such as 19, 20, 21, 22 or 23 nt; composed of alternating 2'-O-methyl modified regions and 2'-fluoro modified regions, the number of consecutive nucleotides in each modified region being any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides; the modification mode of the first modified region from the 5' end and the 3' end is the same; and

(2) Antisense strand: 19-25 nt in length, such as 19, 20, 21, 22, 23, 24 or 25 nt; composed of alternating 2'-O-methyl modified regions, 2'-fluoro modified regions, unmodified regions and/or DNA regions, with the length of consecutive nucleotides in each modified region being any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides; the modification mode of the first modified region from the 5' end and the 3' end is the same;

In the sense strand and the antisense strand, the continuous nucleotide region from the 1st to 2nd, 1st to 3rd, 1st to 4th, 1st to 5th, 1st to 6th, or 1st to 7th positions from the 5' end, and the continuous nucleotide region from the 1st to 2nd, 1st to 3rd, 1st to 4th, 1st to 5th, 1st to 6th, or 1st to 7th positions from the 3' end are all connected by a thiophosphate backbone; preferably, the continuous nucleotide region from the 1st to 3rd positions from the 5' end, and the continuous nucleotide region from the 1st to 3rd positions from the 3' end are all connected by a thiophosphate backbone.

A double-stranded RNAi agent according to any one of claims 1 to 3, wherein the lipophilic modification is one or more lipophilic moieties conjugated to one or more nucleotides on at least one chain of the double-stranded RNAi agent, and the lipophilic moiety is selected from a saturated or unsaturated straight or branched C4-C30 hydrocarbon chain, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl) glycerol, geranyloxyhexanol, glycerol, borneol, menthol, 1,3-propylene glycol, palmitic acid, myristic acid, dimethoxytrityl or phenoxazine.

The double-stranded RNAi agent according to claim 4, wherein the one or more lipophilic moieties are conjugated to one or more of the following positions in the nucleotide: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand; the conjugation is connected to the base or the sugar ring; preferably the lipophilic moiety is connected to the sugar ring; further preferably the lipophilic moiety is connected to the 2' position of the sugar ring.

The double-stranded RNAi agent according to claim 5, wherein the one or more lipophilic moieties are conjugated to the 6th, 7th, and/or 8th nucleotides from the 5' end of the sense strand.

The double-stranded RNAi agent according to claim 5 or 6, wherein the lipophilic part is a hexadecyl group linked to 2'-O.

The double-stranded RNAi agent according to claim 6 or 7, wherein: the 5' position of the 5' terminal nucleotide of the antisense strand is connected to vinyl phosphate.

The double-stranded RNAi agent according to any one of claims 1 to 8, comprising a modified motif selected from any one of the following:

(1) Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm,

Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

(2) Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm,

Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

(3) Sense chain: NmsNmsNmNmNmN(hd)NfNmNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm,

Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; and

(4) Sense chain: NmsNmsNmNmNmN(hd)NfNmNfNfNfNmNmNmNmNmNmNmNmNmsNmsNm,

Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

Among them, Nm represents a ribonucleotide modified by 2’-O-methyl; Nf represents a ribonucleotide modified by 2’-fluoro; (s) represents that the two nucleotides before and after are connected by a phosphorothioate backbone; N(hd) represents a ribonucleotide modified by 2’-O-C16.

The double-stranded RNAi agent according to any one of claims 1 to 9, comprising one or more selected from the following:

(1) a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO:5) and corresponding modifications, and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence CmsAfsGmGmAmUfAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO:6) and corresponding modifications;

(2) a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO:5) and corresponding modifications, and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence CmsAfsGmGmAmUmAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO:7) and corresponding modifications;

(3) a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide sequence AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and corresponding modifications, and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide sequence UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 9) and corresponding modifications; and

(4) a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and corresponding modifications, and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence UmsCfsAmGmGmAmUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 10) and corresponding modifications;

Among them, Am, Um, Cm and Gm represent ribonucleotides A, U, C and G modified by 2’-O-methyl, respectively; Af, Uf, Cf and Gf represent ribonucleotides A, U, C and G modified by 2’-fluoro, respectively; (s) means that the two nucleotides before and after are connected by a phosphorothioate backbone.

The double-stranded RNAi agent according to claim 10, wherein the nucleic acid sequence comprises any one or more selected from the following:

(1) the sense strand nucleic acid sequence consists of UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and another 0-5 nucleotides at the 5' and/or 3' end,

The antisense strand nucleotide sequence consists of CmsAfsGmGmAmUfAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 6) and an additional 0-5 nucleotides at the 5' and/or 3' end;

(2) the sense strand nucleic acid sequence consists of UmsAmsGmCmUmGmUfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 5) and another 0-5 nucleotides at the 5' and/or 3' end,

The antisense strand nucleotide sequence consists of CmsAfsGmGmAmUmAmCmAmUmUmUmCmUfAmCfAmGmCmUmAmsGmsCm (SEQ ID NO: 7) and an additional 0-5 nucleotides at the 5' and/or 3' end;

(3) the sense strand nucleotide sequence consists of AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 8) and another 0-5 nucleotides at the 5' and/or 3' end,

The antisense strand nucleotide sequence consists of UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 9) and an additional 0-5 nucleotides at the 5' and/or 3' end;

(4) The sense strand nucleotide sequence consists of AmsGmsCmUmGmUmAfGmAfAfAfUmGmUmAmU mCmCmUmsGmsAm (SEQ ID NO: 8) and another 0-5 nucleotides at the 5' and/or 3' end,

The antisense strand nucleotide sequence consists of UmsCfsAmGmGmAmUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 10) and an additional 0-5 nucleotides at the 5' and/or 3' end;

(5) the sense strand nucleotide sequence consists of UmsAmsGmCmUmG(hd)UfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 11) and another 0-5 nucleotides at the 5' and/or 3' end,

(6) the sense strand nucleotide sequence consists of UmsAmsGmCmUmG(hd)UfAmGfAfAfAmUmGmUmAmUmCmCmsUmsGm (SEQ ID NO: 11) and 0-5 nucleotides at the 5' and/or 3' end,

(7) the sense strand nucleotide sequence consists of AmsGmsCmUmGmU(hd)AfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 12) and another 0-5 nucleotides at the 5' and/or 3' end,

The antisense strand nucleotide sequence consists of UmsCfsAmGmGmAfUmAmCmAmUmUmUmCfUmAfCmAmGmCmUmsAmsGm (SEQ ID NO: 9) and an additional 0-5 nucleotides at the 5' and/or 3' end; and

(8) the sense strand nucleotide sequence consists of AmsGmsCmUmGmU(hd)AfGmAfAfAfUmGmUmAmUmCmCmUmsGmsAm (SEQ ID NO: 12) and another 0-5 nucleotides at the 5' and/or 3' end,

Among them, Am, Um, Cm and Gm represent ribonucleotides A, U, C and G modified by 2’-O-methyl, respectively; Af, Uf, Cf and Gf represent ribonucleotides A, U, C and G modified by 2’-fluoro, respectively; (s) represents that the two nucleotides before and after are connected by a phosphorothioate backbone; G(hd) and U(hd) represent G and U modified by 2’-O-C16, respectively.

The double-stranded RNAi agent according to claim 10, wherein the nucleic acid sequence is formed by linking vinyl phosphate to the 5' position of the 5' terminal nucleotide of the antisense strand in the double-stranded RNAi agent according to claim 10.

The double-stranded RNAi agent according to claim 6 or 7, wherein: the sense strand structure of the double-stranded RNAi agent is as shown in SEQ ID NO: 12; the antisense strand structure of the double-stranded RNAi agent is as shown in SEQ ID NO: 10 or SEQ ID NO: 13.

A biomaterial comprising:

(A) a reagent or a kit comprising the double-stranded RNAi agent according to any one of claims 1 to 13; or

(B) A pharmaceutical composition, consisting of the double-stranded RNAi agent according to any one of claims 1 to 13 and other pharmaceutically acceptable components.

A use of a double-stranded RNAi agent, selected from any one of the following groups:

(I) Use of the double-stranded RNAi agent according to any one of claims 1 to 13 or the biomaterial according to claim 14 in inhibiting sod1 gene expression or preparing a product for inhibiting sod1 gene expression;

(II) Use of the double-stranded RNAi agent according to any one of claims 1 to 13 or the biomaterial according to claim 14 in reducing sod1 mRNA expression or SOD1 protein concentration in different brain region tissues and spinal cord, or in the preparation of a product for reducing sod1 mRNA expression or SOD1 protein concentration in different brain region tissues and spinal cord;

(III) Use of the double-stranded RNAi agent according to any one of claims 1 to 13 or the biomaterial according to claim 14 in preventing and/or treating a disease mediated by a mutated sod1 gene, or in preparing a product for preventing and/or treating a disease mediated by a mutated sod1 gene;

(IV) Use of the double-stranded RNAi agent according to any one of claims 1 to 13 or the biomaterial according to claim 14 for alleviating the symptoms of a disease mediated by a mutated sod1 gene, or for preparing a product for alleviating the symptoms of a disease mediated by a mutated sod1 gene;

The diseases mediated by the mutated sod1 gene described in (III) and (IV) above include amyotrophic lateral sclerosis (ALS) or neurodegenerative diseases.