Oligonucleotides targeting SOD1

Abstract

The present application relates to siRNA and oligonucleotide agents for use in the prevention or treatment of SOD1-related neurodegenerative diseases or conditions, such as amyotrophic lateral sclerosis, ALS. The oligonucleotide agent comprises a double-stranded targeting oligonucleotide (siRNA) and a non-targeting single-stranded oligonucleotide (ACO), wherein the siRNA targets the mRNA region of the target gene SOD1.

Description

Technical Field

[0001] The present application relates to the field of nucleic acid technology, and in particular to an oligonucleotide agent that reduces SOD1 transcription levels and its pharmaceutical application. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal paralytic disorder caused by the degeneration of motor neurons. ALS is characterized by progressive, adult-onset degeneration of motor neurons in the cranium, brainstem, and spinal cord, leading to death from respiratory failure within 3 to 5 years of diagnosis. ALS occurs in either familial or sporadic forms, depending on whether there is a family history of the disease, with sporadic ALS (sALS) accounting for 90% of ALS patients. In the United States, the following most commonly mutated genes account for approximately 75% of ALS cases: chromosome 9 open reading frame 72 gene (C9orf72; 40%), superoxide dismutase 1 (SOD1; 20%), trans-reactive DNA binding protein 43 (TDP43; 4%), and fusion in sarcoma / translocation in liposarcoma (FUS / TLS; 4%). Mutations in the C9orf72 and SOD1 genes also apparently account for approximately 5% to 8% and approximately 2% to 3% of sALS cases, respectively. To date, there is no effective therapy available for ALS, and new therapies are needed to treat the disease.

[0003] Among the prominent known genes for ALS, the SOD1 gene remains the main cause of fALS and is considered an important ALS drug target. The first description of ALS disease dates back to at least 1824 by Charles Bell. However, SOD1 was not discovered as the first risk gene for ALS until 1993. In 1994, the first SOD1 transgenic mouse model (hSOD1 G93A The establishment of the SOD1 gene therapy (SOD1) marked a new era in ALS research. SOD1 mutants most likely cause disease through gain-of-function, and reducing their levels may be beneficial. Therefore, silencing SOD1 transcription is an important strategy for treating ALS. Summary of the Invention

[0004] To this end, the present application provides an oligonucleotide agent that has a strong inhibitory effect on the expression of superoxide dismutase 1 (SOD1). The oligonucleotide agent includes a small interfering RNA (siRNA), which is used to treat diseases or conditions caused by SOD1 gene mutations, such as ALS, by targeting SOD1 mRNA and subsequently downregulating SOD1 protein levels in cells or individuals through RNA interference (RNAi).

[0005] Specifically, the inventors of the present application have discovered an oligonucleotide agent with potent knockdown of SOD1 transcripts and high central nervous system delivery efficiency, which comprises: (a) siRNA; and (b) a non-targeting single-stranded auxiliary oligonucleotide (ACO), wherein the ACO is 6 to 22 nucleotides in length, wherein the siRNA and ACO are covalently linked (with or without one or more linking components) to form the oligonucleotide agent.

[0006] In some embodiments, the siRNA comprises a sense strand and an antisense strand forming a double-stranded structure, wherein the antisense strand comprises a nucleotide sequence comprising at least 10 consecutive nucleotides with 0, 1, 2 or 3 mismatches, which has at least 85% nucleotide sequence complementarity or homology with a partial nucleotide sequence of SEQ ID NO: 1 (Table 1).

[0007] In some embodiments, the ACO is composed of one or more of RNA, DNA, BNA, LNA, glycerol nucleic acid (GNA) and peptide nucleic acid (PNA). In some embodiments, the length of the ACO is 6 to 18 nucleotides. In some embodiments, the length of the sense strand is at least 10 nucleotides. In some embodiments, the nucleotide length of the sense strand ranges from 10 to 60 nucleotides. In some embodiments, the nucleotide length of the sense strand ranges from 16 to 25 nucleotides. In some embodiments, the nucleotide length of the antisense strand ranges from 15 to 35 nucleotides. In some embodiments, the nucleotide length of the antisense strand ranges from 19 to 25 nucleotides.

[0008] In certain embodiments, one strand of the oligonucleotide sequence disclosed in the present application has at least 85%, at least 90%, or at least 95% homology or complementarity with a nucleotide sequence selected from SEQ ID NOs: 2 to 269. In certain embodiments, the sense strand of the oligonucleotide sequence disclosed in the present application has at least 85%, at least 90%, or at least 95% homology with a nucleotide sequence selected from SEQ ID NOs: 2 to 269. In certain embodiments, the antisense strand of the oligonucleotide sequence disclosed in the present application has at least 85%, at least 90%, or at least 95% complementarity with a nucleotide sequence selected from SEQ ID NOs: 2 to 269.

[0009] In one aspect of the present application, an oligonucleotide agent capable of inhibiting / downregulating SOD1 transcripts in a cell is provided. In some embodiments, the oligonucleotide agent comprises an siRNA, wherein the sense strand of the siRNA has a nucleotide sequence that is at least 85%, at least 90%, or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 270 to 537.

[0010] In some embodiments, an oligonucleotide agent comprising a siRNA is provided, wherein the antisense strand of the siRNA has a nucleotide sequence that is at least 85%, at least 90%, or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 538 to 805.

[0011] In some embodiments, an oligonucleotide agent comprising an siRNA is provided, wherein the siRNA comprises a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence that is at least 85%, at least 90%, or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 270 to 537, and the antisense strand of the siRNA has a nucleotide sequence that is at least 85%, at least 90%, or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 538 to 805.

[0012] In some embodiments, an oligonucleotide agent comprising an siRNA is provided, wherein the siRNA comprises a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence that is at least 85%, at least 90%, or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 808 to 827 and 867, and the antisense strand of the siRNA has a nucleotide sequence that is at least 85%, at least 90%, or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 828 to 849 and 868.

[0013] In another aspect of the present application, an oligonucleotide agent comprising siRNA and a non-targeting ACO is provided, wherein the ACO comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from SEQ ID NO: 865.

[0014] In some embodiments, the ACO is conjugated to a linking component. In some embodiments, the 5' end or 3' end or one or more internal nucleotides of the ACO are conjugated to a linking component. In some embodiments, the siRNA and ACO are covalently conjugated via a linking component. In some embodiments, the sense strand or antisense strand of the siRNA is covalently conjugated to the ACO via a linking component. In some embodiments, the linking component is or includes one or more of the following groups: an ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, RNA, DNA, a carbohydrate, a thiol linking group, a phosphodiester, a thiophosphate, a phosphoramide, an amide, a carbamate, a tetrazole linking group, and a benzimidazole linking group. In some embodiments, the linking component is or includes one or more of the following: a) Spacer phosphoramidite 18 (phosphoramidite acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecan-1-yl(2-cyanoethyl ester). b) Spacer-9 (3-[2-[2-[2-(bis(4-methoxyphenyl)(phenylmethoxy]ethoxy]ethoxy]ethoxy-[di(propyl-2-yl)amino]phosphino]oxypropionitrile); c) spacer phosphoramidite C3 (6-(4,4'-dimethoxytrityl)hexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite); and d) Spacer C6 phosphoramidite (6-(4,4'-dimethoxytrityl)hexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) e) Divalent linker (DIO): 16-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1,1-bis(4-methoxyphenyl)-18-oxo-1-phenyl-2,5,8,11,14,17-hexaoxaheneicosane-CPG.

[0015] In some embodiments, the ACO is covalently linked to the 3' end, 5' end, or both of the 3' and 5' ends of one or more internal nucleotides of the sense strand of the siRNA. In some embodiments, the ACO is covalently linked to the 3' end, 5' end, or both of the 3' and 5' ends of one or more internal nucleotides of the antisense strand of the siRNA. In some embodiments, more than one ACO is covalently linked to the siRNA. In some embodiments, 2 to 10 ACOs are covalently linked to the siRNA.

[0016] In some embodiments, at least one nucleotide of the siRNA is a chemically modified nucleotide. In some embodiments, at least one nucleotide of the ACO is a chemically modified nucleotide. In some embodiments, the ACO has at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or about 100% of the nucleotides are chemically modified nucleotides. In some embodiments, the sense strand of the siRNA has at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or about 100% of the nucleotides are chemically modified nucleotides. In some embodiments, the antisense strand of the siRNA has at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or about 100% of the nucleotides are chemically modified nucleotides. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 2' sugar modification selected from one or more of a 2'-fluoro-2'-deoxynucleoside (2'-F) modification, a 2'-O-methyl (2'-O-Me) modification, and a 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the ACO comprises at least one phosphorothioate (PS) backbone modification. In some embodiments, the ACO comprises 6 to 17 phosphorothioate (PS) backbone modifications. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is the addition of a 5'-phosphate moiety to the 5' end of the nucleotide sequence. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate moiety to the 5' end of the nucleotide sequence. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is the addition of a 5-methylcytosine moiety to the 5' end of the nucleotide sequence.

[0017] In some embodiments, the ACO is conjugated to one or more conjugated groups. In some embodiments, the siRNA is conjugated to one or more conjugated groups. In some embodiments, the sense strand or antisense strand of the siRNA is conjugated to one or more conjugated groups. In some embodiments, the one or more conjugated groups are selected from the group consisting of lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies. In some embodiments, the one or more conjugated groups are selected from the group consisting of cell penetrating peptides, polyethylene glycols, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0018] The present application relates to an oligonucleotide agent comprising an siRNA and a non-targeting ACO, wherein the sense strand has a nucleotide sequence that is at least 85%, at least 90% or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 808 to 827, 850 to 851 and 856 to 857, and the antisense strand of the siRNA has a nucleotide sequence that is at least 85%, at least 90% or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 828 to 849 and 861 to 863.

[0019] The present application includes a vector containing an oligonucleotide agent disclosed in the present application.

[0020] The present application also provides cells comprising the oligonucleotide agents disclosed herein. In one embodiment, the cells are mammalian cells, optionally human cells. In some embodiments, the cells are host cells. The aforementioned cells can be in vitro, such as cell lines or cell strains, or can be present in or taken from a mammal, such as a human body.

[0021] The present application also provides a pharmaceutical composition comprising the oligonucleotide agent disclosed herein. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, a liposome or LNP, a polymer, a micelle, a colloid, a metal nanoparticle, a non-metallic nanoparticle, a bioconjugate, and a polypeptide. The present application also provides a drug kit comprising the oligonucleotide agent or pharmaceutical composition disclosed herein.

[0022] The present application provides a method for reducing SOD1 gene transcription level or SOD1 protein level, comprising administering the pharmaceutical composition disclosed in the present application to a subject.

[0023] This application also provides a method for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS) in a subject, comprising administering a pharmaceutical composition disclosed herein to the subject. In some embodiments, the subject has sporadic amyotrophic lateral sclerosis (sALS). In some embodiments, the subject has familial amyotrophic lateral sclerosis (fALS). In some embodiments, the pharmaceutical composition can reduce SOD1 gene transcript levels or SOD1 protein levels.

[0024] In some embodiments, the ACO of the oligonucleotide agent can improve the stability, bioavailability, biodistribution, and / or cellular uptake of the siRNA compared to an oligonucleotide agent without the ACO.

[0025] In some embodiments, the ACO of the oligonucleotide agent can increase the biodistribution of the siRNA within one or more target tissues compared to an oligonucleotide agent without the ACO.

[0026] In some embodiments, the ACO of the oligonucleotide agent can increase the biodistribution of the siRNA in two or more target tissues compared to an oligonucleotide agent without the ACO.

[0027] In some embodiments, the one or more target tissues are selected from the group consisting of: prefrontal cortex, cerebellum, cerebrum, spinal cord, muscle, lung, eye, liver, and kidney.

[0028] The present application relates in part to siRNAs comprising an oligonucleotide sequence ranging in length from 16 to 35 consecutive nucleotides, wherein the consecutive oligonucleotide sequence comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% homologous or complementary to an equal length portion of SEQ ID NO: 1, wherein the siRNA can inhibit the mRNA transcription level of the SOD1 gene by at least 80% compared to the baseline level of SOD1 mRNA.

[0029] The inventors of the present application also discovered that the optimal target sequence / sense strand for siRNA in the SOD1 gene includes sequences having the following characteristics: (1) a GC content between 35% and 65%; (2) fewer than 5 consecutive identical nucleotides; (3) 3 or fewer dinucleotide repeats; and (4) 3 or fewer trinucleotide repeats. As a beneficial result, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) can inhibit the transcription level of SOD1 mRNA in a cell by at least 80% compared to the baseline level of SOD1 mRNA after interaction with the siRNA. Based at least in part on these findings, the present disclosure features siRNAs, compositions, and pharmaceutical compositions that can inhibit the transcription level of SOD1 mRNA in a cell by at least 80% compared to the baseline level of SOD1 mRNA. The present application also provides methods for preventing or treating diseases or symptoms induced by SOD1 gene mutations or abnormal SOD1 protein levels in individual cells, comprising administering any siRNA, composition, and / or pharmaceutical composition described herein.

[0030] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description, which merely shows and describes illustrative embodiments of the present application. The present application is capable of other different embodiments, and its several details can be modified in various obvious respects, all without departing from the present invention. Therefore, these drawings and descriptions are to be regarded as illustrative, not restrictive. Join by reference

[0031] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into this application to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference into this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the present invention are set forth with particularity in the appended claims. The features and advantages of the present invention may be better understood by reference to the following detailed description, which illustrates an embodiment incorporating the principles of the present invention and is accompanied by the accompanying drawings (also referred to herein as "Figures"), wherein:

[0033] Figures 1A-1E Shown is the screening of siRNAs that can knock down SOD1 in vitro. Figure 1A shows that HEK293A cells were transfected with each siRNA duplex (268 in total) at a concentration of 10 or 0.1 nM for 24 hours, with two replicates per group. The expression level of SOD1 was quantified by RT-qPCR using a gene-specific primer set. TBP amplification was used as an internal reference to normalize the expression data. Shown in the figure are the expression values ​​of SOD1 mRNA relative to the mock treatment (dashed line) for each experimental parallel sample. The mock sample was transfected in the absence of oligonucleotides. Figure 1B shows the knockdown activity and cell viability of the best performing siRNAs in HEK293A cells treated with 6 increasing concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM) and quantified by RT-qPCR and PI staining. Shown in the figure are the SOD1 knockdown and cytotoxicity results of the top 5 siRNAs (i.e., siSOD1-063, 047, 104, 005, and 258) with the best performance relative to the mock treatment (dashed line). Figure 1C shows the dose-response curve of the top 5 siRNAs that performed best in treating SK-N-AS cells at 8 concentrations (i.e., 0.00006, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25, and 1 nM) and measuring them with RT-qPCR. The data are the mean ± standard deviation of 2 experimental parallel samples. Figure 1D shows the results of transfecting SK-N-AS cells for 24 hours with different chemically modified variants of each siRNA or a nonspecific siRNA control (siCon) at 0.1 nM. The knockdown activity relative to the mock treatment was assessed by RT-qPCR. Figure 1E shows dose-response curves generated by treating SK-N-AS cells with various M3 modification variants (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, and 258M3) and measuring them by RT-qPCR. Data are the mean ± standard deviation of three experimental replicates.

[0034] Figures 2A-2BThe secondary screening siRNA in vitro efficacy and poor cytotoxicity are shown. The knockdown activity and cell viability of each of the remaining top 25 siRNAs that performed best in the initial screening at 6 indicated concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM) in HEK293A cells are also shown. RT-qPCR quantification ( Figure 2A ) and PI staining quantification ( Figure 2B ).

[0035] Figure 3 Figure 2 shows the dose-dependent knockdown results of the top five best-performing siRNA candidates in T98G cells. Dose-response curves for each of the top five best-performing siRNAs in T98G cells transfected for 24 hours at eight treatment concentrations (i.e., 0.00006, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25, and 1 nM). The expression level of SOD1 was quantified by RT-qPCR using a gene-specific primer set. TBP amplification was used as an internal reference to normalize the expression data. Shown in the figure are the mean ± standard deviation of two experimental replicates relative to the control group (without oligonucleotide).

[0036] Figures 4A-4B The top 5 siRNA candidates showed poor in vitro cytotoxicity. The top 5 siRNAs were used with SOD1 knockdown inhibitors containing significantly higher IC values ​​than those of the top 5 siRNA candidates. 50 SK-N-AS cells and T98G cells were transfected with four treatment concentrations (i.e., 1.56, 6.25, 25, and 100 nM) of the same oligonucleotide. 100 nM siCon was used as a negative control (Neg Con). Mock samples were transfected in the absence of oligonucleotides. Assessment of adverse cytotoxicity after 72 hours of treatment was performed. Figure 4A To quantify the results of caspase3 / 7 activity, Figure 4B The cell viability was measured using the ST-8W reagent metabolism as a marker. Data are the mean ± SD of two experimental replicates relative to the mock treatment (dashed line).

[0037] Figure 5 The effect of different chemical modification patterns on siRNA knockdown activity in T98G cells is shown. T98G cells were transfected with four different chemically modified variants of various siRNA candidates (i.e., M1, M2, M3, or M4) or a nonspecific siRNA control (siCon) at 0.1 nM for 24 hours. Knockdown activity relative to mock treatment was assessed by RT-qPCR. Data are mean ± SD of two replicates.

[0038] Figure 6The results show the dose-dependent knockdown of M3-modified siRNA in T98G cells. T98G cells were transfected with various M3-modified siRNA candidates (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, 258M3, and 270M3) at a gradient treatment concentration for 24 hours, and a dose-response curve was generated. The expression level of SOD1 was quantified by RT-qPCR using a gene-specific primer set. TBP amplification was used as an internal reference to normalize the expression data. The figure shows the mean ± standard deviation of three experimental replicates relative to the test group (no oligonucleotide).

[0039] Figures 7A-7D Figure 7A shows the knockdown activity of siRNA-ACO conjugates in vitro. Figure 7A is a visual representation of the siRNA-ACO conjugate structure, in which the 14-nt ACO (called AC1) is conjugated to the 3' end of the sense strand via a short L9 linker (i.e., spacer-9 linker). Figure 7B shows the results of HEK293A and T98G cells transfected with exemplary siRNA-ACO (i.e., siSOD1-005M3-AC1) or AC1 alone for 24 hours at a concentration of 0.25 or 2.5 nM. Mock samples were transfected in the absence of oligonucleotides. siCon treatment served as a negative control for knockdown activity. The expression level of SOD1 was quantitatively determined by RT-qPCR using a gene-specific primer set. TBP amplification was used as an internal reference to normalize expression data. Shown in the figure are the expression values ​​of SOD1 mRNA for each experimental replicate relative to the mock treatment (dashed line). Figure 7C compares the dose-response curves of siRNA knockdown activity in T98G cells, respectively, siSOD1-005M3 conjugated with AC1 (siSOD1-005M3-AC1) or not conjugated with AC1 (siSOD1-005M3), tested at 10 treatment concentrations (i.e., 0.0003, 0.0009, 0.0027, 0.0082, 0.024, 0.074, 0.22, 0.67, 2, and 6 nM), evaluated by RT-qPCR. Figure 7D shows the knockdown activity of siSOD1-005M3 relative to published ASO sequences (i.e., ASO SOD1 , SEQ ID NO: 864; see T. Miller et al., New England Journal of Medicine 383, 109-119 (2020)), a dose-response curve was generated in terms of sequence and chemistry, comparing siRNA-ACO with (siSOD1-005M3-AC1 VP ) or without (siSOD1-005M3-AC1) 5'VP modification knockdown activity.

[0040] Figures 8A-8BIn vitro knockdown activity of siRNA-ACO drug candidates was demonstrated. Each 5'VP-modified siRNA-ACO (i.e., siSOD1-063M3-AC1) was generated in SK-N-AS (Figure 8A) and T98G (Figure 8B) cells at gradient treatment concentrations. VP 、047M3-AC1 VP 、104M3-AC1 VP 、005M3-AC1 VP 、258M3-AC1 VP and 270M3-AC1 VP ). SOD1 expression levels were quantified by RT-qPCR using a gene-specific primer set. TBP amplification was used as an internal control to normalize the data. The figure shows SOD1 mRNA expression relative to mock transfection. Data are mean ± SD of two replicates.

[0041] Figures 9A-9B The siRNA-ACO candidate showed poor in vitro cytotoxicity in SK-N-AS and T98G cells at a rate exceeding its IC 50 Several major siRNA-ACO conjugates (i.e., siSOD1-063M3-AC1) were transfected with a concentration gradient of 1.56, 6.25, 25, and 100 nM. VP 、047M3-AC1 VP 、104M3-AC1 VP 、005M3-AC1 VP 、258M3-AC1 VP 100 nM siCon was used as a negative control. Mock samples were transfected in the absence of oligonucleotides. Assessment of adverse cytotoxicity after 72 hours of treatment. Figure 9A To quantify the results of caspase 3 / 7 activity, Figure 9B The cell viability was measured using the ST-8W reagent metabolism as a marker. Data are the mean ± SD of two experimental replicates relative to the mock treatment (dashed line).

[0042] Figure 10 showed that siRNA-ACO can inhibit hSOD1 G93A Activity of adult hSOD1 in mouse central nervous system tissues G93A Mice were injected intracerebroventricularly (ICV) with a fixed molecular dose (20 nmol) of various siRNA-ACO drug candidates (i.e., siSOD1-047M3-AC1 VP and 005M3-AC1 VPaCSF monotherapy was used as a vehicle control to establish baseline expression, while nonspecific siRNA-ACO (i.e., siCON2-AC1 VP ) was used as a negative control. VP and 005M3-AC1 VP ) or its non-conjugated derivative (i.e. siSOD1-047M3 VP and 005M3 VP ) was administered intraventricularly with a fixed molecular dose (20 nmol) 14 days after administration, knockdown activity in brain (i.e., frontal cortex, cerebellum, and cerebrum), spinal cord (i.e., cervical, thoracic, and lumbar vertebrae), and peripheral (i.e., liver) tissues was quantified by RT-qPCR. Mouse Tbp (mTbp or Tbp) amplification was used as an internal control to normalize expression data. Shown in the figure are the mean expression values ​​± standard deviation of human SOD1 (hSOD1 or SOD1) relative to aCSF treatment (n = 2 to 6 mice / group). The gray dotted line represents 80% knockdown relative to the baseline (linear dotted line).

[0043] Figures 11A-11B The dose-dependent relationship between siRNA-ACO knockdown activity and tissue accumulation in different central nervous system tissues is shown. siSOD1-047M3-AC1 was injected intracerebroventricularly with the indicated doses (i.e., 50, 100, 200, or 400 μg) of VP ( FIG. 11A ) or siSOD1-005M3-AC1 VP (Figure 11B) Treatment of adult hSOD1 G93A Mice. hSOD1 knockdown activity was quantified by RT-qPCR in selected central nervous system tissues (i.e., cerebellum, cerebrum, and spinal cord) on day 14 post-treatment. Animals treated with aCSF alone represent baseline expression levels and the amount of drug detectable in the absence of siRNA-ACO treatment (0 μg). Figures 11A-11B The knockdown activity is shown as the percentage (%) of SOD1 inhibition relative to baseline (0 μg). Drug concentration is expressed as the amount of siRNA-ACO relative to the tissue sample mass (μg / g). Data are presented as mean ± SD (n = 3-4 mice / group).

[0044] Figures 12A-12C A single intracerebroventricular injection of siRNA-ACO delayed disease onset and prolonged animal survival. G93A Mice were injected intracerebroventricularly with the indicated doses (i.e., 50, 100, 200, or 400 μg) of siSOD1-047M3-AC1 on postnatal day (PND) 85 or PND 60. VPor siSOD1-005M3-AC1 VP FIG12A is a graph showing growth rate (i.e., percent change in body weight) relative to the first day of treatment (dashed line) to monitor disease progression. FIG12B shows disease onset as the percentage of animals at peak body weight in each treatment group. Animal survival is shown in FIG12C as the percentage of surviving animals in each treatment group. The number of animals (n) is indicated in each figure.

[0045] Figures 13A-13D Figures 13A-13B show the pathogenic SNPs of the target sites corresponding to the main siRNA-ACO candidates. VP ( FIG13A ) and siSOD1-047M3-AC1 VP (Figure 13B) Location of pathogenic SNPs within the target site. Shown is the targeted strand sequence, which includes the "seed" region (highlighted in gray) that is complementary to the target site in the hSOD1 transcript containing the indicated pathogenic SNP. Nucleotide mutations are shown in bold italics, where "R" indicates a purine substitution. Figure 13C shows the location of pathogenic SNPs in HEK293A cells treated with siSOD1-047M3-AC1 at a concentration of 18 nM. VP or nonspecific scrambled control (siCON2-AC1 VP ) were co-transfected with a luciferase reporter gene construct (i.e., pLuc) containing the conserved sequence or one of its pathogenic mutations (i.e., P.E22G and P.F21C). SOD1 、pLuc P.E22G 、pLuc P.F21C 13D ). In FIG13D , the expression of siSOD1-047M3-AC1 in the presence of the indicated concentrations (i.e., 0.03, 0.07, 0.22, 0.67, 2.0, 6.0, and 18 nM) was detected. VP Dose-response curves of luciferase activity were generated after co-transfection. Data are presented as mean ± SEM of two replicates relative to samples without siRNA treatment. The mean values ​​of each data point in the three dose-response curves were compared using Tukey's multiple comparison test, and the results showed statistical significance ( * ).

[0046] Figures 14A-14C Two doses of siRNA-ACO were shown to delay disease onset and prolong survival in male and female adult hSOD1 G93A Mice were treated twice on PND 68 and PND 100: siSOD1-047M3-AC1 was injected intrathecally (IT) with the indicated doses (i.e., 75, 150, or 300 μg) VP Nonspecific control (i.e. siCON3-AC1 VP ) and ASO SOD1Injection at a dose of 150 μg / time. Treatment with aCSF served as a vehicle control. In Figure 14A, body weight was plotted in grams (g) compared to background animals (wild type, WT) to monitor disease progression. In Figure 14B, disease onset was plotted as the percentage of animals at peak body weight in each treatment group. Animal survival was plotted as the percentage of surviving animals in each treatment group, see Figure 14C. The number of animals (n) is indicated in each figure.

[0047] Figures 15A-15D siRNA-ACO treatment was shown to improve hSOD1 G93A Motor function in mice. Male and female hSOD1 G93A Mice were treated twice on PND 68 and PND 100: siSOD1-047M3-AC1 was injected intrathecally with the indicated doses (i.e., 75, 150, or 300 μg) VP hSOD1 in males and females during the day G93A Mice were subjected to the open field test. The total distance moved by each animal within 15 minutes was automatically recorded in centimeters (cm). The data were plotted as the mean ± standard deviation of the distance moved for each treatment group, as shown in Figure 15A. The grip strength of male and female animals was assessed by treatment group. The grip strength test was repeated three times, and the average value of each animal was recorded. The data of grip strength of each treatment group were plotted as the mean ± standard deviation in grams (g), as shown in Figure 15B. Animal fatigue and coordination were assessed by a 5-minute rotating rod test. The experiment was repeated three times, and the longest latency time (seconds) for each animal to fall was recorded, as shown in Figure 15C. Before the open field, rotating rod and / or grip strength test, the motor function of all animals was scored using the ALS TDI neurological score (NS) scale. The experiment was repeated three times, and the longest latency time (seconds) for each animal to fall was recorded, as shown in Figure 15D. The average NS ± standard deviation of each treatment group at the specified time point is shown.

[0048] Figure 16 Comparison of rotarod performance times for each animal after siRNA-ACO treatment (as shown in Figure 14) is shown. The latency to fall (in seconds) of each animal in the corresponding treatment group was measured by the rotarod test at an early time point (i.e., PND 90) compared to performance at the final time point (i.e., the last time point before death, see Table 8). All experiments were performed in triplicate, and the longest latency for each animal was recorded.

[0049] Figure 17siRNA knockdown activity against SOD1 mRNA expression in HeLa cells is shown. The designated siRNAs (i.e., RD-15757, RD-18972, RD-12500, RD-18973, RD-18948, and RD-18949) were added directly to culture medium containing HeLa cells and cultured for 3 days at a concentration of 1500 nM. Cells were transfected in the absence of oligonucleotides as a mock treatment (not shown). RD-11566 (dsCon2M3v) was used as a non-targeted double-stranded control. SOD1 mRNA levels were quantified by two-step RT-qPCR (using a gene-specific primer set set in a single PCR reaction). TBP was amplified as an internal reference. The values ​​(y-axis) represent the SOD1 mRNA levels relative to the mock treatment after normalization by TBP (mean ± SEM of three replicate transfection wells).

[0050] Figures 18A-18B siRNA knockdown activity against SOD1 mRNA expression in SK-N-AS cells is shown. The indicated siRNAs (i.e., RD-12926, RD-15757, RD-12500, RD-18947, RD-18948, RD-18949, RD-18946, RD-18972, and RD-18973) were transfected into SK-N-AS cells at the indicated concentrations (i.e., 0.0002, 0.001, 0.0039, 0.0156, 0.0625, 0.25, 1, and 4 nM) for 24 hours. Cells were mock-treated in the absence of oligonucleotides (not shown). RD-11566 (dsCon2M3v) was used as a non-targeting double-stranded control (not shown). Figures 18A-18B Shown are SOD1 mRNA levels quantified by two-step RT-qPCR (using gene-specific primer sets in individual PCR reactions). TBP was amplified as an internal control. Values ​​(y-axis) represent SOD1 mRNA levels normalized to TBP relative to mock-treated cells (mean ± SEM of triplicate transfected wells). DETAILED DESCRIPTION

[0051] Although the application has shown and described various embodiments of the present invention, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Without departing from the present invention, those skilled in the art may find many variations, changes, and replacements. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0052] The present application includes methods for treating amyotrophic lateral sclerosis (ALS) by administering an effective amount of an oligonucleotide agent comprising a SOD1-targeting siRNA. The oligonucleotide agent interferes with SOD1 mRNA transcription through an RNA silencing mechanism (RNAi). The inventors of the present application have developed a SOD1 siRNA with a potent inhibitory effect and improved delivery, biodistribution, bioavailability, and other pharmacological properties for the treatment of ALS.

[0053] This application is based on research related to oligonucleotide agents, compositions and methods, in which targeted oligonucleotides (such as siRNA) are combined with oligonucleotide delivery vehicles (ODVs) to downregulate / reduce gene expression to improve the therapeutic effect of genetic diseases. The term "oligonucleotide delivery vehicle (ODV)" refers to a structure that facilitates the introduction of molecules into or uptake by cells, tissues or organs of an individual by conjugating "auxiliary oligonucleotides (ACOs)" to molecules (e.g., duplex oligonucleotides).

[0054] The inventors of the present application have discovered that some double-stranded siRNA sequences are more potent at inhibiting SOD1 transcription than prior art sequences with the same or similar target sequences. The inventors of the present application have also discovered that chemical modification of these siRNAs can enhance their in vitro activity. Surprisingly, by conjugating them to a single-stranded helper oligonucleotide (ACO or ODV), the oligonucleotide agents (i.e., ODV-siRNA or siRNA-ACO) achieve central nervous system delivery and desirable biodistribution and bioavailability in central nervous system tissues when administered via intracerebroventricular (ICV) and / or intrathecal (IT) injection into brain or spinal cord tissue.

[0055] definition

[0056] In this application, the relevant terms are defined as follows:

[0057] Every numerical range given throughout this specification will include every narrower numerical range that falls within the broader numerical range, as if such narrower numerical ranges were expressly written into this application.

[0058] The transitional terms / phrases (and grammatical variations thereof) "comprising," "including," and "consisting of" include phrases such as "consisting essentially of," "consisting essentially of," "consisting of," and "consisting of," and are used interchangeably throughout this application. The open-ended term "comprising" also includes the closed-ended term "consisting of." The terms "including," "having," and "comprising" as used in this application are synonymous, and these terms and variations thereof should be construed as non-limiting.

[0059] The term "amyotrophic lateral sclerosis" or "ALS" includes, but is not limited to, familial amyotrophic lateral sclerosis (fALS), sporadic amyotrophic lateral sclerosis (sALS), Lou Gehrig's disease, diseases associated with mutations in the chromosome 9 open reading frame 72 gene (C9orf72; 40%), superoxide dismutase 1 (SOD1; 20%), trans-reactive DNA binding protein 43 (TDP43; 4%), and fusion in sarcoma / translocation in liposarcoma (FUS / TLS; 4%).

[0060] The term "target gene" as used in this application may refer to a nucleic acid sequence, transgene, viral or bacterial sequence, chromosomal or extrachromosomal gene in the form of DNA, RNA or DNA / RNA hybrids that naturally exists in an organism and / or can be transiently or stably transfected or incorporated into a cell and / or its chromatin. The target gene can be a protein-coding gene or a non-protein-coding gene (such as a microRNA gene and a long non-coding RNA gene), or a transcript of a protein-coding gene, such as a messenger RNA (mRNA) or complementary DNA (cDNA) of a protein-coding gene. "Target sequence," "target site," or "target" are used interchangeably and refer to a continuous oligonucleotide sequence in a target gene sequence, such as an mRNA or cDNA of a target gene, that is homologous or complementary to the sense strand or antisense strand of an siRNA with or without one or more mismatched base pairs.

[0061] As used herein, the terms "SOD1" and "SOD1 gene" are used interchangeably and refer to a gene encoding a SOD1 protein, preferably a mammalian gene, more preferably a human gene. As used herein, the term "SOD1 mRNA" refers to messenger RNA (mRNA) produced by expression of the SOD1 gene or transcription of the SOD1 gene. As used herein, the term "SOD1 cDNA" refers to complementary DNA (cDNA) produced by reverse transcription of SOD1 mRNA. As used herein, the term "SOD1 protein" refers to a protein produced by expression of the SOD1 gene or translation of SOD1 mRNA.

[0062] As used herein, the terms "baseline expression of the SOD1 gene" or "baseline level of SOD1 mRNA" are used interchangeably and refer to the expression of the SOD1 gene in a parallel reference (such as a cell or individual) before or without siRNA treatment.

[0063] The term "oligonucleotide agent" or "oligonucleotide" is used interchangeably and refers to a polymer of nucleotides, including but not limited to single-stranded or double-stranded nucleic acid molecules of DNA, RNA or DNA / RNA hybrids, oligonucleotide chains containing regular and irregular alternating deoxyribose and ribose moieties, and modifications of such oligonucleotides and naturally or non-naturally occurring frameworks. The oligonucleotides described herein that inhibit target gene mRNA transcription levels are small inhibitory nucleic acid molecules (siRNA), antisense oligonucleotide molecules (ASOs) or siRNA molecules (siRNA-ACOs) conjugated to oligonucleotide delivery vehicles (ODVs).

[0064] As used herein, the terms "oligonucleotide chain," "chain," and "oligonucleotide sequence" are used interchangeably and refer to a short nucleotide sequence (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) having less than 35 bases. In one non-limiting embodiment, the length of the chain can be any length from 16 to 25 nucleotides.

[0065] The terms "subject" and "individual" as used herein are used interchangeably and refer to any organism that can be treated with the drugs described herein. The term "patient" refers to a human subject or individual, including infants, children, and adults as described herein.

[0066] A "therapeutically effective amount" of a composition refers to an amount sufficient to achieve the desired therapeutic effect, and therefore does not require a cure or complete remission. In the embodiments of the present application, therapeutic effect refers to an improvement in any indicator of the disease, and a therapeutically effective amount is sufficient to improve clinically significant symptoms / symptoms in the individual being treated. The phrases "therapeutically effective amount" and "effective amount" in this application refer to an amount sufficient to reduce by at least about 15%, preferably at least 50%, more preferably at least 90%, or to reduce by at least about 50%, at least about 100%, at least about 200%, more preferably at least about 500%, and most preferably to prevent clinically significant deficits in the activity, function, and response of the individual being treated.

[0067] The effective amount may vary depending on factors such as the subject's size and weight, the type of illness, or the specific drug described herein. For example, the choice of different drugs described herein may affect the "effective amount." One of ordinary skill in the art would be able to study the factors involved and determine the effective amount of a drug described herein without undue experimentation.

[0068] The dosage regimen can affect the effective dose. The drugs described herein can be administered to a subject before or after diagnosis of a disease or the onset of a condition. In addition, the drugs can be administered daily, weekly, monthly, quarterly, or in sequence in several doses and staggered administration, or the dosage can be continuously infused or bolused. In addition, the dosage of the drugs described herein (one or more) can be increased or decreased proportionally, depending on the urgency of the treatment or prevention.

[0069] The term "treatment" as used herein has a meaning generally understood in the medical field and does not require a cure or complete remission, and includes any beneficial or desired clinical outcome. Non-limiting examples of such beneficial or desired clinical outcomes include: prolonged survival compared to the expected survival without treatment, alleviation of symptoms (including one or more of the following symptoms): proximal skeletal muscle weakness and atrophy, inability to sit or walk independently, difficulty swallowing, difficulty breathing, etc.

[0070] As used herein, the term "preventing" or "delaying" a disease means inhibiting the full development of the disease.

[0071] The term "biological sample" refers to any tissue, cell, fluid, or other material derived from an organism (e.g., a human subject). In some embodiments, the biological sample refers to serum or blood.

[0072] The term "identity" or "homology" used in this application refers to that an oligonucleotide chain (sense chain or antisense chain) of siRNA has sequence similarity with the coding chain or template chain in the target gene region. As used in this application, "identity" or "homology" can be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95% or 99%. In some embodiments, the siRNA has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues that are different from the reference sequence. In order to determine the identity percentage of two nucleic acid sequences, the sequences can be compared to achieve the best comparison purpose (for example, a room can be introduced in one or both of the first and second nucleic acid sequences for the best comparison, and for the purpose of comparison, non-homologous sequences can be ignored). Subsequently, the nucleotides on the corresponding nucleotide positions are compared. When a certain position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules on this position are identical. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0073] Mathematical algorithms can be used to compare sequences and determine the percent identity between two sequences, for example, the algorithm proposed by Needleman and Wunsch (1970, J. Mol. Biol. 48: 444-453), which has been incorporated into the GAP program of the GCG software package (available at www.gcg.com), can be used. The percent identity between two nucleotide sequences can be determined using the algorithm proposed by E. Meyers and W. Miller (1989, CABIOS, 4: 11-17), in combination with the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, which has been incorporated into the ALIGN program (version 2.0). It will be understood that the molecules described herein may have additional conservative or non-essential nucleic acid substitutions that have no substantial effect on their function.

[0074] In embodiments of the present application, a target gene is SOD1. "Target sequence" refers to a sequence segment to which the sense oligonucleotide strand or antisense oligonucleotide of the siRNA is homologous or complementary. For example, in some embodiments, the SOD1 siRNA is homologous or complementary to a target sequence in a human SOD1 transcript.

[0075] The term "non-targeting" used in this application refers to that the auxiliary oligonucleotide (ACO) conjugated to a targeting oligonucleotide (e.g., siRNA, saRNA, etc.) is not specifically complementary to the target sequence on which the targeting oligonucleotide works, and / or the target sequence of the oligonucleotide (i.e., ACO) is different from the target sequence on which the targeting oligonucleotide (e.g., siRNA, saRNA, etc.) specifically works. The targeting oligonucleotide disclosed in this application is a nucleic acid sequence specifically complementary to a target sequence or its region. In some embodiments, the term "non-targeting oligonucleotide" may include any of the oligonucleotides except the "targeting sequence". In some cases, "specific complementarity" may refer to a complementarity between a targeting oligonucleotide and a target sequence or its region of at least about 95%. Non-targeting oligonucleotides (i.e., ACO) do not cause biological activity by any known mechanism, nor are they used to cause activity indicating the function of an ASO (i.e., "mixed polymers" or "gap polymers") to a complementary nucleic acid sequence (i.e., mRNA) in a specific subject, subject organ, subject tissue, or subject cell when the oligonucleotide is administered. A non-targeting oligonucleotide (i.e., ACO) is used to facilitate the introduction of the bound targeting oligonucleotide (such as siRNA, saRNA, etc.) into a specific subject, subject organ, subject tissue, subject cell, or subject cell nucleus when the oligonucleotide conjugate is administered.

[0076] The term "gap polymer" used in this application refers to a short DNA antisense oligonucleotide (ASO) structure with modified RNA fragments on both sides of the central DNA structure. In some embodiments, at least one of the modified RNA fragments comprises one or more nucleotides selected from locked nucleic acid (LNA) and 2'-OMe or 2'-F modified to increase affinity to the target, increase nuclease resistance, reduce immunogenicity and / or reduce toxicity. In some embodiments, the gap polymer comprises at least one nucleotide modified with a thiophosphate (PS) group. In some embodiments, the gap polymer is designed to hybridize with the RNA target fragment and silence the gene transcript by inducing RNase H cleavage. For example, the ASO drug "Tofersen" is a gap polymer that knocks down SOD1mRNA to treat amyotrophic lateral sclerosis. A possible embodiment of a dual-acting oligonucleotide (DAO) with a gap polymer ASO disclosed in this application can be a "siSOD1-Tofersen conjugate".

[0077] The term "mixed polymer" used in this application refers to an antisense oligonucleotide (ASO) characterized in that it is a mixture of DNA and structurally chemically modified nucleic acid analogs. Alternatively, the mixed polymer is composed of fully modified nucleotides or nucleic acid analogs. In some embodiments, the mixed polymer is designed to bind and mask complementary RNA sequences to spatially block the interaction of proteins, factors or other RNAs with the targeted RNA. In some embodiments, the mixed polymer is designed to change pre-mRNA splicing by replacing the spliceosome. In some embodiments, the mixed polymer is designed to bind and isolate microRNA (miRNA), in which case it is also referred to as "miRNA antagonist" or "anti-miR".

[0078] The terms "sense strand" and "sense oligonucleotide strand" used herein are used interchangeably. The sense oligonucleotide strand of an siRNA molecule may include, for example, a first nucleic acid strand of an siRNA comprising a fragment of a sequence in the human genome or a sequence of a target gene.

[0079] As used herein, the terms "antisense strand" and "antisense oligonucleotide strand" are used interchangeably. The antisense oligonucleotide strand of an siRNA molecule may include, for example, a second nucleic acid strand in the siRNA duplex that is complementary to the sense oligonucleotide strand. The antisense strand of an siRNA may be complementary to a continuous segment of a target gene sequence and may bind to the continuous segment with 0, 1, 2, 3, 4, or 5 mismatches without affecting the function of the siRNA.

[0080] As used herein, the term "coding strand" refers to the DNA strand of the target gene that cannot be transcribed, whose nucleotide sequence is identical to the sequence of the RNA produced by transcription (in the RNA, T in DNA is replaced by U). The coding strand of the double-stranded DNA sequence of the target gene promoter described herein refers to the promoter sequence located on the same DNA strand as the coding strand of the target gene DNA.

[0081] The term "template strand" as used in this application refers to the other strand in the double-stranded DNA of the target gene, which is complementary to the coding strand and can be transcribed into RNA as a template and is complementary to the bases of the transcribed RNA (AU, GC). During transcription, RNA polymerase binds to the template strand, moves along the 3'→5' direction of the template strand, and catalyzes RNA synthesis in the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter described in this application refers to the promoter sequence located on the same DNA strand as the target gene DNA template strand.

[0082] The term "overhang" as used in this application refers to an oligonucleotide chain end (5' or 3') with no base-paired nucleotides, which is another chain extending beyond a chain within the siRNA. The single-stranded region extending beyond the 3' and / or 5' ends of the duplex is referred to as an overhang. In some embodiments, the length of the overhang is 0 to 6 nucleotides. It should be understood that an overhang of 0 nucleotides means that there is no overhang.

[0083] As used herein, the term "natural overhang" refers to an overhang consisting of one or more nucleotides identical or complementary to corresponding positions on the target sequence. A natural overhang on the sense strand consists of one or more nucleotides identical to corresponding positions on the target mRNA. A natural overhang on the antisense strand consists of one or more nucleotides complementary to corresponding positions on the target mRNA.

[0084] As used herein, the terms "gene silencing," "gene knockdown," "gene downregulation," "reduction of gene expression," and "downregulation of gene expression" are used interchangeably and refer to the determination of a reduction or downregulation of the transcription, translation, expression, or activity of a nucleic acid sequence by measuring the level of transcription, mRNA, protein level, enzyme activity, methylation state, chromatin state or configuration, translation level, or activity or state within a cell or genetic biological system. These activities or states can be determined directly or indirectly. In addition, "downregulation of gene" or "downregulation of gene expression" refers to a decrease in activity associated with a nucleic acid sequence, regardless of the mechanism of such downregulation. For example, downregulation of gene includes both transcriptional and post-transcriptional levels, reduced transcription into RNA, which in turn reduces RNA levels, which translates into protein levels that are lower than baseline levels, thereby reducing protein expression.

[0085] The terms "short interfering RNA," "siRNA," and "silencing RNA" are used interchangeably herein to refer to RNA molecules that can downregulate, knockdown, or silence target gene expression. These molecules can be double-stranded. siRNA primarily binds to target mRNA in the cytoplasm, downregulating gene expression post-transcriptionally through the RNA interference (RNAi) mechanism.

[0086] SiRNA may contain natural nucleotides or chemically modified nucleotides. Modification may increase the stability and / or cellular efficacy of the nuclease. Examples of chemical modifications include phosphorothioate backbone modifications, 2'-deoxynucleotides, ribonucleotides containing 2'-OCH3, 2'-F-ribonucleotides, 2'-methoxyethyl ribonucleotides, and combinations thereof. The siRNA may have different lengths (e.g., 10 to 200 bp) and structures (e.g., hairpin structures, single / double strands, protrusions, gaps / gaps, mismatches), and may be processed in cells to knock down target mRNA. The number of nucleotides in each chain (blunt end) or asymmetric end (overhang) of the double-stranded siRNA may be the same. For example, an overhang of 1 to 2 nucleotides may be present on the sense strand and / or antisense strand, or may be present at the 5' and / or 3' end of a given strand.

[0087] The length of the siRNA molecule is typically about 10 to about 60, about 10 to about 50, about 15 to about 30, about 17 to about 29, about 18 to about 28, about 19 to about 27, about 20 to about 26, about 21 to about 25, and about 22 to about 24 base pairs, and is typically about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 23, about 25, about 30, about 40, or about 50 base pairs. In addition, the terms "small interfering RNA," "silencing RNA," and "siRNA" also encompass nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogs.

[0088] The term "equal length portion" refers to a portion of a sequence that is compared to a target sequence (eg, a contiguous oligonucleotide sequence from the siRNA) and has an equal length (equal number of bases) to the target sequence.

[0089] The term "sequence-specific pattern" as used in this application refers to the way in which two nucleic acid fragments bind or hybridize based on their nucleotide sequences, such as the Watson-Crick pattern (e.g., A to T, A to U, and C to G) or any other pattern that allows the formation of a duplex (e.g., Hoogsteen or reverse Hoogsteen base pairing).

[0090] As used herein, the terms "isolated target site," "target site," and "isolated polynucleotide" are used interchangeably to refer to a nucleic acid target site to which an siRNA is complementary or hybridizes. For example, an isolated nucleic acid sequence of a target site can include a nucleic acid sequence that is complementary to or hybridizes to a region of an siRNA.

[0091] As used herein, the term "complementarity" refers to the ability to form base pairs between two oligonucleotide chains. Base pairs are typically formed by hydrogen bonding between nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains can be paired in a Watson-Crick manner (e.g., A with T, A with U, and C with G), or in any other manner that allows duplex formation (e.g., Hoogsteen or reverse Hoogsteen base pairing).

[0092] Complementarity includes complete complementarity and incomplete complementarity. "Complete complementarity" or "100% complementarity" means that each nucleotide from the first oligonucleotide chain can form hydrogen bonds with the nucleotides at the corresponding position in the second oligonucleotide chain in the double-stranded region of the siRNA molecule, and there are no base pair "mismatches". "Incomplete complementarity" or "mismatch" means that not all nucleotide units of the two chains are bound to each other by hydrogen bonds. For example, for two oligonucleotide chains with a double-stranded region of 20 nucleotides in length, if only two base pairs in the double-stranded region can form hydrogen bonds, the oligonucleotide chains have 10% complementarity. In the same embodiment, if there are 18 base pairs in the double-stranded region that can form hydrogen bonds, the oligonucleotide chains have 90% complementarity. Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95% or 99% complementarity.

[0093] As used herein, the terms "ODV" and "oligonucleotide delivery vector" are used interchangeably to refer to an oligonucleotide molecule comprising a double-stranded or double-stranded RNA (e.g., siRNA or saRNA) and an ACO covalently linked to the double-stranded RNA via a linker, as described in more detail below.

[0094] As used herein, the terms "covalent linker," "linker," and "linking component" are used interchangeably to refer to an organic moiety that connects two parts of a compound. For example, one or more of a single-stranded oligonucleotide (e.g., ACO) and a dsRNA (e.g., siRNA), two dsRNAs, etc., are covalently linked via, for example, a nucleic acid linker, a peptide linker, and the like, including disulfide linkers.

[0095] The term "synthetic" as used in this application refers to the method of synthesizing oligonucleotides, including any method capable of synthesizing or chemically modifying RNA, such as chemical synthesis, in vitro transcription, vector expression, etc.

[0096] The terms "oligonucleotide modulator" and "oligonucleotide agent" are used interchangeably and refer to an oligonucleotide-containing substance comprising at least one or more siRNAs of the present invention or consisting thereof, which has the activity of modulating target gene expression or enhancing the effect of the siRNA, and may further comprise other oligonucleotide moieties / components (such as ASOs or auxiliary oligonucleotides (ACOs)) or non-oligonucleotide moieties / components conjugated, bound, or mixed with the siRNA. In certain embodiments, the oligonucleotide modulator comprises RNA (e.g., the siRNA of the present invention), DNA, BNA, LNA, GNA, or PNA.

[0097] The term "LNA" as used in this application refers to a locked nucleic acid in which the 2'-oxygen atom and the 4'-carbon atom are connected by an additional bridge. The term "BNA" as used in this application refers to a nucleic acid bridged by 2'-O and 4'-aminoethylene, which may contain a five-membered or six-membered bridge structure with an N-O bond. The term "PNA" as used in this application refers to a nucleic acid mimic having a pseudopeptide backbone consisting of N-(2-aminoethyl)glycine units in which the nucleobases are connected to the glycine nitrogen via a carbonyl methylene linker. The term "GNA" as used in this application is also called glycerol nucleic acid, a nucleic acid similar to DNA or RNA, but with a different composition of its sugar-phosphodiester backbone, using propylene glycol instead of ribose or deoxyribose.

[0098] In this application, singular forms such as "a," "an," "the," and "the" include plural referents unless the context clearly dictates otherwise.

[0099] Unless defined otherwise, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. siRNA

[0100] The embodiments of the present application are based in part on the following surprising discovery: oligonucleotide agents (e.g., siRNA, also referred to herein as "SOD1 gene siRNA" or "SOD1 siRNA") can inhibit or downregulate the expression of the SOD1 gene in cells. Following administration of the oligonucleotide agents described herein, the reduction of functional SOD1 gene transcripts can significantly reduce or downregulate SOD1 mRNA and SOD1 protein levels in cells or mammals.

[0101] Specifically, the inventors of the present application have discovered functional oligonucleotide agents capable of inhibiting the expression of superoxide dismutase 1 (SOD1), wherein the functional oligonucleotide agent includes a small interfering RNA (siRNA), wherein the siRNA comprises a sense strand and an antisense strand forming a double strand, wherein the antisense strand comprises a nucleotide sequence containing at least 10 consecutive nucleotides with 0, 1, 2 or 3 mismatches, and the nucleotide sequence has at least 85% nucleotide sequence complementarity or homology with a partial nucleotide sequence of SOD1 mRNA.

[0102] As a favorable result, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) can inhibit / downregulate SOD1 mRNA transcripts by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 93%, at least 96%, at least 99%, or about 100%, compared to baseline levels of SOD1 mRNA, when interacting with the siRNA. In one embodiment, SOD1 mRNA is reduced by at least 80%. Based at least in part on these findings, the present application features siRNAs, compositions, and pharmaceutical compositions that inhibit / downregulate SOD1 mRNA transcription by at least 10%, compared to baseline levels of SOD1 mRNA. The present application also provides methods for preventing or treating diseases or conditions induced by overexpression of SOD1 protein, mutation of the SOD1 gene, and / or elevated or abnormal levels of SOD1 in an individual, comprising administering to the individual any of the siRNAs, compositions, and / or pharmaceutical compositions described herein.

[0103] In certain embodiments, the oligonucleotide sequences disclosed in the present application have at least 85%, at least 90%, or at least 95% homology or complementarity with a nucleotide sequence selected from SEQ ID NOs: 2 to 269. In certain embodiments, the sense strand of the oligonucleotide sequences disclosed in the present application has at least 85%, at least 90%, or at least 95% homology with a nucleotide sequence selected from SEQ ID NOs: 2 to 269. In certain embodiments, the antisense strand of the oligonucleotide sequences disclosed in the present application has at least 85%, at least 90%, or at least 95% complementarity with a nucleotide sequence selected from SEQ ID NOs: 2 to 269.

[0104] Embodiments of the present application are also based in part on the discovery that a SOD1 mRNA inhibitory oligonucleotide agent comprises an siRNA having a sense strand that is at least 85%, at least 90%, or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 270-537.

[0105] In some other embodiments, the SOD1 mRNA inhibitory oligonucleotide agent comprises an siRNA whose antisense strand is at least 85%, at least 90%, or at least 95% identical to a nucleotide sequence selected from SEQ ID NOs: 538 to 805.

[0106] In some embodiments, the SOD1 mRNA inhibitory oligonucleotide agent comprises an siRNA, wherein the sense and antisense strands of the siRNA have nucleotide sequences that are at least 85%, at least 90%, or at least 95% homologous to a nucleotide sequence selected from SEQ ID NOs: 808 to 849, 867, and 868, respectively.

[0107] The siRNA oligonucleotides described herein comprise an RNA strand (antisense strand) having a region of 60 nucleotides or less, i.e., 15 to 40 nucleotides, typically 19 to 25 nucleotides, that is substantially complementary to at least a portion of the mRNA transcript of the SOD1 gene. These siRNAs can be used to target and inhibit mRNAs of genes involved in pathological processes associated with SOD1 expression in mammals. In particular, extremely low doses of SOD1 siRNA can specifically and effectively mediate RNAi, thereby significantly inhibiting the expression of the SOD1 gene. The inventors of the present application have demonstrated using cell-based assays that siRNAs targeting SOD1 can specifically and effectively mediate RNAi, thereby significantly inhibiting the expression of the SOD1 gene. Therefore, methods and oligonucleotides comprising these siRNAs can be used to treat pathological processes that can be regulated by downregulating SOD1, such as for treating disorders that cause elevated SOD1 levels, such as amyotrophic lateral sclerosis (ALS). The following detailed description discloses how to prepare and use oligonucleotide agents containing siRNA to inhibit SOD1 gene expression, as well as oligonucleotide agents and methods for treating diseases and disorders caused by the expression of this gene.

[0108] On the one hand, RNA interference agents include single-stranded RNA that interacts with the target RNA sequence to guide the cracking of the target RNA. Without wishing to be bound by theory, long double-stranded RNA is introduced into plant and invertebrate cells and decomposed into siRNA using a type III endonuclease called Dicer (Sharp et al., Genes Dev. 2001, 15: 485). Dicer is an enzyme similar to ribonuclease III that can process dsRNA into short interfering RNAs of 19-23 base pairs, with characteristic double base 3' overhangs (Bernstein et al., (2001) Nature 409: 363). Then, the siRNA is integrated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strands, enabling the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107: 309). After binding to the corresponding target mRNA, one or more endonucleases within the RISC unravel the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188). Therefore, one aspect of the present invention relates to a single-stranded RNA that promotes the formation of the RISC complex to achieve silencing of the target gene.

[0109] In some embodiments, the contiguous oligonucleotide sequence of the siRNA has five or fewer nucleotide differences or mismatches relative to an equal length portion of the SOD1 mRNA, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches. In some embodiments, the contiguous oligonucleotide sequence of the siRNA sense strand has three or fewer nucleotide differences or mismatches relative to an equal length portion of the SOD1 mRNA, i.e., 3, 2, 1, or 0 nucleotide differences or mismatches. In some embodiments, the contiguous oligonucleotide sequence of the siRNA antisense strand has three or fewer nucleotide differences or mismatches relative to an equal length portion of the SOD1 mRNA, i.e., 3, 2, 1, or 0 nucleotide differences or mismatches.

[0110] In some embodiments, the SOD1 mRNA disclosed herein does not contain a nucleotide mutation. In some embodiments, the SOD1 mRNA disclosed herein contains at least one nucleotide mutation. In some embodiments, the SOD1 mRNA disclosed herein contains at least one nucleotide mutation at the target site of the siRNA. In some embodiments, the SOD1 mRNA disclosed herein contains at least one nucleotide mutation upstream and / or downstream of the target site of the siRNA.

[0111] In some embodiments, the difference or mismatch is located in the middle or 3' end of the siRNA oligonucleotide sequence. The methods and principles of siRNA molecule design are well known to those skilled in the art and are described in detail in, for example, Place et al., Molecular Therapy-Nucleic Acids (2012) 1, e15 and Li et al., PNAS, 2006, vol. 103, no. 46, 17337-17342, all of which are incorporated herein by reference.

[0112] In some embodiments, the siRNA disclosed in the present application includes a sense strand and an antisense strand. The sense strand and the antisense strand contain complementary regions capable of forming a double-stranded nucleic acid structure, and the double-stranded nucleic acid structure reduces the SOD1 transcription level in the cell through the RNAi mechanism. The RNAi mechanism (also referred to as RNA interference) used in this application refers to the mechanism by which a double-stranded nucleic acid structure can downregulate the target gene in a sequence-specific manner at the transcriptional level. The sense strand and antisense strand of the siRNA may be present on two different nucleic acid chains or on the same nucleic acid chain (e.g., a continuous nucleic acid sequence). When the sense strand and antisense strand of the siRNA are located on two different chains, at least one of the chains has a 3' overhang of 0 to 6 nucleotides in length, thereby forming an overhang of 0, 1, 2, 3, 4, 5, or 6 nucleotides in length, and in some cases, both chains have a 3' overhang of 2 or 3 nucleotides in length. In some cases, the nucleotides of the overhang are thymidine deoxynucleotides (dT), or in some cases are natural overhangs selected from or complementary to the nucleotides at the corresponding positions on its DNA target. When the sense strand and the antisense strand are located on a nucleic acid chain, in some cases, the siRNA is a hairpin single-stranded nucleic acid molecule, wherein the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure with each other. In the siRNA disclosed in the present application, in some embodiments, the sense strand has a length ranging from 10 to 60 nucleotides. For example, in some embodiments, the sense strand and the antisense strand include 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides respectively. In some embodiments, the antisense strand has a length ranging from 10 to 60 nucleotides. For example, in some embodiments, the sense strand and the antisense strand comprise 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides, respectively.

[0113] In some embodiments, the antisense strand disclosed herein is capable of interacting with a target nucleic acid sequence on an SOD1 gene mRNA in a sequence-specific manner, i.e., the antisense strand is capable of hybridizing to the target nucleic acid via hydrogen bonding. In some embodiments, the antisense strand has a nucleotide sequence that, when written in a 5' to 3' orientation, comprises the reverse complement of the target portion of the target nucleic acid to which it is targeted. In certain such embodiments, the antisense strand has a nucleotide sequence that, when written in a 5' to 3' orientation, comprises the reverse complement of the target portion of a SOD1 gene transcript fragment. ACO

[0114] Although some previous studies have demonstrated that siRNA capable of inhibiting SOD1 mRNA and reducing SOD1 protein expression can be used to treat SOD1 protein-related diseases (e.g., amyotrophic lateral sclerosis (ALS) patients), the inventors of the present application have found that there are two unresolved problems: one is the lack of efficacy of SOD1 siRNA molecules, and the other is the lack of an effective delivery method for delivering the siRNA molecules to cells of target organs or tissues (e.g., the central nervous system).

[0115] "Delivering" an siRNA into a cell refers to effective uptake or absorption by the cell as understood by those skilled in the art. Absorption or uptake of the siRNA can occur by unassisted diffusion or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an siRNA can also be "introduced" into a cell that is part of a living organism. In this case, introduction into a cell would include delivery to the organism. For example, for in vivo delivery, the siRNA can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Other methods not known in the art are described below.

[0116] When the siRNA agent is conjugated to a non-targeted single-stranded auxiliary oligonucleotide (ACO), the bioavailability, biodistribution and / or cellular uptake and in vivo efficacy of the siRNA are significantly improved compared to an oligonucleotide agent without ACO. In particular, in some in vivo embodiments of the present application, the ACO of the oligonucleotide agent increases the biodistribution of the siRNA in one or two or more target tissues compared to an oligonucleotide agent without ACO.

[0117] Therefore, the present application also relates to an oligonucleotide agent comprising small interfering RNA (siRNA) and ACO and capable of inhibiting the expression of superoxide dismutase 1 (SOD1).

[0118] In some embodiments, oligonucleotide agents comprising one or more conjugated ACOs can enhance their biodistribution in the specific tissue to which they are administered and can increase their permeability and permeability across membranes such as the blood-brain barrier.

[0119] In some embodiments, the ACO is an oligonucleotide comprising a 5' end and a 3' end.

[0120] In some embodiments, the siRNA and ACO are covalently linked into an oligonucleotide agent with or without one or more linking components.

[0121] In some embodiments, the length of the ACO comprises a length of 6 to 22 nucleotides, such as 6 or more nucleotides, 7 or more nucleotides, 8 or more nucleotides, 9 or more nucleotides, 10 or more nucleotides, 11 or more nucleotides, 12 or more nucleotides, 13 or more nucleotides, 14 or more nucleotides, 15 or more nucleotides, 16 or more nucleotides, 17 or more nucleotides, 18 or more nucleotides, 19 or more nucleotides, 20 or more nucleotides, 21 or more nucleotides, 22 or more nucleotides. In some embodiments, the length of the ACO is 6 to 18 consecutive oligonucleotides.

[0122] In some embodiments, the length of the ACO can regulate the activity and / or biodistribution of the oligonucleotide agent in the target tissue or target cell. For example, the inventors of the present application found that shorter ACOs showed activity throughout the central nervous system, while longer ACOs showed activity only in specific areas of the brain (such as the cerebellum).

[0123] In another aspect of the present application, an oligonucleotide agent comprising an siRNA and a non-targeting ACO is also provided, wherein the ACO comprises a single-stranded oligonucleotide sequence comprising a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to a nucleotide sequence selected from SEQ ID NO: 865. In some embodiments, the ACO comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from SEQ ID NO: 865.

[0124] In some embodiments, the oligonucleotide agent described herein comprises more than one ACO covalently linked to siRNA, such as 2, 3, 4, 5, 6, 7, 9, 10 ACOs, and there are or do not have one or more linkers between the ACO and the siRNA. The number of ACOs can vary from 1 to 4, 2 to 10, and is connected to the siRNA in a branched or linear form by a multivalent linker (such as a polymer linker). In some embodiments, a plurality of ACOs of a medicament are covalently linked to 2 or more siRNAs, such as 2, 3, 4, 5, 6, 7, 9, 10 or more siRNAs. The PCT application numbered WO2023280190A1 is incorporated herein by reference in its entirety for all purposes, and the PCT application describes the principles of ACO design and the ACO embodiments conjugated to the siRNA to improve pharmacokinetic properties (including assisted delivery of double-stranded RNA to cells). Chemical modification

[0125] In the siRNA or ACO disclosed in the present application, all nucleotides can be natural or non-chemically modified nucleotides, or at least one nucleotide can be a chemically modified nucleotide. Non-limiting examples of chemical modifications include one or more combinations of the following: 1) Modification of the phosphodiester bonds of the nucleotides in the siRNA or ACO nucleotide sequence; 2) modification of the 2'-OH group of the ribose in the siRNA or ACO nucleotide sequence; 3) modification of bases in the siRNA or ACO nucleotides; 4) at least one nucleotide in the nucleotide sequence of the siRNA or ACO is BNA, LNA, GNA or PNA, and 5) At least one nucleotide in the ACO nucleotide sequence is a deoxyribonucleotide (DNA).

[0126] Chemical modifications described herein are well known to those skilled in the art, wherein modification of phosphodiester bonds refers to modification of oxygen in phosphodiester bonds, including phosphorothioate modification and borane phosphate modification. The modifications disclosed herein can stabilize the siRNA structure, maintaining high specificity and high affinity for base pairing. The modifications disclosed herein can also stabilize the ACO structure and maintain its delivery-assisting properties, such as bioavailability, biodistribution and / or cellular uptake of oligonucleotide agents in various tissues such as the prefrontal cortex, cerebellum, brain, spinal cord (e.g., cervical, thoracic, lumbar), muscle, lung, eye, liver and kidney.

[0127] In some embodiments, the chemical modification is to replace the phosphodiester bond on the nucleotide sequence backbone of the oligonucleotide agent disclosed herein with a phosphorothioate (PS) bond. In some embodiments, the oligonucleotide agent disclosed herein comprises at least one phosphorothioate backbone modification. In some embodiments, the ACO comprises at least one PS backbone modification. In some embodiments, the ACO comprises 6 to 17 phosphorothioate backbone modifications.

[0128] In some embodiments, the siRNA or ACO described herein includes at least one chemically modified nucleotide, which is modified at the 2'-OH position in the pentose of the nucleotide, i.e., certain substituents are introduced at the hydroxyl position of the ribose, such as 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylenemethoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification or 2'-deoxy modification, such as 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides.

[0129] In some embodiments, the siRNA or ACO described herein includes at least one chemically modified nucleotide, which is modified at the base of the nucleotide, for example, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.

[0130] In some embodiments, the chemical modification of the siRNA or ACO is the addition of an (E)-vinylphosphonate moiety to the 5' end of the sense or antisense sequence. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is the addition of a 5-methylcytosine moiety to the 5' end of the sense or antisense sequence.

[0131] In some embodiments, at least one nucleotide in the nucleotide sequence of the siRNA or ACO nucleic acid described herein is chemically modified, such as a locked nucleotide, an abasic nucleotide, a glycerol nucleic acid (GNA), a morpholino nucleotide, a phosphoramide, and a nucleotide containing a non-natural base. In some embodiments, the siRNA disclosed herein includes "internal light" modifications of nucleotides having 2'-O-methyl modifications and nucleotides containing 5'-phosphorothioate groups.

[0132] In some embodiments, the siRNA or ACO described herein is chemically modified to enhance stability or other beneficial properties. The nucleic acids described herein can be synthesized and / or modified by conventional methods, such as those described in "Current protocols in nucleic acid chemistry" (Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), the contents of which are incorporated herein by reference. Modifications include, but are not limited to, (a) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, trans-linking, etc.), 3' terminal modifications (conjugation, DNA nucleotides, trans-linking, etc.); (b) base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that pair with an expanded partner library, removal of bases (abasic nucleotides), or conjugation of bases; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacement; and (d) backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of siRNA molecules that can be used in the present application include, but are not limited to, RNAs that contain modified backbones or that do not contain natural internucleoside bonds. In some embodiments, RNAs with modified backbones include RNAs that do not have a phosphorus atom in the backbone, etc. In some embodiments, modified RNAs that do not have a phosphorus atom in the internucleoside backbone can also be oligonucleosides. In some embodiments, modified oligonucleotides will have a phosphorus atom in their internucleoside backbone.

[0133] Modified oligonucleotide backbones include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramides (including 3'-aminophosphoramides and aminoalkylphosphoramides), thiophosphoramides, thioalkylphosphonates, thioalkylphosphotriesters, and borophosphates with normal 3'-5' linkages and their 2'-5' linkage analogs, as well as antisense compounds with reversed polarity, wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0134] In some embodiments, the siRNA or ACO consists of one or more of RNA, DNA, BNA, LNA, GNA, or PNA. Covalent attachment

[0135] The present application includes oligonucleotide agents comprising a covalently linked double-stranded targeting oligonucleotide (ie, siRNA) and an ACO.

[0136] In some embodiments, any oligonucleotide in the oligonucleotide agent described herein includes a linking component. In some embodiments, the siRNA and ACO are covalently linked by a linking component. In some embodiments, the siRNA and ACO are connected with a covalent linker. Various combinations of chains can be connected, for example, the first and second dsRNA sense strands are covalently linked, or for example, the first and second dsRNA antisense strands are covalently linked.

[0137] In some embodiments, the sense strand of the siRNA is covalently linked to the ACO. In some embodiments, the antisense strand of the siRNA is covalently linked to the ACO. In some embodiments, the ACO is covalently linked to the 3' end, the 5' end, or both the 3' end and the 5' end of the sense strand of the siRNA. In some embodiments, the ACO is covalently linked to the 3' end, the 5' end, or both the 3' end and the 5' end of the antisense strand of the siRNA. In some embodiments, more than one ACO is covalently linked to the siRNA. In some embodiments, 2 to 10 ACOs are covalently linked to the siRNA. In some embodiments, more than one siRNA is covalently linked to the ACO. In some embodiments, 2 to 10 siRNAs are covalently linked to the ACO.

[0138] In some embodiments, the ACO is conjugated to a linking component. In some embodiments, the 5' end or the 3' end of the ACO is conjugated to a linking component. In some embodiments, the siRNA and the ACO are covalently conjugated via a linking component. In some embodiments, the sense strand or the antisense strand of the siRNA is covalently conjugated to the ACO via a linking component.

[0139] Linkers typically include direct bonds or atoms such as oxygen or sulfur, and linking units (e.g., NR 1 、C(O)、C(O)O、C(O)NR 1, SO, SO2, SO2NH) or a chain of atoms such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl alkyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R')2, C(O), a cleavable linker, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclyl, wherein R 1 is hydrogen, acyl, aliphatic or substituted aliphatic.

[0140] Various types of linker functions may be included in the conjugate, including but not limited to cleavable and non-cleavable linkers, and reversible and irreversible linkers.

[0141] In some embodiments, the linker is a cleavable linker. A cleavable linker refers to a linker that is released by the process in the target cell that binds the two parts of the linker together (such as the ACO and the dsRNA), such as reduction in the cytoplasm, exposure to the acidic conditions of lysosomes or endosomes, or cleavage by specific enzymes (such as proteases) in the cell. Therefore, a cleavable linker allows the release of the dsRNA in its original form after the conjugate is internalized and processed in the target cell. Cleavable linkers include, but are not limited to, linkers whose bonds can be cleaved by enzymes (such as peptide linkers), reducing conditions (such as disulfide linkers), or acidic conditions (such as hydrazones and carbonates).

[0142] In some embodiments, the linking component is selected from one or more of an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a carbohydrate, a thiol linking group, a phosphodiester, a phosphorothioate, a phosphoramide, an amide, and a carbamate. In some embodiments, the linking component includes, but is not limited to: spacer phosphoramidite 18 (phosphoramidite acid, N,N-bis(1-methylethyl)-, 19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxa nonadecan-1-yl(2-cyanoethyl ester); spacer-9 (3-[2-[2-[2-(bis(4-methoxyphenyl)(phenylmethoxy]ethoxy]ethoxy]ethoxy-[di(propyl-2-yl)amino]phosphino]oxypropionitrile); spacer phosphoramidite C3 (6-(4,4'-dimethoxytrityl)hexyl-1 -[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite); spacer C6 phosphoramidite (6-(4,4'-dimethoxytrityl)hexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite); and a divalent linker (DIO)16-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1,1-bis(4-methoxyphenyl)-18-oxo-1-phenyl-2,5,8,11,14,17-hexaoxaheneicosane-CPG. In some embodiments, the linking component includes the compound structures shown in Table 15.

[0143] In some embodiments, the linking component is spacer phosphoramidite 18 (phosphoramidite acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecan-1-yl(2-cyanoethyl ester)).

[0144] Table 15. Linkers used in oligonucleotide reagents

[0145] In some embodiments, the siRNA and ACO are covalently linked via a phosphodiester bond. In some embodiments, the siRNA and ACO are covalently linked via a phosphorothioate bond.

[0146] In some embodiments, the siRNA comprises a sense strand covalently linked to the ACO. In some embodiments, the siRNA comprises an antisense strand covalently linked to the ACO.

[0147] In some embodiments, the siRNA and ACO are covalently linked through one or more nucleotides.

[0148] Non-limiting examples of covalent linkers can be found in U.S. Patent Application Publication No. 20200332292, the entire contents of which are incorporated herein by reference. The covalent linker can connect the siRNA and ACO.

[0149] In some embodiments, the covalent linker comprises RNA and / or DNA and / or a peptide. The linker can be single-stranded, double-stranded, partially single-stranded, or partially double-stranded. In some embodiments, the linker comprises a disulfide bond. The linker can be cleavable or non-cleavable.

[0150] In some embodiments, the covalent linker comprises a disulfide bond, optionally a dihexyl disulfide linker. In one embodiment, the disulfide linker is

[0151] In some embodiments, the covalent linker comprises a peptide bond, for example, comprising amino acids. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably comprising 4-5 amino acids, optionally X-Gly-Phe-Gly-Y, wherein X and Y represent any amino acids.

[0152] In some embodiments, the covalent linker comprises HEG, a hexaethylene glycol linker. ODV-siRNA oligonucleotides

[0153] In some embodiments, the oligonucleotide agent can reduce the expression of the SOD1 gene or SOD1 protein. Oligonucleotide agents can be applied to patients to treat or delay the onset of ALS, such as familial or sporadic ALS or Leu Lou Gehrig's disease. In some embodiments, the oligonucleotide agent reduces the amount of SOD1 protein by, for example, lowering the level of SOD1 transcription or reducing the amount of full-length SOD1 mRNA. In some embodiments, SOD1 mRNA is reduced by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%). In some embodiments, SOD1 mRNA is reduced by at least 80%. In some embodiments, the amount of SOD1 protein is reduced to a level sufficient to alleviate the symptoms associated with ALS. In some embodiments, SOD1 protein is reduced by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). In some embodiments, SOD1 protein is reduced by at least 80%.

[0154] In some embodiments, the oligonucleotide agent that reduces the expression of the SOD1 gene or SOD1 protein is a siRNA-ACO conjugate (or ODV-siRNA). The SOD1 siRNA-ACO conjugate can reduce or downregulate the expression of the SOD1 gene in cells with abnormal or overexpressed SOD1 gene.

[0155] In typical embodiments, the first strand of the SOD1 siRNA in the oligonucleotide agent comprises a fragment having at least 75% sequence identity or sequence complementarity with a 6-60 nucleotide fragment of the selected target region of the SOD1 gene, thereby achieving inactivation or downregulation of gene expression.

[0156] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to the nucleotide sequence of siSOD1-047M3-AC1, the antisense strand of which has a nucleotide sequence of SEQ ID NO: 834 that is complementary to a fragment of the ODV structured sense strand of SEQ ID NO: 850.

[0157] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to the nucleotide sequence of siSOD1-005M3-AC1, the antisense strand of which has a nucleotide sequence of SEQ ID NO: 842 that is complementary to a fragment of the ODV structured sense strand of SEQ ID NO: 851.

[0158] In some embodiments, the oligonucleotide agent has a similar affinity to siCON1-AC1. VP The siCON1-AC1 comprises a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of VP The antisense strand has a nucleotide sequence of SEQ ID NO:858, which is complementary to a fragment of the ODV structured sense strand of SEQ ID NO:852.

[0159] In some embodiments, the oligonucleotide agent has a similar affinity to siCON2-AC1. VP The siCON2-AC1 comprises a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to a nucleotide sequence of VP The antisense strand has a nucleotide sequence of SEQ ID NO:859, which is complementary to a fragment of the ODV structured sense strand of SEQ ID NO:853.

[0160] In some embodiments, the oligonucleotide agent has a similar affinity to siCON3-AC1. VP The siCON3-AC1 comprises a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to a nucleotide sequence of VP The antisense strand has a nucleotide sequence of SEQ ID NO:860, which is complementary to a fragment of the ODV structured sense strand of SEQ ID NO:854.

[0161] In some embodiments, the oligonucleotide agent has a similar affinity to siSOD1-063M3-AC1 VPThe siSOD1-063M3-AC1 comprises a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to a nucleotide sequence of VP The antisense strand has a nucleotide sequence of SEQ ID NO: 861, which is complementary to a fragment of the ODV structured sense strand of SEQ ID NO: 855.

[0162] In some embodiments, the oligonucleotide agent has a similar affinity to siSOD1-047M3-AC1 VP The siSOD1-047M3-AC1 comprises a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to a nucleotide sequence of VP The antisense strand has a nucleotide sequence of SEQ ID NO: 848, which is complementary to a fragment of the ODV structured sense strand of SEQ ID NO: 850.

[0163] In some embodiments, the oligonucleotide agent has a similar affinity to siSOD1-104M3-AC1 VP The siSOD1-104M3-AC1 comprises a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to a nucleotide sequence of VP The antisense strand has a nucleotide sequence of SEQ ID NO: 862, which is complementary to a fragment of the ODV structured sense strand of SEQ ID NO: 856.

[0164] In some embodiments, the oligonucleotide agent has a similar affinity to siSOD1-005M3-AC1 VP The nucleotide sequence of siSOD1-005M3-AC1 is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of VP The antisense strand has a nucleotide sequence of SEQ ID NO: 849, which is complementary to a fragment of the ODV structured sense strand of SEQ ID NO: 852.

[0165] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to the nucleotide sequence of siSOD1-258M3-AC1VP, the antisense strand of which has a nucleotide sequence of SEQ ID NO: 863 that is complementary to a fragment of the ODV structured sense strand of SEQ ID NO: 857.

[0166] In addition, in order to promote the entry of the siRNA into cells, a chemical conjugation group other than the ACO disclosed in the present application can be introduced at the end of the sense or antisense chain of the siRNA on the basis of the above-mentioned modification to promote the passage through the cell membrane composed of the lipid bilayer, as well as the nuclear membrane and the mRNA region within the cell nucleus.

[0167] In some embodiments, the siRNA disclosed herein is covalently linked to one or more conjugated groups. In some embodiments, the conjugated groups alter one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance. In some embodiments, the conjugated groups impart new properties to the attached oligonucleotide, such as a fluorophore or reporter group capable of detecting the oligonucleotide.Certain conjugated groups and conjugated moieties have been described previously, for example: cholesterol groups (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as 10-decanediol or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-triethylamine phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), a palmitoyl group of adamantaneacetic acid (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexamidocarbonyloxycholesterol group (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740) or a GalNAc cluster (e.g., WO 2014 / 179620).

[0168] In some embodiments, the siRNA described herein relates to the sense strand or antisense strand of the siRNA, which is conjugated to one or more conjugation groups selected from the group consisting of an intercalator, a reporter molecule, a polyamine, a polyamide, a peptide, a carbohydrate, a vitamin moiety, a polyethylene glycol, a thioether, a polyether, cholesterol, a thiocholesterol, a bile acid moiety, a folic acid, a lipid, a phospholipid, biotin, a phenazine, a phenanthridine, an anthraquinone, an adamantane, an acridine, a fluorescein, a rhodamine, a coumarin, a fluorophore, and a dye.

[0169] In some embodiments, the conjugate group includes an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazine, chlorothiazide, diazepine, indomethacin, barbiturates, cephalosporins, sulfonamides, antidiabetic drugs, antibacterial drugs, or antibiotics.

[0170] In some embodiments, the siRNA described herein is conjugated to one or more conjugating groups selected from the group consisting of a lipid, a fatty acid, a fluorophore, a ligand, a carbohydrate, a peptide, and an antibody.

[0171] In some embodiments, the siRNA described herein involves the siRNA sense strand or antisense strand being conjugated to one or more coupling groups selected from the group consisting of cell penetrating peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0172] In some embodiments, a siRNA conjugated to one or more conjugating groups disclosed in the embodiments is directly contacted, transferred, delivered, or administered to a cell or subject. Cells containing siRNA

[0173] After contact with a cell, the oligonucleotide agents disclosed herein can effectively inhibit or downregulate the expression of the SOD1 gene in the cell, for example, downregulate expression by at least 10% (eg, compared to a baseline level of SOD1 transcript).

[0174] In some embodiments, the application relates to cells comprising the oligonucleotide agents disclosed herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells, such as human cells in various tissues such as the prefrontal cortex, cerebellum, spinal cord (e.g., cervical, thoracic, lumbar), muscle, liver, and kidney.

[0175] The cells disclosed herein can be in vitro or ex vivo, such as cell lines or cell strains, or can be present in a mammal, such as a human. The human disclosed herein is a subject suffering from a disease or symptom caused by a SOD1 gene mutation, abnormal SOD1 mRNA levels, and / or overexpression of SOD1 protein in the central nervous system.

[0176] In some embodiments, the cell is from central nervous system tissue of a subject with ALS. In some embodiments, the cell is from a subject with ALS. In some embodiments, the cell is from a subject with Alzheimer's disease (AD), Parkinson's disease (PD), and Down syndrome (DS). Compositions containing siRNA

[0177] Another aspect of the present application provides a composition or pharmaceutical composition capable of downregulating the level of SOD1 mRNA transcripts through the mechanism of action (MoA) of RNA interference, comprising the oligonucleotide agent disclosed in the present application, for treating or preventing the onset of SOD1-related diseases (particularly amyotrophic lateral sclerosis).

[0178] In some embodiments, the present application relates to a composition or pharmaceutical composition comprising the siRNA of the present application.

[0179] In some embodiments, the present application relates to a composition or pharmaceutical composition comprising siRNA and ACO as described herein. In some embodiments, the present application relates to a composition or pharmaceutical composition comprising siRNA and ACO covalently linked by a linking component as described herein.

[0180] In one embodiment, the pharmaceutically acceptable carrier comprises one or more of an aqueous carrier, a liposome or LNP, a polymer, a micelle, a colloid, a metal nanoparticle, a non-metallic nanoparticle, a bioconjugate (e.g., GalNAc), and a polypeptide. In one embodiment, the aqueous carrier can be, for example, RNase-free water or an RNase-free buffer. According to the present application, the composition can contain 1 to 150 nM (e.g., 1 to 100 nM, 1 to 50 nM, 1 to 20 nM, 10 to 100 nM, 10 to 50 nM, 20 to 50 nM, 20 to 100 nM, 50 nM) of the oligonucleotide agent or a nucleic acid encoding the full length or portion of the oligonucleotide agent.

[0181] In some embodiments, the composition comprises 1 to 150 nM of an oligonucleotide agent described herein.

[0182] Another embodiment provides pharmaceutical compositions or medicaments comprising the oligonucleotide agents described herein and a therapeutically inert carrier, diluent, or pharmaceutically acceptable excipient, as well as methods of preparing these compositions and medicaments using the oligonucleotide agents described herein.

[0183] A typical formulation can be prepared by mixing the drug described herein with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, for example, Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004; Lippincott, Williams & Wilkins, Philadelphia); Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000; Lippincott, Williams & Wilkins, Philadelphia); and Rowe RC, Handbook of Pharmaceutical Excipients (2005; Pharmaceutical Press, Chicago). The formulation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavorings, diluents, and other known additives to better present the drug (i.e., the agent of the present application or its pharmaceutical composition) or to facilitate the production of the drug (i.e., the drug).

[0184] The compositions described herein are formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the dosing schedule, and other factors known to physicians.

[0185] The oligonucleotide compositions described herein can be delivered by parenteral infusion, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal, or subcutaneous administration; or by oral, intranasal, inhalation, vaginal, or rectal administration.

[0186] In another aspect, the present application provides use of an oligonucleotide agent according to any embodiment described herein or a composition according to any embodiment described herein in the preparation of a medicament for treating a gene or protein-related disease in an individual. According to certain embodiments of the use, the condition may include a condition associated with SOD1, including ALS, AD, PD, and / or DS. Also provided are uses of certain embodiments, wherein the individual is a mammal, preferably a human. medicine box

[0187] On the other hand, any composition described in the present application can provide in the form of one or more medicine boxes, optionally including the instructions for use of the composition. That is to say, the medicine box can include the instructions for use of oligonucleotide agent or composition or pharmaceutical composition in any method described in the present application. " medicine box " used in the present application generally refers to a packaging, assembly or container (such as an insulated container), including one or more components or embodiments of the present application, and / or other components relevant to the present application, for example, as previously mentioned. Any medicament or component of the medicine box can provide in liquid form (such as solution) or solid form (such as dry powder, cryogen etc.).

[0188] In some cases, the kit includes one or more components, which can be in the same container, two or more containers, and / or any combination thereof. The container can hold a liquid, and non-limiting examples include bottles, vials, jars, test tubes, flasks, beakers, etc. In some cases, the container is spill-proof (when closed, liquid cannot flow out of the container regardless of the container's orientation).

[0189] Examples of other compositions or components associated with the agents, compositions, and methods described herein include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, and the like, such as those used to administer, modify, assemble, store, package, prepare, mix, dilute, and / or preserve components for a particular application. In embodiments where a liquid form of any component is used, the liquid may be in a concentrated or ready-to-use form.

[0190] In other embodiments, the medicine box can provide instructions for using the medicine box and the components and / or methods described herein, or include a website or any form of other resources indicating such information. For example, the instructions can include instructions for use, modification, mixing, dilution, preservation, assembly, storage, packaging and / or preparation of the components and / or other components relevant to the medicine box. In some cases, the instructions can also include instructions for transporting the components, for example, transporting or storing under conditions such as room temperature, subzero temperatures, low temperatures. The instructions can be provided in any form useful to the medicine box user, such as written or oral (e.g., telephone), digital, optical, visual (e.g., videotape, DVD, etc.) and / or electronic communication (including the Internet or network-based communication). How to use

[0191] Another aspect of the present application relates to oligonucleotide agents described herein for use in therapeutic methods for treating diseases such as ALS.

[0192] By way of non-limiting embodiment, the present application provides a method for reducing SOD1 gene transcription level or SOD1 protein level, comprising administering the pharmaceutical composition disclosed in the present application to a subject.

[0193] In some embodiments, the present application relates to a method for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS) in a subject, comprising administering a pharmaceutical composition disclosed herein to the subject. In some embodiments, the subject has sporadic amyotrophic lateral sclerosis (sALS). In some embodiments, the subject has familial amyotrophic lateral sclerosis (fALS). In some embodiments, the pharmaceutical composition can reduce SOD1 gene transcript levels or SOD1 protein levels.

[0194] In some embodiments, the ACO of the oligonucleotide agent can improve the stability, bioavailability, biodistribution, and / or cellular uptake of the siRNA compared to an oligonucleotide agent without the ACO.

[0195] In some embodiments, the ACO of the oligonucleotide agent can increase the biodistribution of the siRNA within one or more target tissues compared to an oligonucleotide agent without the ACO.

[0196] In some embodiments, the ACO of the oligonucleotide agent can increase the biodistribution of the siRNA in two or more target tissues compared to an oligonucleotide agent without the ACO.

[0197] In some embodiments, the one or more target tissues are selected from the group consisting of: prefrontal cortex, cerebellum, cerebrum, spinal cord, muscle, lung, eye, liver, and kidney.

[0198] In some embodiments, the oligonucleotide agents described herein can achieve a reduction in full-length SOD1 protein to a lower amount than the same amount of a double-stranded oligonucleotide (e.g., an siRNA agent) without an ODV structure administered alone, demonstrating greater efficacy with reduced toxicity or unwanted side effects. In some embodiments, the oligonucleotide agents described herein can achieve a reduction in full-length SOD1 protein to a lower amount than the additive effect achieved by the same amount of siRNA administered alone.

[0199] Specifically, the oligonucleotide agents described herein can inhibit / downregulate SOD1 mRNA transcripts by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% compared to baseline SOD1 mRNA transcripts). In some embodiments, for an in vitro cell line, when an oligonucleotide agent disclosed in the embodiments is administered to a cell or subject at a concentration of 10 nM, the resulting SOD1 mRNA transcripts are inhibited / downregulated by at least 50%, 60%, 70%, 77%, 79%, 81%, 84%, 85%, and 88% compared to baseline SOD1 mRNA transcripts in a control group. In some embodiments, the oligonucleotide agent can inhibit or downregulate SOD1 mRNA transcripts by about 80%.

[0200] In some embodiments, the expression of the SOD1 gene is inhibited / downregulated by administering to cells the oligonucleotide agents disclosed in the embodiments at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM or 150 nM. In some embodiments, by administering to cells or objects the oligonucleotide agents disclosed in the embodiments, the SOD1 gene encoding protein (SOD1 protein) is inhibited / downregulated. Compared to baseline expression of SOD1 protein, the SOD1 protein is knocked down by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100%). In some embodiments, the oligonucleotide agent can inhibit or downregulate the expression of SOD1 protein by about 80%. In some embodiments, the SOD1 protein is inhibited / downregulated by administering an oligonucleotide agent disclosed in the embodiments to a cell at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM or 150 nM.

[0201] In some embodiments, the oligonucleotide agent disclosed in the embodiments has a dose-dependent knockdown activity in cells. In some embodiments, the oligonucleotide agent knocks down the IC of SOD1 mRNA transcript in cells.50 Less than 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.8 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.08 nM, 0.06 nM, 0.04 nM, 0.02 nM, 0.01 nM, 0.008 nM or 0.005 nM.

[0202] Another aspect of the present application relates to a method for preventing or treating a disorder or condition induced by overexpression of SOD1 protein, SOD1 gene mutation and / or high SOD1 mRNA levels in an individual, comprising administering to the individual an effective amount of the siRNA, oligonucleotide agent, or an oligonucleotide agent composition disclosed herein. In some embodiments, the effective amount of the siRNA disclosed herein can be a concentration within the range of 0.01nM to 50nM, such as 0.01nM, 0.02nM, 0.05nM, 0.08nM, 0.1nM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM, 0.8nM, 1nM, 5nM, 10nM, 25nM, 50nM, 75nM, 100nM, or 150nM. In some embodiments, the disorder or condition is amyotrophic lateral sclerosis. In some embodiments, the individual is a mammal. In some embodiments, the individual is human.

[0203] In any embodiment provided herein, such cells can be isolated cells, such as cell lines, or can exist in a mammal, such as a human. In some embodiments, the human is a patient or individual suffering from a SOD1 protein-related disease or ALS, AD, PD or DS.

[0204] Another aspect of the present application relates to administering an effective amount of an oligonucleotide agent or a composition thereof to an individual using a route of administration as described herein. In some embodiments, the route of administration is selected from one or more of the following: parenteral infusion, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration. In some embodiments, the route of administration is selected from one or more of the following: intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, and subcutaneous administration. Dosage regimen and route of administration

[0205] Various aspects of this application relate to pharmaceutical compositions comprising the oligonucleotide agents of this application. In some embodiments, the pharmaceutical compositions comprise the oligonucleotide agents of this application and a pharmaceutically acceptable carrier, a therapeutically inert carrier, a diluent, or a pharmaceutically acceptable excipient. The pharmaceutical compositions disclosed herein are intended for development into medicaments for the prevention or treatment of SOD1 protein-related diseases or ALS.

[0206] Aspects of the present application also relate to methods of using the oligonucleotide agents of the present application to prepare such compositions.

[0207] Another aspect of the present application relates to the use of the oligonucleotide agent of the present application in the production of the pharmaceutical composition disclosed in the present application.

[0208] Another aspect of the present application relates to the use of an oligonucleotide agent according to any embodiment described herein or a composition according to any embodiment described herein in the manufacture of a medicament for preventing or treating a gene or protein-related symptom induced by overexpression of SOD1 protein, SOD1 gene mutation, and / or high SOD1 protein levels in an individual. According to certain embodiments of the use, the disease may include a disorder or condition related to SOD1 protein / mutation, including ALS. According to certain embodiments of the use, the symptom induced by overexpression of abnormal SOD1 protein is ALS. Also related to certain embodiments of the use, wherein the individual is a mammal, such as a human.

[0209] The dosage ranges of the various oligonucleotide agents or compositions of the present application can vary widely and are tailored to the individual needs of each case. In some embodiments, the pharmaceutical composition of the present application is administered for the first time when the subject is less than 1 week old, less than 1 month old, less than 3 months old, less than 6 months old, less than 1 year old, less than 2 years old, less than 15 years old, or greater than 15 years old.

[0210] The single dose range of the oligonucleotide agent is 0.01 mg / kg to 1000 mg / kg, for example, about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 40, 50, 75, 100, 120, 150, 200, 250, 300, 400, 500, 750 or 1000 mg / kg. The dosage described herein can include two or more of any oligonucleotide agent sequence described herein.

[0211] In some embodiments, the proposed dosing frequency is approximate. For example, in some embodiments, if the proposed dosing frequency is a first dose on day 1 and a second dose on day 29, the ALS patient may receive the second dose 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 days after receiving the first dose. In some embodiments, if the proposed dosing frequency is a first dose on day 1 and a second dose on day 15, the ALS patient may receive the second dose 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after receiving the first dose. In some embodiments, if the proposed dosing frequency is a first dose on day 1 and a second dose on day 85, the ALS patient may receive the second dose 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days after receiving the first dose.

[0212] In some embodiments, the injection dose and / or volume will be adjusted based on the subject's age, the subject's weight, and / or other factors that may require adjustment of injection parameters.

[0213] In some embodiments, the pharmaceutical composition includes a co-solvent system. For example, such a co-solvent system includes benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such a co-solvent system is used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is an absolute ethanol solution containing 3% w / v benzyl alcohol, 8% w / v of the non-polar surfactant polysorbate 80. TM and 65% w / v polyethylene glycol 300. The proportions of such cosolvent systems can vary considerably without significantly changing their solubility and toxicity characteristics. In addition, the types of cosolvent components can be varied: for example, other surfactants can be used instead of polysorbate 80. TM ; The fraction sizes of polyethylene glycol vary; other biocompatible polymers can replace polyethylene glycol, such as polyvinylpyrrolidone; other sugars or polysaccharides can replace glucose.

[0214] Examples of other compositions or components associated with the oligonucleotide agents, compositions, pharmaceutical compositions, and methods described herein include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, and the like, such as those used to administer, modify, assemble, store, package, prepare, mix, dilute, and / or preserve components for a particular application. In embodiments where a liquid form of any component is used, the liquid may be in a concentrated or ready-to-use form.

[0215] In some embodiments, the lipid moiety for nucleic acid therapy can be applied in the present application to deliver the oligonucleotide agent molecules disclosed in the present application. In these methods, nucleic acid (such as one or more oligonucleotide agents described in the present application) is introduced into a preformed liposome or lipid complex made from a mixture of cationic lipids and neutral lipids. In some methods, in the absence of neutral lipids, an oligonucleotide agent complex with a single cation or multiple cation lipids is formed. In some embodiments, lipid moieties are selected to increase the distribution of medicament to specific cells or tissues. In some embodiments, lipid moieties are selected to increase the distribution of medicament in adipose tissue. In some embodiments, lipid moieties are selected to increase the distribution of medicament in specific cells or muscle tissue.

[0216] In some embodiments, the pharmaceutical composition includes a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to prepare certain pharmaceutical compositions, including compositions comprising hydrophobic compounds. In some embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0217] In some embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more agents of the present invention to a specific tissue or cell type. For example, in some embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0218] In some embodiments, oligonucleotide agents can be delivered or administered by a vector. Any vector that can be used for gene delivery can be used. In some embodiments, viral vectors can be used. Non-limiting examples of viral vectors that can be used for the application include but are not limited to human immunodeficiency virus, herpes simplex virus (HSV), Moloney murine sarcoma virus (MMSV), mouse stem cell virus (MSCV), Semliki Forest virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), vesicular stomatitis virus (VSV), vaccinia virus (VV), adeno-associated virus (AAV), adenovirus, lentivirus and retrovirus.

[0219] In some embodiments, the vector is a recombinant AAV vector (rAAV). AAV vectors are relatively small DNA viruses that can be integrated into the genome of the cells they infect in a stable and site-specific manner. They are able to infect many types of cells without any effect on cell growth, morphology or differentiation, and do not seem to be associated with human pathology. The AAV genome has been cloned, sequenced and characterized. It contains approximately 4,700 bases, with an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the starting point for viral replication. The rest of the genome is divided into two basic regions with encapsidation function: the left part of the genome, which contains the rep gene involved in viral replication and viral gene expression; and the right part of the genome, which contains the cap gene encoding the viral capsid protein.

[0220] The formulations, pharmaceutical compositions or medicaments of the present disclosure are formulated, dosed and administered in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual subject, the cause of the condition, the site of delivery of the agent, the method of administration, the dosing schedule and other factors known to physicians.

[0221] The formulations, pharmaceutical compositions or medicaments of the present disclosure may be delivered by parenteral infusion, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal or subcutaneous administration; or by oral, intranasal, inhalation, vaginal or rectal administration.

[0222] Typical formulations of the oligonucleotide modulators of the present disclosure are prepared by mixing the siRNA of the present disclosure with a carrier or adjuvant. Suitable carriers and adjuvants are well known to those skilled in the art and are described in detail, for example, in Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004; Lippincott, Williams & Wilkins, Philadelphia); Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000; Lippincott, Williams & Wilkins, Philadelphia); and Rowe RC, Handbook of Pharmaceutical Excipients (2005; Pharmaceutical Press, Chicago). The formulation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, aromas, flavorings, diluents and other known additives to present the drug (i.e., the siRNA of the present disclosure or its pharmaceutical composition) well or to facilitate the production of a pharmaceutical product (i.e., a drug). Example

[0223] The following examples are intended to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention and are not intended to limit the scope of the invention to which this application relates, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, "parts" are by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, such as bp: base pairs; kb: kilobases; pl: picoliters; s or sec: seconds; min: minutes; h or hr: hours; aa: amino acids; nt: nucleotides; im: intramuscular; ip: intraperitoneal; sc: subcutaneous; icv or ICV: intracerebroventricular, etc. Example 1. Development of siRNA drug candidates for knockdown of human SOD1

[0224] The human SOD1 transcript sequence (NM_000454.5) was retrieved from the NCBI nucleotide database. The sequence includes a 465 bp open reading frame (ORF) located between nucleotides 78 and 542, which was used as a template for siRNA design (Table 1). Table 1. SOD1 cDNA sequences used for siRNA design

[0225] A total of 268 siRNA duplexes were designed and synthesized, each with a length of 21 nucleotides (nt), no more than 4 consecutive repeat nucleotides and a GC content between 35% and 65%. The target sites and homologous siRNA chain sequences are shown in Table 2. The knockdown activity of each siRNA in HEK293A cells was evaluated using high-throughput RT-qPCR at concentrations of 0.1 and 10 nM. The data were sorted according to the average knockdown activity, of which 121 siRNAs reduced SOD1 by more than 90% at 10 nM (Figure 1A). As an indicator of efficacy, 69 and 15 siRNAs reduced SOD1 levels by more than 50% and 75%, respectively, when treated with a 0.1 nM concentration. In HEK293A cells, 25 siRNAs were screened in an additional round at six concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM) to demonstrate dose-dependent activity, with propidium iodide (PI) incorporated into sample preparations to monitor changes in nucleic acid content as an indicator of adverse cytotoxicity (Figure 2). Figure 1B shows data for the top five siRNAs with the greatest knockdown activity (i.e., siSOD1-063, 047, 104, 005, and 258), which also demonstrate the absence of significant cytotoxicity (i.e., <20% reduction in PI staining).

[0226] Dose-response curves were then generated for each of the five siRNA candidates to validate their efficacy in model cell lines representing neuronal diseases, including SK-N-AS (Figure 1C) and T98G ( Figure 3 As shown in Table 3, the in vitro potency of each duplex in both cell lines was in the low picomolar range. 50 Adverse cytotoxicity was assessed after treatment with a concentration 200 times the value for 72 hours. Figure 4A As shown in Figure 2B, only siSOD1-047 and 005 had no detectable effect on apoptosis or cell number in SK-N-AS or T98G cells, while the remaining candidates (i.e., siSOD1-063, 104, and 258) showed a dose-dependent response to caspase 3 / 7 activity in T98G cells, which was negatively correlated with cell viability. SK-N-AS cells appeared to be more resistant to treatment, with similar patterns of cell viability observed.

[0227] Several medicinal chemistry patterns (referred to as M1, M2, M3, or M4) representing different duplex lengths (i.e., 20, 21, 22, and 23 nt, respectively) were applied to each lead candidate, including phosphorothioate (PS) backbone modifications and 2'-O-methylation (2'Ome) or 2'-fluoro (2'F) substitutions at selected positions of each nucleotide, and screened for their knockdown activity against the target mRNA. As shown in Figure 1D, at a treatment concentration of 0.1 nM in SK-N-AS cells, the M3 variants (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, and 258M3) generally had better knockdown activity than other chemically modified siRNAs. Almost the same pattern was also observed in T98G cells ( Figure 5 To further characterize the efficacy, the SK-N-AS (Figure 1E) and T98G ( Figure 6 Dose-response curves for M3-modified duplexes (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, 258M3, and 270M3) were generated in 1447 cells. As shown in Table 3, chemical modifications generally retained good in vitro potency.

[0228] By sharing medicinal chemistry that is generally not tolerated by classic siRNAs, auxiliary oligonucleotide (ACO) conjugates were developed to confer self-delivery properties similar to ASOs. Thus, the positive strand of each M3 variant (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, and 258M3) was covalently linked to a 14-nucleotide ACO (referred to as AC1) via a short linker (L9, i.e., a spacer-9 linker), wherein each position within the ACO had a PS backbone substitution and a 2'-O-methoxyethyl (2'-MOE) modification (Figure 7A). As shown in Figure 7B, at concentrations of 0.25 and 2.5 nM, no SOD1 knockdown activity was detected in vitro with AC1 treatment alone, while activity was only observed when it was conjugated to siRNA. Additional dose-response analysis showed that AC1 conjugation (i.e., siSOD1-005M3-AC1) resulted in an approximately 10-fold loss in siRNA potency compared to the chemically modified duplex alone (i.e., siSOD1-005M3) ( FIG7C ). However, after modification of the 5′ end of the guide strand with 5′-(E)-vinylphosphonate (5′VP) (i.e., siSOD1-005M3-AC1), the siRNA potency was significantly decreased. VP ), the efficacy was restored (Figure 7D). An ASO (i.e., ASO) similar in sequence and chemical properties to Tofersen (an antisense oligonucleotide ASO undergoing clinical research that has shown therapeutic benefits in the treatment of ALS by inhibiting the level of mutant SOD1 mRNA; see T. Miller et al., New England Journal of Medicine 383, 109-119 (2020)) was added. SOD1, SEQ ID NO: 864) as a comparison, two siRNA-ACO variants (i.e., siSOD1-005M3-AC1 or siSOD1-005M3-AC1 VP )'s SOD1 knockdown effect was greater than that of ASO SOD1 stronger, regardless of their 5' VP modification (Fig. 7D).

[0229] Subsequently, M3 chemical modification and 5'VP modification (i.e., siSOD1-063M3-AC1 VP 、047M3-AC1 VP 、104M3-AC1 VP 、005M3-AC1 VP 、258M3-AC1 VP and 270M3-AC1 VP ) to synthesize siRNA-ACO conjugates for downstream in vivo screening. Before administration, dose-response curves were generated in SK-N-AS and T98G cells to verify the activity of each siRNA-ACO candidate in inhibiting mutant SOD1 mRNA levels ( Figures 8A-8B As shown in Table 3, the in vitro potency was generally well preserved compared to the non-conjugated form. Apoptosis and cell viability were also quantitatively determined 72 hours after treatment to measure whether the chemical modification and ACO conjugation caused any changes in cytotoxicity. Figures 9A-9B As shown, in SK-N-AS and T98G cells, siSOD1-047M3-AC1 VP and 005M3-AC1 VP Usually unaffected and with minimal impact on cell health, siSOD1-104M3-AC1 VP Retaining poor signs of cytotoxicity, the remaining candidates (i.e., siSOD1-063M3-AC1 VP and 258M3-AC1 VP ) showed improved in vitro safety (i.e., reduced caspase 3 / 7 activity) compared to its unmodified form ( Figure 4A -B). Table 3. In vitro potency of drug candidates * IC 50 Values ​​± standard deviation, siRNA concentration that knocked down the SOD1 gene by half; “ / ” means not detected. Example 2. In vivo screening of siRNA-ACO drug candidates

[0230] hSOD1 G93A Transgenic hemizygous mice express mutant human SOD1 and exhibit disease phenotypes similar to ALS, including progressive loss of motor function and shortened lifespan due to neuronal damage (P. Weydt et al., Neuroreport 14, 1051-1054 (2003); PH Tu et al., Proc Natl Acad Sci USA 93, 3155-60 (1996)). To detect in vivo knockdown activity, siRNA-ACO (i.e., siSOD1-047M3-AC1) was injected intracerebroventricularly with an equal molar amount (i.e., 20 nmol / dose) of siRNA-ACO. VP or siSOD1-005M3-AC1 VP ) to treat hSOD1 G93A mice, and treated with non-conjugate (i.e. siSOD1-047M3 VP or siSOD1-005M3 VP ) was used as a control to demonstrate that AC1 conjugation can promote in vivo knockdown activity. All siRNA-ACOs were prepared in aCSF, which was treated with aCSF alone as a vehicle control to establish baseline expression, while siCON2-AC1 VP As a negative control for siRNA-ACO activity. Figure 10 As shown, both siRNA-ACO duplexes had higher knockdown activity in all tissues of the brain (i.e., frontal cortex, cerebellum, and cerebrum) and spinal cord (i.e., cervical, thoracic, and lumbar vertebrae), compared to their unconjugated homologs, while their activity in peripheral tissues (i.e., liver) was very low. SOD1 In comparison, siSOD1-005M3-AC1 was also characterized VP Durability of knockdown relative to its non-5'VP control (ie, siSOD1-005M3-AC1) In summary, the combination of AC1 conjugation and 5'VP modification provides our siRNA with the enhanced activity required for a durable effect in vivo. Example 3. Single intracerebroventricular injection of siRNA-ACO can delay hSOD1 G93A Disease progression and prolonged survival in mice

[0231] On PND 85 or 60, siRNA-ACO candidates (i.e., siSOD1-047M3-AC1) were injected intracerebroventricularly at 50, 100, 200, or 400 mg / dose, respectively. VP or siSOD1-005M3-AC1 VP ) treated with adult hSOD1 G93AOn day 14 after administration, the drug concentration and hSOD1 expression level in CNS tissues (i.e., cerebellum, cerebrum, and spinal cord) were quantitatively determined in a subgroup of animals. As shown in Figures 11A-B, siSOD1-047M3-AC1 VP and siSOD1-005M3-AC1 VP The activity and tissue accumulation of siRNA-ACO were dose-dependent, with SOD1 knockdown negatively correlated with increased siRNA-ACO concentrations in the CNS tissues. 50 The drug concentrations that responded (median effective dose) are summarized in Table 4. Table 4. ED of siRNA-ACOs that triggered median responses 50 and tissue concentrations *Mass of siRNA-ACO (mg) per gram (g) of tissue. ND: Not determined

[0232] In the remaining animals, body weight changes were plotted to monitor growth rate and disease progression. As shown in Figure 12A, compared with aCSF treatment, siSOD1-047M3-AC1 VP or siSOD1-005M3-AC1 VP All treated groups continued to gain weight. In addition, as shown by the time required for growth rate to return to starting weight (dashed line), disease-related weight loss was delayed in a dose-dependent manner. Disease progression was confirmed for each animal when a peak weight loss of 10% was recorded. Data were plotted using Kaplan-Meier curves to show when animals in each treatment group progressed to disease (Figure 12B). Data were collected until the animals inevitably succumbed to the disease, and survival curves were generated (Figure 12C). In summary, siSOD1-047M3-AC1 VP (Table 5) and siSOD1-005M3-AC1 VP (Table 6) Treatment delayed disease progression and prolonged animal survival, with the highest dose (i.e., 400 mg) extending lifespan by 70 days and 111.5 days, respectively, compared to the vehicle control group. Table 5. Single-dose administration of siSOD1-047M3-AC1 at PND 85 VP Median age of onset and survival of animals *Compared with the aCSF group, P < 0.001 Compared with the aCSF group, P < 0.01 Table 6. Single-dose administration of siSOD1-005M3-AC1 on PND 60VP Median age of onset and survival of animals *Compared with the aCSF group, P < 0.001 Example 4. Pathogenic mutations at the siRNA-ACO target site affect lead selection

[0233] siSOD1-005M3-AC1 VP Computer simulation analysis of pathogenic single nucleotide polymorphisms (SNPs) within the target site revealed a total of five SNPs, four of which were located within the region complementary to its "seed" sequence (Figure 13A). Mismatches in this region are known to inhibit siRNA activity, which could exclude approximately 8.9-12.4% of ALS cases worldwide from its therapeutic repertoire. SOD1 Patients (O. Abel et al., Hum Mutat 33, 1345-51 (2012); https: / / alsod.ac.uk / , Amyotrophic Lateral Sclerosis Online Database - ALSoD). In contrast, siSOD1-047M3-AC1 VP There are only two pathogenic SNPs reported in its target sites (i.e., P.E22G and P.F21C), accounting for approximately SOD1 4.04% and 2.70% of the population (Figure 13B). VP The luciferase reporter gene construct (i.e., pLuc) contains a conserved sequence or one of the pathogenic mutations (i.e., P.E22G and P.F21C) that is completely complementary to the guide strand. SOD1 、pLuc P.E22G and pLuc P.F21C ) were co-transfected with siRNA-ACO into HEK293A cells. VP Specificity is achieved because the scrambled sequence control (i.e., siCON2-AC1 VP ) transfection did not reduce reporter gene expression (Figure 13C). The dose-response data showed that the P.E22G mutation had a significant effect on siSOD1-047M3-AC1 compared with the conserved sequence of the target site. VP Knockdown did not have any significant impact on activity / potency, whereas P.F21C was partially resistant to treatment, exhibiting incomplete knockdown and lower potency ( FIG. 13D ). Example 5. Delaying hSOD1 by IT injection of siRNA-ACO G93A Disease progression, prolonged survival, and improved motor function in mice

[0234] Based on the expected patient population, siSOD1-047M3-AC1 was selected VP For further in vivo analysis, male and female hSOD1 G93A Mice received two consecutive siSOD1-047M3-AC1 injections VP The dose was 75, 150 or 300 μg / dose. Non-specific siRNA-ACO (i.e. siCON3-AC1 VP ) was used as a negative control for efficacy. The body weight of the animals was monitored and compared with wild-type animals (WT), all doses of siSOD1-047M3-AC1 VP The benefits of treatment were similar in male mice, while weight gain in female mice appeared to be more pronounced in a dose-dependent manner (Figure 14A). Disease progression (i.e., 10% loss of peak body weight) and animal survival were also plotted, where siSOD1-047M3-AC1 VP Treatment delayed disease progression and prolonged survival in both male and female mice (Figure 14B-C). The disease progression and median survival days of male and female animals treated with IT siRNA-ACO are summarized in Table 7. Overall, siCON3-AC1 VP No therapeutic benefit was provided, whereas an equivalent dose of siSOD1-047M3-AC1 VP (i.e. 150 mg) extended the survival of male and female animals by 61 days and 29 days, respectively. Table 7. siSOD1-047M3-AC1 administered by IT injection VP Median age of onset and survival in male and female mice *Compared with the aCSF group, P < 0.001 Compared with the aCSF group, P < 0.01

[0235] Neuromuscular performance was also assessed in both male and female mouse groups. siSOD1-047M3-AC1 showed significant differences in distance traveled as measured by open field walking ( FIG. 15A ), rotarod test ( FIG. 15B ), and grip strength ( FIG. 15C ). VP Treatment significantly improved hSOD1 G93A The motor function of mice was improved and generally persisted until the end of the study. Comparison of the rotarod performance of each animal at an early time point before measurable weight loss (i.e., PND 90) with its respective last time point further demonstrated that the rotarod performance was improved compared to the control group or ASO group. SOD1 Compared with siSOD1-047M3-AC1VP Treatment preserved or improved neuromuscular performance in most animals ( Figure 16 The latency of each animal's rotarod performance is summarized in Table 8.

[0236] Before the open field, rotarod, and / or grip strength tests, all animals were scored for motor function using the ALS Therapeutic Development Institute (ALS TDI) Neurological Score (NS) system. VP In the control group, all mice developed abnormal opening (i.e., NS2) at approximately PND 130, and the severity of the opening continued to increase over time (i.e., ≥NS3). In contrast, all mice treated with siSOD1-047M3-AC1 showed abnormal opening at any time point during the study. VP The mean scores of all doses did not exceed NS2, especially in the highest dose group (i.e., 300 mg), where the mean NS remained around NS1. VP Compared with the control group, ASO SOD1 NS also improved in the treatment group. However, by PND 150, despite the ASO SOD1 Compared to siSOD1-047M3-AC1 VP The molecule was in approximately 3-fold excess, but NS began to increase with a slope similar to that of the control group.

[0237] In summary, the siRNA-ACO conjugate provides SOD1 siRNA with the pharmacological properties required for clinical development, including delivery to the CNS and good tissue biodistribution providing potent and sustained activity. G93A "These results clearly demonstrate that siRNA offers a significant improvement in targeting and knocking down SOD1 for the treatment of ALS compared to current clinical treatments (i.e., tofersen). Example 6. Knockdown activity of siRNA on SOD1 mRNA expression in HeLa and SK-N-AS cells

[0238] To evaluate the knockdown activity of siRNA, the designated siRNAs (i.e., RD-15757, RD-18972, RD-12500, RD-18973, RD-18948, and RD-18949) were added directly to the culture medium of HeLa cells at a concentration of 1500 nM and transfected for 3 days. Cells without any treatment were used as mock controls, and cells treated with RD-11566 were used as double-stranded controls. Figure 17 As shown, all siRNAs caused varying degrees of reduction in SOD1 mRNA levels compared with RD-11566 (ranging from 1-32%), with RD-12500 causing the greatest reduction (32%).

[0239] To further evaluate the dose-dependent knockdown activity of siRNA, the indicated siRNAs (i.e., RD-12926, RD-15757, RD-12500, RD-18947, RD-18948, RD-18949, RD-18946, RD-18972, and RD-18973) were added to SK-N-AS cells at the indicated concentrations (i.e., 0.0002, 0.001, 0.0039, 0.0156, 0.0625, 0.25, 1, and 4 nM) and transfected for 24 h. SOD1 mRNA levels were quantified by two-step RT-qPCR, see Figure 18A and 18B Extrapolation to obtain EC 50 EC values ​​after siRNA treatment in SK-N-AS cells were used to determine the potency at which each tested siRNA achieved maximal activity in terms of dose-dependent knockdown of SOD1 mRNA. 50 The values ​​are summarized in Table 9. Table 9. ECs after siRNA treatment in SK-N-AS cells 50 value Materials and Methods High-throughput screening of siRNA targeting human SOD1 siRNAs were designed using the open reading frame (ORF) of the human SOD1 (hSOD1) cDNA sequence (NM_000454.5) as a template using an in-house algorithm. A total of 268 19-nucleotide duplexes were synthesized without medicinal chemistry (Table 2). HEK293A cells were plated and transfected in 96-well plates, with each well containing 32 siRNAs and 8 quality control treatments at two concentrations (i.e., 0.1 and 10 nM) in duplicate. Cells were cultured for 24 hours and automatically lysed using a Fluent System 780 liquid handling system (Tecan, Hombrechtikon, Switzerland) using an optimized formulation containing propidium iodide (PI) based on the cell lysis (CL) buffer for one-step RT-qPCR, as previously described (K. Shatzkes et al., SciRep 4, 4659 (2015)). PI was added during sample preparation to monitor changes in cell number (e.g., adverse cytotoxicity) by staining the total nucleic acid content in the crude lysate. Staining was quantified by optical density (OD) at 535 nm excitation and 615 nm emission wavelengths on an Infinite M200 Pro microplate reader (Tecan). The samples were then transferred to 384-well plates and RT-qPCR analysis was performed on a 480 real-time PCR system (Roche, Basel, Switzerland) using a one-step TB Green PrimeScript RT-PCR kit II (Takara, Kyoto, Japan). PCR reactions were automatically prepared using an Echo 525 acoustic liquid handler (Beckman Coulter, Brea, California, USA). Only the top 30 best siRNAs were subsequently screened at 6 concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM). All samples were amplified in triplicate. siRNA synthesis

[0240] Oligonucleotide sequences were synthesized internally on a solid support using an HJ-12 synthesizer (Beijing Haijing High-Tech Co., Ltd., China) at Ractigen Therapeutics (Rudong, China) and subsequently purified by RP-HPLC using an acetonitrile gradient on a UniPS column (Suzhou Nano Micro Technology Co., Ltd., China). Each sequence was redissolved in sterile water by buffer exchange. The chain mixture was briefly heated and cooled to room temperature, and each chain of equimolar amount was annealed to form a corresponding double chain. Single bands were separated by gel electrophoresis at the predicted molecular weight to confirm the formation of duplexes. Duplexes were identified using ESI-MS, and overall purity was analyzed by SEC-HPLC using an XBridge Protein BEH SEC 125A column (Waters Corporation, Milford, Massachusetts, USA). Endotoxin levels in each batch were quantitatively determined by enzyme factor C using an endpoint chromogenic endotoxin quantification kit (Bioendo, Xiamen, China). All control duplexes and chemically modified sequences are shown in Table 10. Cell culture and treatment

[0241] HEK293A cells (Kebai, Nanjing, China, catalog number: CBP60436) and SK-N-AS cells (Punosai, Wuhan, China, catalog number: CL-0621) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% bovine serum (Sigma-Aldrich), penicillin (100 U / ml, Gibco), and streptomycin (100 mg / ml, Gibco). T98G cells (Cobioer, catalog number: CBP60301) were cultured in MEM supplemented with 10% FBS, 1% NEAA, sodium pyruvate (1 mM), penicillin (100 U / ml), and streptomycin (100 μg / ml). Human cervical cancer cell line HeLa (ATCC) cells were cultured in modified RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 10% bovine serum and 1% penicillin / streptomycin. All cell lines were cultured at 37° C. in a humidified atmosphere with 5% CO 2 . Transfections were performed using Lipofectamine RNAiMax (ThermoFisher, Waltham, MA, USA) according to the manufacturer's protocol in growth medium without antibiotics. RT-qPCR One-step reverse transcription-quantitative polymerase chain reaction (one-step RT-qPCR)

[0242] After transfection, the culture medium was discarded and the cells were washed once with 150 μL of PBS per well. After discarding the PBS, 100 μL of cell lysis buffer was added to each well and incubated at room temperature for 5 minutes. 0.5 μL of cell lysate was taken from each well and RT-qPCR analysis was performed using the One-Step TB Green™ PrimeScrip™ RT-PCR Kit II (Takara, RR086A) in a Roche Lightcycler 480 Real-Time PCR instrument. PCR reactions were prepared using an Echo 525 Acoustic Liquid Handler (Beckman Coulter). Three replicate wells were set up for amplification for each transfected sample. PCR reaction conditions are shown in Table 11. Table 11. PCR reaction preparation

[0243] Reaction conditions were as follows: Reverse transcription (Phase 1): 42°C for 5 minutes, 95°C for 10 seconds; PCR (Phase 2): 95°C for 5 seconds, 59°C for 20 seconds, 72°C for 10 seconds, 40 cycles; Melting curve analysis (Phase 3). The human SOD1 gene was amplified as the target gene. Human TBP or mouse TBP was used as a reference gene and amplified as an internal control for RNA loading. Primer sequences are shown in Table 12. Table 12. Primer sequences for RT-qPCR detection Two-step RT-qPCR

[0244] Animal tissues frozen in RNALater (Sigma-Aldrich, St. Louis, MO, USA) were homogenized using a Bioprep-24 homogenizer (Allsheng, Hangzhou, China) in a total RNA isolation reagent (Biosharp, Hefei, China). Chloroform was added to the homogenate after removing the aqueous phase and mixed with isopropanol. Total RNA was extracted from the tissue preparation using the RNeasy RNA kit (Qiagen) according to the manufacturer's protocol. RNA was extracted from cell cultures using the Auto-Pure 96A (Allsheng) nucleic acid extraction system. Reverse transcription (RT) reactions were performed using 1 μg of total RNA using the PrimeScript RT kit with gDNAEraser (Takara, Shlomo, Japan). cDNA was amplified in triplicate using SYBR Premix Ex Taq II (Takara, Shlomo, Japan) on a Roche LightCycler 480 Multiwell Plate 384 (Roche, Reference No. 4729749001, USA) using a primer set specific for human SOD1 (hSOD1) and an internal control from human (i.e., TBP) or mouse (i.e., mTBP) samples. A melting curve was generated after amplification to confirm primer specificity. Reaction conditions were as follows: reverse transcription (stage 1): 42°C for 5 minutes, 95°C for 10 seconds; PCR (stage 2): 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 10 seconds; 40 amplification cycles; and melting curve analysis (stage 3). PCR reaction conditions are shown in Tables 13 and 14. Primer sequences are shown in Table 12. Table 13. RT reaction Table 14. RT-qPCR reaction

[0245] To calculate the expression level (Erel) of SOD1 mRNA in siRNA-transfected samples relative to the control treatment (mock), the average Ct value of the target gene and the reference gene was substituted into Formula 1, E rel =2 (CtTm-CtTs) / 2 (CtRm-CtRs) (Formula 1) Among them, CtT m is the Ct value of the target gene in the simulated sample; CtT s is the Ct value of the target gene in the siRNA-treated sample; CtR m is the Ct value of the internal reference gene in the simulated sample; CtR sis the Ct value of the internal reference gene in the siRNA-treated sample. Caspase3 / 7 activity assay

[0246] Caspase 3 / 7 activity was quantitatively measured in cell culture using the Caspase-Glo 3 / 7 Assay System (Promega, Madison, WI, USA). Briefly, the luminescent substrate was added directly to the culture medium and incubated at 37°C for 20 minutes. Luminescence was then measured on an Infinite M200 Pro microplate reader (Tecan). The luminescence value for each well was subtracted from the blank background signal and the data were normalized to the untreated (mock) control group to calculate the relative caspase 3 / 7 activity. Cell viability assay

[0247] In vitro cell viability was determined using the CCK-8 assay (Dojindo, Mashiki-machi, Japan) according to the manufacturer's protocol. Briefly, fresh culture medium containing WST-8 substrate was added to each well of the tissue culture plate and incubated at 37°C for at least 1 hour. The absorbance was measured at 450 nm on an Infinite M200 Pro microplate reader (Tecan). The background signal of the blank control was subtracted from the OD value of each well, and the data were normalized by the untreated (mock) control group to calculate relative activity. Luciferase reporter constructs and knockdown assessment

[0248] The target sequence containing the P.E22G or P.F21C mutant SNP was cloned into the multiple cloning site (MCS) of the luciferase reporter gene vector pmirGLO (Promega) between the NheI and SalI restriction endonuclease (RE) sites downstream of the firefly luciferase gene (luc2) to generate constructs pLuc P.E22G and pLuc P.F21C A control reporter gene construct (pLuc SOD1 ), which contains siSOD1-047M3-AC1 VPThe guide strand was fully complementary to the conserved sequence of hSOD1. All constructs were subcloned in DH5a bacteria (Tolobio, Shanghai, China) and colonies were selected for DNA sequencing to confirm the insertion of the target sequence. Example colonies were amplified by midiPrep (Qiagen, Hilden, Germany) for plasmid isolation. HEK293A cells were plated at 30,000 cells / well in 96-well cell culture plates in the absence of antibiotics. One of the reporter gene plasmids (i.e., pLuc) was added to the PCR product using 0.3 μl Lipofectamine 2000 (ThermoFisher) at 100 ng / well. P.E22G 、pLuc P.F21C or pLuc SOD1 ) and the indicated concentrations of siSOD1-047M3-AC1 VP Or scrambled control cells were co-transfected. The wells to which the test substance (0 nM) was not added were used as untreated controls. The cells were cultured for 24 hours and luciferase activity was quantitatively determined using the Dual-Glo luciferase assay system (Promega) according to the manufacturer's protocol. Briefly, cells were lysed in 50 μl passive lysis buffer (Promega), 20 μl of the lysate was mixed with 20 μl of Dual-Glo luciferase reagent and incubated at room temperature for 10 minutes. Luminescence was subsequently measured on an Infinite 200Pro microplate reader (Tecan) to quantify luciferase activity. After measurement, 20 μl of Dual-Glo Stop&Glo reagent (Promega) was added to each well and incubated for another 10 minutes at room temperature. Luminescence was measured again to quantitatively determine Renilla luciferase activity for normalization of luciferase reporter gene results. The results were calculated as the percentage of knockdown (% KD) of the reporter gene data after normalization with the Renilla luciferase data relative to the untreated control group data = 1-(ratio of the siRNA-treated group / ratio of the untreated group) * 100. The ratio of the siRNA-treated group = firefly luciferase activity of the siRNA-treated group / Renilla luciferase activity of the siRNA-treated group; the ratio of the untreated group = firefly luciferase activity of the untreated group / Renilla luciferase activity of the untreated group. Animal handling and grouping

[0249] Parental transgenic hSOD1 G93AMice (strain ID: 004435) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and imported into China through Nantong University (Nantong, Jiangsu, China). Mice were transported to the animal facility at 6 weeks of age and subsequently maintained at Nantong University, which provided the animals for this study. All animal procedures were approved by the Nantong University Laboratory Animal Care and Use Committee. Animal dosing preparations were prepared fresh before use by dissolving lyophilized oligonucleotides in aCSF to prepare stock solutions that were diluted to the intended dosing concentration. Animals were randomly assigned to study groups based on body weight and sex. Any animals that were in poor health or had obvious abnormalities were removed from the experiment. Randomized groups were analyzed by a common one-way analysis of variance model using GraphPad Prism (Windows) version 8.3.0 (GraphPad Software, San Diego, CA, USA). Female hSOD1 G93A Mice typically weigh about 20-25% less than their male littermates. Intracerebroventricular (ICV) injection

[0250] Avertin (1.2%) was freshly prepared and sterilized through a 0.2 μm filter. Mice were administered an intraperitoneal (IP) injection at a dose of 0.30-0.35 ml / 10 g in a stereotaxic apparatus to rapidly induce anesthesia for up to 30 minutes. An approximately 11.5 mm incision was made in the animal's scalp, and a 25-gauge needle was connected to a Hamilton syringe containing the appropriate siRNA formulation and placed at the level of bregma. The needle was moved to the appropriate anterior / posterior and medial / lateral coordinates (0.2 mm anterior / posterior, 1 mm medial / lateral on the right side). A total of 10 μl was injected into the lateral ventricle at a rate of approximately 1 μl / s. After injection, the needle was slowly withdrawn and the wound sutured. Intrathecal (IT) injection

[0251] Anesthesia was initiated with 3.0% isoflurane in an anesthesia induction chamber for 10 minutes. The hair surrounding the injection site at the base of the tail was shaved and cleaned with 75% ethanol. The interspinous space between L5 and L6 was identified, and a 30-gauge needle connected to a microliter syringe containing the appropriate drug preparation was slowly inserted into the dura mater until a tail flick was observed. The needle was then positioned securely, and a total volume of 10 μl of the solution was injected over 1 minute. Quantitative analysis of siRNA-ACO in animal tissues

[0252] Tissue lysates were prepared in lysis buffer (0.5% CA-630, 1 mM EDTA, 150 mM NaCl) using a Bioprep-24 homogenizer (Allsheng). Samples were then heated to 95°C to inactivate sample proteins. Untreated lysates supplemented with siRNA-ACO were serially diluted to eight concentration levels, and a standard curve was plotted. Reverse transcription (RT) reactions were performed using the PrimeScript RT kit (Takara) conjugated with custom stem-loop primers specific for the siRNA guide strand. Each sample was amplified in triplicate using the SYBR Premix Ex Taq II (Takara) reaction mixture and primers specific for the guide strand cDNA on a 480 Real-Time PCR system (Roche). After amplification, melting curves were plotted to confirm primer specificity. The absolute amount of siRNA was extrapolated by linear regression based on the corresponding standard curve. Tissue concentration was calculated as the ratio of the absolute mass of siRNA (ng) to the total weight of the tissue sample used for lysis (g). Clinical observations and endpoint criteria

[0253] Animals were observed for up to 4 hours after injection and daily thereafter until endpoint. Body weight was measured before and at recorded time points after test article administration. Animals with a >20% loss of initial body weight compared to the day of treatment or a neurological score of NS4 met endpoint criteria. Neurological function score

[0254] For animals dosed by IT injection, the ALS Therapeutic Development Institute (ALS TDI) Neurological Score (NS) system was used to assess motor deficits in mice. G93A Hindlimb dysfunction, common in mice, is an objective assessment of disease progression (T. Hatzipetros et al., Journal of Visualized Experiments (2015), https: / / doi.org / 10.3791 / 53257). NS is assigned according to the following 4-point scale: 0 for no signs of motor dysfunction (i.e., presymptomatic), 1 for obvious hindlimb tremor when suspended by the tail (i.e., initial symptom), 2 for gait abnormalities (i.e., onset of paresis), 3 for dragging of at least one hindlimb (i.e., partial paralysis), and 4 for failure to recover spontaneously within 10 seconds (i.e., terminal paralysis). Open field test

[0255] During the day, each mouse was placed in a corner of an open field (50 cm long × 50 cm wide × 50 cm high) and allowed to roam freely for 15 minutes. An overhead camera recorded the path of each animal. Video footage was analyzed using the automated tracking software Samart 3.0 (Bioseb, Vitrolles, France) to calculate the total distance traveled. Rotarod test

[0256] Animals were trained for three days before data collection. Mice were placed on a 60 mm diameter rotarod apparatus (Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China). The rotational speed was accelerated from 0 to 30 rpm over 300 seconds. The latency was recorded as the time required for each animal to fall from the rotarod. Each animal was tested three times, and the maximum latency was selected. Grip strength test

[0257] Mice were placed on a gridded plate and allowed to grip the grid with their front and hind paws. The tail was gently pulled, and maximum muscle force was measured in units of mass on an XR501 Grip Dynamometer (Xinruan Information Technology) until the animal released its grip. The test was repeated three times for each animal, and the average value was used to represent the grip strength. Statistical analysis

[0258] Data were analyzed using GraphPad Prism (Windows) version 8.3.0. Dose-response curves and IC values ​​were extrapolated using a four-parameter concentration-inhibition model using nonlinear regression. 50 Where indicated, Tukey's multiple comparison test was used to compare means to determine statistical differences between different dose-response curves. The amount of drug in tissue associated with knockdown activity (including ED) was calculated using a 3-parameter concentration-response model using nonlinear regression. 50 Time-stratified data (i.e., peak weight analysis and animal survival) were plotted using Kaplan-Meier plots, and statistical significance was verified using the Mantel-Cox test.

[0259] Although the present application has shown and described preferred embodiments of the present invention, it will be apparent to those skilled in the art that these embodiments are provided as examples only. The present invention is not limited to the specific embodiments in the specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments do not have a restrictive meaning. Without departing from the present invention, many changes, modifications and substitutions will now occur to those skilled in the art. In addition, it should be understood that the various aspects of the present invention are not limited to the specific description, configuration or relative proportions of the various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described in this application can be adopted in the implementation of the present invention. Therefore, it is expected that the present invention will also cover any such substitutions, modifications, variations or equivalents. The following claims define the scope of the invention and cover methods, structures and their equivalents within the scope of these claims.

Claims

1. An siRNA comprising a sense strand and an antisense strand forming a double-stranded structure, wherein at least one of the two strands comprises a nucleotide sequence having at least 85% complementarity or homology with a portion of the nucleotide molecule of SEQ ID NO: 1, and wherein the siRNA is capable of inhibiting / downregulating superoxide dismutase 1 (SOD1) gene transcription in cells.

2. The siRNA of claim 1, wherein at least one of the two strands has 0, 1, 2 or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO: 1; and / or wherein a portion of the nucleotide sequence comprises any one of SEQ ID NOs: 2 to 269 columns; and / or wherein the sense strand has at least 85% nucleotide sequence homology to any one of SEQ ID NOs: 270 to 537; and / or wherein the antisense strand has at least 85% nucleotide sequence homology to any one of SEQ ID NOs: 538 to 805; and / or wherein the siRNA is capable of inhibiting / downregulating superoxide dismutase 1 (SOD1) gene transcription in cells, reducing it by at least 10% compared to the SOD1 mRNA baseline level; and / or wherein the sense strand comprises at least 10 consecutive nucleotides; and / or wherein the antisense strand comprises at least 10 consecutive nucleotides; and / or The sense strand and the antisense strand comprise 0, 1, 2 or 3 mismatches.

3. The siRNA of claim 1, wherein the siRNA comprises a sense strand comprising SEQ ID NO: n and an antisense strand comprising SEQ ID NO: n+268, where n is any integer between 270 and 537.

4. The siRNA of claim 1 , wherein at least one nucleotide of the siRNA is a chemically modified nucleotide located in the sense strand, the antisense strand, or both strands; and / or wherein the chemically modified nucleotides are nucleotides modified at the 5' end, the 3' end, both ends or within the chain; and / or wherein at least 50% of the nucleotides in the sense strand, the antisense strand, and / or both strands of the siRNA are chemically modified.

5. The siRNA of claim 4, wherein the chemically modified nucleotides are one or more selected from the group consisting of: 2' sugar modification, base modification, phosphorothioate (PS) backbone modification, addition of a 5'-phosphate moiety or a 5-methylcytosine moiety at the 5' end of the nucleotide sequence.

6. The siRNA of claim 5, wherein the 2' sugar modification is one or more selected from the group consisting of a 2'-fluoro-2'-deoxynucleoside (2'-F) modification, a 2'-O-methyl (2'-O-Me) modification, and a 2'-O-(2-methoxyethyl) (2'-O-MOE) modification; and / or The addition of the 5'-phosphate moiety adds one or more (E)-vinylphosphonate moieties to the 5' end of the nucleotide sequence.

7. The siRNA of claim 4, wherein the sense strand has at least 85% nucleotide sequence homology to the sequence of SEQ ID NOs: 808 to 827, 867 (with or without an (E)-vinylphosphonate moiety added to the 5' end); and / or The antisense strand has at least 85% nucleotide sequence homology to the sequences of SEQ ID NOs: 828 to 849, 868 (with or without the (E)-vinylphosphonate moiety added to the 5' end).

8. The siRNA of claim 4, wherein the siRNA comprises a sense strand comprising SEQ ID NO: m and an antisense strand comprising SEQ ID NO: m+20, where m is any integer between 808 and 827; or wherein the siRNA comprises a sense strand comprising SEQ ID NO: 814 and an antisense strand comprising SEQ ID NO: 848; a sense strand comprising SEQ ID NO: 822 and an antisense strand comprising SEQ ID NO: 849; or a sense strand comprising SEQ ID NO: 814 and an antisense strand comprising SEQ ID NO:

866.

9. An oligonucleotide agent comprising: (a) the small interfering RNA (siRNA) according to any one of claims 1 to 8; and (b) a non-targeting single-stranded oligonucleotide (auxiliary oligonucleotide, ACO), The ACO is about 6 to 22 nucleotides in length, wherein the siRNA and the ACO are covalently linked, without or with one or more linking components, to form the oligonucleotide agent.

10. The oligonucleotide agent of claim 9, wherein the ACO consists of one or more RNA, DNA, BNA, LNA, GNA, and PNA. The oligonucleotide agent of claim 9 , wherein the ACO is about 6 to 18 nucleotides in length.

12. The oligonucleotide agent of claim 11, wherein the ACO is about 8 to 16 nucleotides in length.

13. The oligonucleotide agent of claim 9, wherein the siRNA comprises a sense strand ranging from about 16 to 25 nucleotides in length; and / or The siRNA comprises an antisense strand with a length ranging from about 19 to 25 nucleotides.

14. The oligonucleotide agent of claim 9, wherein the oligonucleotide agent is capable of inhibiting / downregulating the transcription level of superoxide dismutase 1 (SOD1) gene in a cell by at least 50%.

15. The oligonucleotide agent of claim 9, wherein the ACO comprises a 5' end and a 3' end, and wherein the 5' end or the 3' end of the ACO is conjugated to a linking component.

16. The oligonucleotide agent of claim 9, wherein the sense strand and / or the antisense strand of the siRNA is covalently linked to the ACO via one or more linking components.

17. The oligonucleotide agent of claim 9, wherein the linking component is selected from one or more of the following linking forms: an ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, RNA, DNA, a carbohydrate, a thiol linking group, a phosphodiester, a phosphorothioate, a phosphoramide, an amide, a carbamate, a tetrazole linking group, and a benzimidazole linking group.

18. The oligonucleotide agent of claim 17, wherein the linking component is selected from the group consisting of: a) Spacer phosphoramidite 18 (phosphoramidite acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecan-1-yl(2-cyanoethyl ester); b) Spacer-9 (3-[2-[2-[2-(bis(4-methoxyphenyl)(phenylmethoxy]ethoxy]ethoxy]ethoxy-[di(propyl-2-yl)amino]phosphino]oxypropionitrile); c) spacer phosphoramidite C3 (6-(4,4'-dimethoxytrityl)hexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite); and d) Spacer C6 phosphoramidite (6-(4,4'-dimethoxytrityl)hexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) e) Divalent linker (DIO)16-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 1,1-Bis(4-methoxyphenyl)-18-oxo-1-phenyl-2,5,8,11,14,17-hexaoxaheneicosane- CPG.

19. The oligonucleotide agent of claim 9, wherein the ACO is covalently linked to the 3' end, or the 5' end, or both the 3' end and the 5' end of the sense strand of the siRNA; and / or wherein the ACO is covalently linked to the 3' end, or the 5' end, or to both the 3' end and the 5' end of the antisense strand of the siRNA; and / or wherein the ACO is covalently linked to one or more internal nucleotides of the siRNA.

20. The oligonucleotide agent of claim 9, wherein one, two or more ACOs are covalently linked to the siRNA.

21. The oligonucleotide agent of any one of claims 9 to 20, wherein at least one nucleotide of the ACO is chemically modified.

22. The oligonucleotide agent of any one of claims 9 to 20, wherein at least about 50% of the nucleotides of the ACO are chemically modified nucleotides.

23. The oligonucleotide agent of claim 21, wherein the at least one chemically modified nucleotide has a 2′-sugar modification selected from one or more of a 2′-fluoro-2′-deoxynucleoside (2′-F) modification, a 2′-O-methyl (2′-O-Me) modification, and a 2′-O-(2-methoxyethyl) (2′-O-MOE) modification.

24. The oligonucleotide agent of claim 21, wherein the chemical modification of the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification.

25. The oligonucleotide agent of claim 24, wherein the ACO comprises 6 to 17 phosphorothioate (PS) backbone modifications.

26. The oligonucleotide agent of claim 13, wherein the antisense strand comprises an (E)-vinylphosphonate moiety added to the 5' end of the nucleotide sequence.

27. The oligonucleotide agent of claim 21, wherein the chemical modification of the at least one chemically modified nucleotide is the addition of a 5-methylcytosine moiety to the 5' end of the nucleotide sequence.

28. The oligonucleotide agent of claim 10, wherein the ACO and / or the siRNA are conjugated to one or more conjugating groups.

29. The oligonucleotide agent of claim 38, wherein the sense strand and / or the antisense strand of the siRNA is conjugated to one or more conjugating groups.

30. The oligonucleotide agent of claim 38, wherein the one or more conjugated groups are selected from the group consisting of: a lipid, a fatty acid, a fluorophore, a ligand, a sugar, a peptide, and an antibody.

31. The oligonucleotide agent of claim 38, wherein the one or more conjugated groups are selected from the group consisting of: cell penetrating peptides, polyethylene glycol, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

32. The oligonucleotide agent of any one of claims 10 to 38, wherein the sense strand and the antisense strand of the siRNA have at least 85% homology to a nucleotide sequence selected from the group consisting of: a) RD-12926 (SEQ ID NO:867 and SEQ ID NO:868), b) siSOD1-063M1 (SEQ ID NO: 808 and SEQ ID NO: 828), c) siSOD1-063M2 (SEQ ID NO: 809 and SEQ ID NO: 829), d) siSOD1-063M3 (SEQ ID NO: 810 and SEQ ID NO: 830), e) siSOD1-063M4 (SEQ ID NO: 811 and SEQ ID NO: 831), f) siSOD1-047M1 (SEQ ID NO: 812 and SEQ ID NO: 832), g) siSOD1-047M2 (SEQ ID NO:813 and SEQ ID NO:833), h) siSOD1-047M3 (SEQ ID NO: 814 and SEQ ID NO: 834), i) siSOD1-047M4 (SEQ ID NO: 815 and SEQ ID NO: 835), j) siSOD1-104M1 (SEQ ID NO: 816 and SEQ ID NO: 836), k) siSOD1-104M2 (SEQ ID NO: 817 and SEQ ID NO: 837), 1) siSOD1-104M3 (SEQ ID NO: 818 and SEQ ID NO: 838), m) siSOD1-104M4 (SEQ ID NO: 819 and SEQ ID NO: 839), n) siSOD1-005M1 (SEQ ID NO: 820 and SEQ ID NO: 840), o) siSOD1-005M2 (SEQ ID NO: 821 and SEQ ID NO: 841), p) siSOD1-005M3 (SEQ ID NO: 822 and SEQ ID NO: 842), q) siSOD1-005M4 (SEQ ID NO: 823 and SEQ ID NO: 843), r) siSOD1-258M1 (SEQ ID NO: 824 and SEQ ID NO: 844), s) siSOD1-258M2 (SEQ ID NO: 825 and SEQ ID NO: 845), t) siSOD1-258M3 (SEQ ID NO: 826 and SEQ ID NO: 846), u) siSOD1-258M4 (SEQ ID NO: 827 and SEQ ID NO: 847), v) siSOD1-270M3 (SEQ ID NO: 814 and SEQ ID NO: 866), w)siSOD1-047M3 VP (SEQ ID NO:814 and SEQ ID NO:848), x)siSOD1-005M3 VP (SEQ ID NO:822 and SEQ ID NO:847), y) siSOD1-047M3-AC1 (SEQ ID NO: 850 and SEQ ID NO: 834), z) siSOD1-005M3-AC1 (SEQ ID NO: 851 and SEQ ID NO: 842), aa) siCON1-AC1VP (SEQ ID NO: 852 and SEQ ID NO: 858), bb) siCON2-AC1VP (SEQ ID NO: 853 and SEQ ID NO: 859), cc) siCON3-AC1VP (SEQ ID NO: 854 and SEQ ID NO: 860), dd) siSOD1-063M3-AC1VP (SEQ ID NO: 855 and SEQ ID NO: 861), ee) siSOD1-047M3-AC1 VP (SEQ ID NO:850 and SEQ ID NO:848), ff)siSOD1-104M3-AC1 VP (SEQ ID NO:856 and SEQ ID NO:862), gg)siSOD1-005M3-AC1 VP (SEQ ID NO:852 and SEQ ID NO:847), hh)siSOD1-258M3-AC1 VP (SEQ ID NO:857 and SEQ ID NO:863), and ii)siSOD1-270M3-AC1 VP (SEQ ID NO: 850 and SEQ ID NO:866).

33. The oligonucleotide agent of claim 10, wherein the ACO of the oligonucleotide agent improves the stability, bioavailability, biodistribution and / or cellular uptake of the siRNA compared to an oligonucleotide agent without the ACO.

34. The oligonucleotide agent of claim 10, wherein the ACO of the oligonucleotide agent increases biodistribution of siRNA in one or more target tissues compared to an oligonucleotide agent without the ACO.

35. The oligonucleotide agent of claim 44, wherein the target tissue is selected from the group consisting of: prefrontal cortex, cerebrum, cerebellum, spinal cord, muscle, lung, eye, liver, and kidney.

36. A vector comprising the siRNA according to any one of claims 1 to 9 and / or the oligonucleotide agent according to any one of claims 10 to 35.

37. A cell comprising the siRNA according to any one of claims 1 to 9, the oligonucleotide agent according to any one of claims 10 to 35, and / or the vector according to claim 36.

38. The cell of claim 37, wherein the cell is present in a mammal.

39. The cell of claim 37, wherein the cell is present in a human.

40. The cell of claim 37, wherein the cell is a host cell in vitro, in vivo, or ex vivo.

41. A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 9, the oligonucleotide agent according to any one of claims 10 to 35, the vector according to claim 36 and / or the cell according to any one of claims 37 to 40, and a pharmaceutically acceptable carrier.

42. The pharmaceutical composition of claim 41, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers selected from aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metallic nanoparticles, non-metallic nanoparticles, bioconjugates, and polypeptides.

43. The pharmaceutical composition of claim 41, wherein the pharmaceutical composition reduces SOD1 gene transcription level or SOD1 protein level.

44. A kit comprising the siRNA according to any one of claims 1 to 9, the oligonucleotide agent according to any one of claims 10 to 35, the vector according to claim 36, the cell according to any one of claims 37 to 40, and / or the pharmaceutical composition according to any one of claims 41 to 43.

45. A method for reducing SOD1 gene transcription level or SOD1 protein level, the method comprising administering the pharmaceutical composition of any one of claims 41 to 43 to a subject.

46. ​​A method for treating or delaying the onset or progression of a neurodegenerative disease or symptom associated with SOD1 gene mutation, abnormal SOD1 gene expression, or abnormal SOD1 protein accumulation, the method comprising: The pharmaceutical composition of any one of claims 41 to 43 is administered to a subject.

47. The method of claim 46, wherein the neurodegenerative disease or disorder is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down syndrome (DS).

48. The method of claim 46, wherein the pharmaceutical composition is administered to the subject intrathecally or intracerebroventricularly.

49. The method of claim 57, wherein the ACO of the oligonucleotide agent improves the stability, bioavailability, biodistribution and / or cellular uptake of the siRNA compared to an oligonucleotide agent without the ACO.

50. Use of the siRNA according to any one of claims 1 to 9, the oligonucleotide agent according to any one of claims 10 to 35, the vector according to claim 36, the cell according to any one of claims 37 to 40, and / or the pharmaceutical composition according to any one of claims 41 to 43 in the manufacture of a medicament for treating or delaying the onset or progression of a SOD1-related neurodegenerative disease or symptom.

51. The use of claim 64, wherein the SOD1-related neurodegenerative disease or condition is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD) and Down syndrome (DS).

52. The siRNA according to any one of claims 1 to 9, the oligonucleotide agent according to any one of claims 10 to 35, the vector according to claim 36, the cell according to any one of claims 37 to 40, and / or the pharmaceutical composition according to any one of claims 41 to 43 for use in treating or delaying the onset or progression of a SOD1-related neurodegenerative disease or symptom.

53. The siRNA, oligonucleotide agent, vector, cell and / or pharmaceutical composition of claim 52, wherein the neurological disease or disorder is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD) and Down syndrome (DS).

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