Oligonucleotides that target SOD1

Abstract

This application relates to siRNA and oligonucleotide agents for the prevention or treatment of SOD1-related neurodegenerative diseases or their symptoms (e.g., amyotrophic lateral sclerosis (ALS)). The oligonucleotide agent comprises a double-stranded oligonucleotide (siRNA) that targets the mRNA region of the target gene SOD1 and a non-targeting single-stranded oligonucleotide (ACO), wherein the ACO is covalently linked to the siRNA.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acids, specifically, oligonucleotide agents that reduce the SOD1 transcription level and their pharmaceutical uses.

Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is an adult-onset lethal paralytic disease caused by the degeneration of motor neurons. ALS is characterized by the adult-onset progressive degeneration of motor neurons in the brain, brainstem, and spinal cord, and leads to death due to respiratory failure within 3 to 5 years after diagnosis. ALS is divided into familial and sporadic types depending on the presence or absence of a family history, and sporadic ALS (sALS) accounts for 90% of ALS patients. The most common mutated genes account for about 75% of ALS in the United States: chromosome 9 open reading frame 72 gene (C9orf72; 40%), superoxide dismutase 1 (SOD1; 20%), transactive response DNA-binding protein 43 (TDP43; 4%), sarcoma / fatty sarcoma metastatic fusion type (FUS / TLS; 4%). Mutations in the C9orf72 gene and the SOD1 gene also account for about 5-8% and about 2-3% of sALS, respectively. At present, there is no effective treatment for ALS, and new treatment methods are needed.

[0003] Among the known genes underlying ALS, the SOD1 gene is the main cause of fALS and has been considered an important target for ALS therapeutic drugs. The first description of ALS dates back at least to Charles Bell in 1824, but SOD1 as the first risk gene for ALS was discovered in 1993. In 1994, the first SOD1 transgenic mouse model (hSOD1 G93A ) was established, and ALS research entered a new era. SOD1 variants are thought to cause the disease, probably by gain of function, and reducing their levels is considered beneficial. Therefore, silencing the SOD1 transcription level is an important strategy for the treatment of ALS.

Summary of the Invention

Problems to be Solved by the Invention

[0004] To address this problem, the present invention provides an oligonucleotide agent that potently inhibits the expression of superoxide dismutase 1 (SOD1). The oligonucleotide agent targets SOD1 mRNA and contains short interfering RNA (siRNA) that downregulates the SOD1 protein level in cells or in vivo by RNA interference (RNAi) for treating diseases or conditions such as ALS caused by SOD1 gene mutations.

Means for Solving the Problems

[0005] In particular, the inventors have discovered that an oligonucleotide agent having a potent efficacy to knockdown SOD1 transcript and a high CNS delivery efficiency comprises the following: (a) siRNA; and (b) a non-target single-stranded auxiliary oligonucleotide (ACO), where the ACO has a length of 6 to 22 nucleotides, and the siRNA and the ACO covalently bond to form the oligonucleotide agent, regardless of the presence or absence of one or more linking components.

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

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

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

[0009] One aspect of this application provides an oligonucleotide agent capable of inhibiting / downregulating intracellular SOD1 transcription. In some embodiments, the oligonucleotide agent includes siRNA, where the sense strand of the siRNA has a nucleotide sequence having at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from SEQ ID NOs: 270-537.

[0010] In some embodiments, oligonucleotide agents comprising siRNA are provided, wherein the antisense strand of the siRNA has a nucleotide sequence having at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from SEQ ID NOs: 538-805.

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

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

[0013] In another aspect of this application, an oligonucleotide agent comprising an siRNA and a non-targeted 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 bound to a linking component. In some embodiments, the 5' end, 3' end, or one or more internal nucleotides of the ACO are bound to the linking component. In some embodiments, the siRNA and the ACO are covalently linked by the linking component. In some embodiments, the sense or antisense strand of the siRNA is covalently linked to the ACO by the linking component. In some embodiments, the linking component is one or more selected from the group consisting of ethylene glycol chains, alkyl chains, alkenyl chains, alkynyl chains, peptides, RNA, DNA, carbohydrates, thiol bonds, phosphodiesters, phosphothioates, phosphoramidates, amides, carbamates, tetrazole bonds, and benzimidazole bonds, or includes them. In some embodiments, the linking component is one or more selected from the group consisting of: a) Spacer phosphoramidite 18 (phosphoramidic acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecyl-1-yl 2-cyanoethyl ester). b) Spacer-9 (3-[2-[2-[2-[bis(4-methoxyphenyl)phenylmethoxy]ethoxy]ethoxy]ethoxy-[di(propan-2-yl)amino]phosphoranyl]oxypropanil) 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-hexaoxahenicosan-CPG.

[0015] In some embodiments, the ACO is covalently ligated to the 3' end, 5' end, or both the 3' and 5' ends of one or more internal nucleotides on the sense strand of the siRNA. In some embodiments, the ACO is covalently ligated to the 3' end, 5' end, both the 3' and 5' ends, or one or more internal nucleotides on the antisense strand of the siRNA. In some embodiments, one or more ACOs are covalently ligated to the siRNA. In some embodiments, 2–10 ACOs are covalently ligated 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, 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 of the ACO are chemically modified nucleotides. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or about 100% of the nucleotides of the sense strand of the siRNA are chemically modified nucleotides. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or about 100% of the nucleotides of the antisense strand of the siRNA are chemically modified nucleotides. In some embodiments, the chemical modification of at least one chemically modified nucleotide is a 2' sugar modification selected from one or more of the following: 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. In some embodiments, the chemical modification of at least one chemically modified nucleotide is a phosphorothioate (PS) skeleton modification. In some embodiments, ACO comprises at least one phosphorothioate (PS) skeleton modification. In some embodiments, ACO comprises 6 to 17 phosphorothioate (PS) skeleton modifications. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5'-phosphate moiety at the 5' end of the nucleotide sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate moiety at the 5' end of the nucleotide sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5-methylcytosine moiety at 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 or antisense strand of the siRNA is conjugated to one or more conjugated groups. In some embodiments, one or more conjugated groups are selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies. In some embodiments, one or more conjugated groups are selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0018] Aspects of this application relate to oligonucleotide agents comprising siRNA and untargeted ACO, wherein the sense strand has a nucleotide sequence that is at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from SEQ ID NOs. 808-827, 850-851, and 856-857, and the antisense strand of the siRNA has a nucleotide sequence that is at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from SEQ ID NOs. 828-849 and 861-863.

[0019] Aspects of this application include vectors containing the oligonucleotide agents disclosed herein.

[0020] Another aspect of this application provides cells containing oligonucleotide agents disclosed herein. In one embodiment, the cells are mammalian cells, selectively human cells. In some embodiments, the cells are host cells. The aforementioned cells may be cell lines or in vitro like cell lines, or they may be present in or obtained from the body of a mammal, such as the body of a human.

[0021] A further aspect of this application provides a pharmaceutical composition comprising an oligonucleotide agent disclosed herein. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier selected from aqueous carriers, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates, and polypeptides. Also provided herein are kits comprising an oligonucleotide agent or a pharmaceutical composition disclosed herein.

[0022] This application provides a method for reducing the transcript of the SOD1 gene or SOD1 protein, comprising administering a pharmaceutical composition disclosed herein to a subject.

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

[0024] In some embodiments, ACOs in oligonucleotide agents improve the stability, bioavailability, in vivo distribution, and / or cellular uptake of siRNAs compared to oligonucleotide agents without ACOs.

[0025] In some embodiments, ACOs in oligonucleotide agents increase the in vivo distribution of siRNA within one or more target tissues compared to oligonucleotide agents without ACOs.

[0026] In some embodiments, ACOs in oligonucleotide agents increase the in vivo distribution of siRNA in two or more target tissues compared to oligonucleotide agents without ACOs.

[0027] In some embodiments, one or more target tissues are selected from the prefrontal cortex, cerebellum, cerebrum, spinal cord, muscles, lungs, eyes, liver, and kidneys.

[0028] This application relates in part to an siRNA comprising an oligonucleotide sequence having a length in the range of 16–35 consecutive nucleotides, wherein the consecutive oligonucleotide sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homology or complementarity with equal-length portions of SEQ ID NO: 1, wherein the siRNA inhibits the mRNA transcript of the SOD1 gene by at least 80% compared to baseline SOD1 mRNA levels.

[0029] The inventors have also discovered that the optimal target sequence / sense strand of siRNA within the SOD1 gene contains a sequence having: (1) a GC content of 35% to 65%; (2) five or fewer consecutive identical nucleotides; (3) three or fewer dinucleotide repeats; and (4) three or fewer trinucleotide repeats. As a beneficial result, the target sequence (e.g., an isolated nucleic acid sequence containing the target sequence), when interacting with siRNA, can inhibit intracellular SOD1 mRNA transcript levels by at least 80% compared to baseline levels of SOD1 mRNA. Based at least in part on these findings, the disclosure features siRNA, compositions, and pharmaceutical compositions that inhibit intracellular SOD1 mRNA transcription levels by at least 80% compared to baseline levels of SOD1 mRNA. Also provided herein are methods for preventing or treating diseases or conditions induced by SOD1 gene mutations or abnormal levels of SOD1 protein in the cells of an individual, comprising administering any of the siRNA, compositions, and / or pharmaceutical compositions described herein.

[0030] Other aspects and advantages of this application will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of this application. As will be understood, other different embodiments are possible, and some of their details are modifiable in various obvious ways without departing from the disclosure. Accordingly, the drawings and description should be considered exemplary in nature and not limiting. <Reference>

[0031] All publications, patents, and patent applications described herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is explicitly and individually indicated to be incorporated by reference.

[0032] Novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are employed, and to the appended drawings (also referred to as "Figures" in this specification). [Brief explanation of the drawing]

[0033] [Figure 1]Figures 1A–1E show siRNA screening for SOD1 knockdown in vitro. Figure A shows HEK293A cells double-transfected with each siRNA double-stranded (268 in total) at concentrations of 10 or 0.1 nM for 24 hours. SOD1 expression levels were quantified by RT-qPCR using gene-specific primer sets. TBP was amplified as an internal reference used to normalize expression data. SOD1 mRNA expression values ​​for each experimental replica against mock treatment (dotted line) are shown. Mock samples were transfected in the absence of oligonucleotides. Figure 1B shows the knockdown activity and cell viability of top-performance siRNAs quantified in HEK293A cells at six incremental concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM), respectively, via RT-qPCR and PI staining. Figure 1C shows the results for both SOD1 knockdown and cytotoxicity of the top five execution siRNAs (i.e., siSOD1-063, 047, 104, 005, and 258) compared to mock treatment (dotted line). Figure 1C shows that dose-response curves were generated in SK-N-AS cells via RT-qPCR for each of the top five siRNAs at eight treatment concentrations (i.e., 6e-05, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25, and 1 nM). Data represent the mean ± standard deviation from two replicate experiments. Figure 1D shows that SK-N-AS cells were transfected for 24 hours at 0.1 nM with different chemically modified mutants or non-specific siRNA controls (siCon) for each siRNA. Knockdown activity was assessed via RT-qPCR compared to mock treatment. Figure 1E shows that dose-response curves were generated in SK-N-AS cells for each of the M3-modified mutants (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, and 258M3) via RT-qPCR. Data represent the mean ± standard deviation from three replicate experiments. [Figure 2]Figures 2A–2B show secondary screening of siRNA activity and adverse cytotoxicity in vitro. Knockdown activity and cell viability in HEK293A cells of the remaining top 25 run siRNAs identified in the initial screening are shown after transfection at six indicated concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM). SOD1 knockdown and adverse cytotoxicity were quantified by RT-qPCR in Figure 2A and PI staining in Figure 2B, respectively, compared to mock treatment. [Figure 3] Figure 3 shows dose-dependent knockdown of the top five candidate siRNAs in T98G cells. Dose-response curves were generated in T98G cells after 24-hour transfection with each of the top five siRNAs at eight treatment concentrations (i.e., 6e-05, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25, and 1 nM). SOD1 expression levels were quantified by RT-qPCR using gene-specific primer sets. TBP was amplified as an internal reference used to normalize expression data. Mean ± SD from two experimental replicates compared to a control treated in the absence of oligonucleotides are shown. [Figure 4] Figures 4A–4B show the adverse cytotoxicity of the top five siRNA candidates in vitro. SK-N-AS and T98G cells were transfected with four treatment concentrations (1.56, 6.25, 25, and 100 nM) that significantly exceeded the IC50 values ​​for SOD1 knockdown. 100 nM siCon treatment with each of the top five siRNAs served as a negative control (Neg Con). Mock samples were transfected in the absence of oligonucleotides. Unfavorable cytotoxicity was assessed 72 hours after treatment by quantifying both caspase 3 / 7 activity in Figure 4A and the metabolism of the WST-8 reagent as a marker of cell viability in Figure 4B. Data are shown as mean ± SD from two experimental replicates associated with mock treatment (dotted line). [Figure 5]Figure 5 shows the effect of different chemical modification patterns on siRNA knockdown activity in T98G cells. T98G cells were transfected for 24 hours at 0.1 nM with four different chemically modified mutants (M1, M2, M3, or M4) of each candidate siRNA or with a non-specific siRNA control (siCon). Knockdown activity was assessed via RT-qPCR compared to mock treatment. Data represent the mean ± standard deviation from two replicate experiments. [Figure 6] Figure 6 shows dose-dependent knockdown of M3-modified siRNAs in T98G cells. Dose-response curves were generated for T98G cells after 24-hour transfection with each M3-modified siRNA candidate (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, 258M3, and 270M3) at gradient treatment concentrations. SOD1 expression levels were quantified by RT-qPCR using gene-specific primer sets. TBP was amplified as an internal reference used to normalize expression data. Mean ± SD values ​​from three experimental replicates for samples treated in the absence of oligonucleotides are shown. [Figure 7]Figures 7A–7D show the knockdown activity of siRNA-ACO conjugates in vitro. Figure 7A shows a visual representation of the siRNA-ACO conjugate structure in which a 14-base ACO (i.e., spacer-9 linker) is conjugated to the 3' end of the sense strand via a short L9 linker. Figure 7B shows HEK293A and TG cells transfected with exemplary siRNA-ACO (i.e., siSOD1-005M3-AC1) or AC1 for only 24 hours at concentrations of 0.25 or 2.5 nM. Mock samples were transfected in the absence of oligonucleotides. Treatment with siCon was used as a negative control for knockdown activity. SOD1 expression levels were quantified by RT-qPCR using gene-specific primer sets. TBP was amplified as an internal reference used to normalize expression data. SOD1 mRNA expression values ​​for each experimental replication against mock treatment (dotted line) are shown. Figure 7C shows that dose-response curves were generated in T98G cells via RT-qPCR comparing the siRNA knockdown activity of siSOD1-005M3 with AC1 (siSOD1-005M3-AC1) or siSOD1-005M3 without AC1 junction (siSOD1-005M3-AC1) 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). Figure 7D shows that the knockdown activity of siRNA-ACO without (siSOD1-005M3-AC1VP) or (siSOD1-005M3-AC1)5'VP modification was compared to the published ASO sequence in both sequence and chemical (i.e., ASOSOD1, SEQ ID NO: 864) compared to the published ASO sequence (T. Miller et al., New England Journal of Medicine 383, 109-119 (2020)), and dose-response curves were generated. [Figure 8]Figures 8A-8B show the in vitro knockdown activity of candidate siRNA-ACO drugs. Dose-response curves were constructed in SK-N-AS (Figure 8A) and T98G (Figure 8B) cells for each 5'VP-modified siRNA-ACO (i.e., siSOD1-063M3-AC1VP, 047M3-AC1VP, 104M3-AC1VP, 005M3-AC1VP, 258M3-ACVP, and 270M3-AC1VP) at gradient concentrations. SOD1 expression levels were quantified by RT-qPCR using gene-specific primer sets. TBP was amplified as an internal reference to normalize expression data. SOD1 mRNA expression values ​​against mock transfection are shown. Data represent the mean ± standard deviation from two replicate experiments. [Figure 9] Figures 9A–9B show the adverse cytotoxicity of siRNA-ACO drug candidates in vitro. SK-N-AS and T98G cells were transfected with lead siRNA-ACO conjugates (i.e., siSOD1-063M3-AC1VP, 047M3-AC1VP, 104M3-AC1VP, 005M3-AC1VP, 258M3-AC1VP) at gradient treatment concentrations exceeding their IC50 values ​​(i.e., 1.56, 6.25, 25, and 100 nM). Treatment with 100 nM siCon served as a negative control. Mock samples were transfected in the absence of oligonucleotides. Unfavorable cytotoxicity was assessed 72 hours after treatment by quantifying both caspase 3 / 7 activity in Figure 9A and the metabolism of WST-8 reagent as a marker of cell viability in Figure 9B. Data are shown as mean ± SD from two experimental replicates associated with mock treatment (dotted line). [Figure 10]Figure 10 shows siRNA-ACO activity in CNS tissue of hSOD1G93A mice. Adult hSOD1G93A mice were administered intraventricularly (ICV) with each siRNA-ACO candidate drug (i.e., siSOD1-047M3-AC1VP and 005M3-AC1VP) at a fixed molecular weight (20 nmole). To establish baseline expression, aCSF treatment alone was used as a solvent control, and a nonspecific siRNA-ACO (i.e., siCON2-AC1VP) was used as a negative control. Knockdown activity was quantified by RT-qPCR in brain (i.e., prefrontal cortex, cerebellum, cerebrum), spinal cord (i.e., cervical, thoracic, and lumbar vertebrae), and peripheral (i.e., liver) tissues 14 days after administration of fixed molecular weight (20 nmole) ICV using siRNA-ACO candidates (i.e., siSOD1-047M3-AC1VP and 005M3VP) or their unconjugated derivatives (i.e., siSOD1-047M3VP and 005M3VP). Expression data were normalized by amplification using mouse Tbp (mTbp or Tbp) as an internal reference. The mean expression value ± SD (n=2-6 mice / group) of human SOD1 (hSOD1 or SOD1) against aCSF treatment is shown. The gray dotted line indicates 80% knockdown compared to baseline (dashed line). [Figure 11]Figures 11A-11B show the dose-dependent relationship between siRNA-ACO knockdown activity and tissue accumulation in different CNS tissues. Adult hSODG93A mice were treated with either siSOD1-047M3-AC1VP (Figure 11A) or siSOD1-005M3-AC1VP (Figure 11B) at indicated doses (i.e., 50, 100, 200, or 400 μg) by ICV injection. The knockdown activity of hSOD1 was quantified via RT-qPCR in selected CNS tissues (i.e., cerebellum, cerebrum, and spinal cord) 14 days post-treatment. Animals treated with aCSF alone represent baseline expression levels and detectable drug amounts in the absence of siRNA-ACO treatment (0 μg). Figures 11A-11B show knockdown activity as the percentage inhibition of SOD1 compared to baseline (0 μg). Drug concentration is expressed as the amount of siRNA-ACO relative to tissue sample mass (μg / g). Data represent mean ± SD (n=3-4 mice / group). [Figure 12] Figures 12A-12C show that siRNA-ACO delays disease onset and extends animal survival via a single ICV injection. Adult hSOD1G93A mice were treated with siSOD1-047M3-AC1VP or siSOD1-005M3-AC1VP via ICV injection at postnatal day 85 or postnatal day 60, respectively, at the prescribed dose (i.e., 50, 100, 200, or 400 μg). Disease progression was monitored in Figure 12A by plotting growth rate (i.e., percentage change in body weight) against day 1 (dotted line). Disease onset is plotted as the percentage of animals with peak body weight in each treatment group in Figure 12B. Animal survival is plotted as the percentage of surviving animals in each treatment group in Figure 12C. The number of animals (n) is shown in each graph. [Figure 13]Figures 13A-13D show pathogenic SNPs at the target sites of candidate lead siRNA-ACOs. Figures 13A-13B show the locations of pathogenic SNPs within the target sites of siSOD1-005M3-AC1VP (Figure 13A) and siSOD1-047M3-AC1VP (Figure 13B). Guide chain sequences containing a complementary "seed" region (highlighted in gray) to the target site of the hSOD1 transcript containing the indicated pathogenic SNPs are shown. Nucleotide mutations are shown in bold in italics, and "R" indicates a purine substitution. Figure 13C shows that luciferase reporter constructs containing either consensus sequences or pathogenic mutations (i.e., P.E22G and P.F21C) (i.e., pLucSOD1, pLucP.E22G, pLucP.F21C) were co-transfected in HEK293A cells at a concentration of 18 nM with siSOD1-047M3-AC1VP or a nonspecific scrambled control (siCON2-AC1VP). Dose-response curves of luciferase activity were generated after co-transfection with siSOD1-047M3-AC1VP at concentrations shown in Figure 13D (i.e., 0.03, 0.07, 0.22, 0.67, 2.0, 6.0, and 18 nM). Data represent the mean ± SEM from two experimental replications of samples treated in the absence of siRNA. Statistical significance (*) was determined using Tukey's multiple comparison test to compare the mean values ​​of each data point within the three dose-response curves. [Figure 14]Figures 14A–14C show that two doses of siRNA-ACO delay disease onset and extend survival. Adult male and female hSOD1G93A mice were treated twice with siSOD1-047M3-AC1VP at indicated doses (i.e., 75, 150, or 300 μg) by intrathecal (IT) injection into PND68 and PND100. A non-specific control (i.e., siCON3-AC1VP) and ASOSOD1 were administered at 150 μg / injection. aCSF administration was used as a solvent control group. Disease progression was monitored in Figure 14A by plotting body weight in grams (g) compared to background animals (wild-type, WT). Disease onset was plotted as the percentage of animals with peak body weight in each treatment group in Figure 14B. Animal survival rates were plotted as the percentage of surviving animals in each treatment group in Figure 14C. The number of animals (n) is shown in each graph. [Figure 15] Figures 15A–15D show that siRNA-ACO treatment improves motor function in hSOD1G93A mice. Male and female hSOD1 mice were treated twice with siRNA-ACO at the indicated doses (i.e., 75, 150, or 300 μg) via IT to PND68 and PND100. Male and female hSOD1G93A mice were used as subjects in a daytime open-field study. The total distance traveled by each animal was autonomously recorded in centimeters (cm) over 15 minutes. Figure 15A plots the mean distance traveled ± SD for each treatment group. Grip strength was assessed separately for male and female animals by treatment group. Grip strength tests were performed in triplicates, and the mean value was recorded for each animal. The data are plotted in grams (g) as the mean value ± SD of grip strength for each treatment group in Figure 15B. Animal fatigue and coordination were assessed for 5 minutes using a rotarod test. The experiment was conducted in three sets, and the longest latency time to fall for each animal was recorded in seconds, as shown in Figure 15C. Motor function was scored using the ALS TDI Neurological Score (NS) scale for all animals before the open field, rotarod, and / or grip strength tests. The experiment was conducted in three sets, and the longest latency time to fall for each animal was recorded in seconds (Figure 15D). The mean NS ± SD is the value at the indicated time in each treatment group. [Figure 16] Figure 16 shows a temporal comparison of rotarod performance for each individual after siRNA-ACO treatment as shown in Figure 14. For each animal in the corresponding treatment group at the initial time (i.e., PND90), the waiting time to fall measured in the rotarod test (seconds) is shown, compared to the performance at the final time (i.e., the last time before death, see Table 8). All tests were performed in sets of three, and the longest latency time was recorded for each animal. [Figure 17] Figure 17 shows the knockdown activity of siRNAs against SOD1 mRNA expression in HeLa cells. The siRNAs shown (i.e., RD-15757, RD-18972, RD-12500, RD-18973, RD-18948, and RD-18949) were directly added to the culture medium containing HeLa cells at 1500 nM for 3 days. Cells were transfected in the absence of oligonucleotides as a mock treatment (not shown). RD-11566 (dsCon2M3v) was used as a non-targeted double-strand control. SOD1 mRNA levels were quantified by two-step RT-qPCR using gene-specific primer sets in each PCR reaction. TBP was amplified as an internal reference. The values ​​(y-axis) represent the SOD1 mRNA levels after mock treatment normalized with TBP (mean ± SEM of 3 replicate transfection wells). [Figure 18]Figures 18A-18B show the knockdown activity of siRNAs against SOD1 mRNA expression in SK-N-AS cells. The siRNAs shown (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 concentrations shown (i.e., 0.0002, 0.001, 0.0039, 0.0156, 0.0625, 0.25, 1, and 4 nM) for 24 hours. Cells were transfected in the absence of oligonucleotides as a mock treatment (not shown). RD-11566 (dsCon2M3v) was used as a non-targeted double-strand control (not shown). Figures 18A-18B show the SOD1 mRNA levels quantified by two-step RT-qPCR using gene-specific primer sets in individual PCR reactions. TBP was amplified as an internal reference. The values ​​(y-axis) represent the SOD1 mRNA levels after mock treatment normalized by TBP (mean ± SEM of 3 replicate transfection wells). [Modes for carrying out the invention]

[0034] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous modifications, alterations, and substitutions can be made to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be adopted.

[0035] Aspects of this application include a method for treating amyotrophic lateral sclerosis (ALS) by administering an effective dose of an oligonucleotide agent containing SOD1-targeted siRNA. The oligonucleotide agent inhibits SOD1 mRNA transcription via the RNA silencing mechanism (RNAi). The inventors have developed SOD1 siRNAs for use in the treatment of ALS that have potent inhibitory effects, improved delivery, biodistribution, bioavailability, and other pharmacological properties.

[0036] This application is based on research on oligonucleotide agents, compositions, and methods in which target gene oligonucleotides (such as siRNA) combined with oligonucleotide delivery vehicles (ODVs) can downregulate / reduce gene expression and improve the therapeutic effect of genetic diseases. The term "oligonucleotide delivery vehicle (ODV)" refers to a structure that facilitates the introduction or uptake of molecules into cells, tissues, or organs of an organism by conjugating "auxiliary oligonucleotides (ACOs)" to molecules, such as double-stranded oligonucleotides.

[0037] The inventors have found that several siRNA double-stranded sequences exhibit superior inhibitory efficacy against SOD1 transcripts compared to sequences with identical or similar target sequences in the prior art. The inventors have also found that chemical modifications to siRNA improve its in vitro activity. Surprisingly, by conjugating with single-stranded accessory oligonucleotides (ACOs, or ODVs), oligonucleotide agents (i.e., ODV-siRNA or siRNA-ACO) achieved CNS delivery, desired biodistribution, and bioavailability in CNS tissues when administered to either brain or spinal cord tissue via intracerebral injection (ICV) and / or intrathecal injection (IT). definition In this specification, the following terms are defined:

[0038] All numerical ranges provided herein include all narrow numerical ranges that fall within such broad numerical ranges, as if all such narrow numerical ranges were explicitly described herein.

[0039] The transitional terms / phrases (and their grammatical variations) “comprises,” “comprises,” and “comprise” include the phrases “consisting essentially of,” “consisting essentially of,” “consisting of,” and “consisting of,” and are interchangeable throughout this application. The open term “comprise” also optionally includes the closed term “consisting of.” As used herein, the terms “contains,” “have,” and “consist of” are used synonymously, and these terms and their variants are intended to be interpreted non-restrictively.

[0040] The term "amyotrophic lateral sclerosis" or "ALS" includes, but is not limited to, familial ALS (FALS), sporadic ALS (sALS), Lou Gehrig's disease, diseases associated with the mutant gene chromosome 9 open reading frame 72 (C9 orf72; 40%), superoxide dismutase 1 (SOD1; 20%), transactive response DNA-binding protein 43 (TDP43; 4%), and intrasarcoma fusion / liposarcoma fusion (FUS / TLS; 4%).

[0041] As used herein, the term “target gene” may refer to a nucleic acid sequence in the form of DNA, RNA or DNA / RNA hybrid, transgene, viral or bacterial sequence, chromosome or extrachromosomal gene, which is naturally present in an organism and / or can be transfected or stably incorporated into one or more cells and / or their chromatin. A target gene may be a protein-coding gene or a non-protein-coding gene (such as a microRNA gene or a long non-coding RNA gene), or a transcript of a protein-coding gene, e.g., message RNA (mRNA) or complementary DNA (cDNA) of a protein-coding gene. “Target sequence,” “target site,” or “target” refers to a contiguous oligonucleotide sequence in the mRNA or cDNA sequence of a target gene that is homologous or complementary to the sense or antisense strand of an siRNA having or not having one or more mismatch base pairs, e.g., a contiguous oligonucleotide sequence in the mRNA or cDNA sequence of a target gene.

[0042] As used herein, the terms “SOD1” and “SOD1 gene” are interchangeable and refer to the gene encoding the SOD1 protein, preferably a mammalian gene, more preferably a human gene. As used herein, the term “SOD1 mRNA” refers to the message RNA (mRNA) produced from the expression of the SOD1 gene or the transcription of the SOD1 gene. As used herein, the term “SOD1 cDNA” refers to the complementary DNA (cDNA) produced from the reverse transcription of the SOD1 mRNA. As used herein, the term “SOD1 protein” refers to the protein produced from the expression of the SOD1 gene or the translation of the SOD1 mRNA.

[0043] As used herein, the terms “baseline expression of the SOD1 gene” or “baseline level of SOD1 mRNA” refer to the expression of the SOD1 gene in a parallel reference (such as one or more cells) without or before siRNA treatment.

[0044] The terms “oligonucleotide agent” or “oligonucleotide” can be used interchangeably and refer to polymers of nucleotides, including, but not limited to, single-stranded or double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl and ribosyl moieties, and modified naturally occurring or unnaturally occurring frameworks for such oligonucleotides. Oligonucleotide agents for inhibiting mRNA transcription of target genes as described herein are small inhibitory nucleic acid molecules (siRNA), antisense oligonucleotide molecules (ASO), or oligonucleotide delivery carrier (ODV)-conjugated siRNA molecules (siRNA-ACO).

[0045] As used herein, the terms “oligonucleotide chain,” “strand,” and “oligonucleotide sequence” refer to short nucleotide sequences of less than 35 bases (including nucleotides of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) and can be used interchangeably. In non-limiting examples, the length of the chain may be any length ranging from 16 to 25 nucleotides.

[0046] Where used herein, the terms “subject” and “individual” are used interchangeably herein to mean any living organism that can be treated with the agent of this application. The term “patient” refers to a human subject or individual, including infants, children and adults, who are subject to disclosure.

[0047] The “therapeutic effective amount” of a composition is an amount sufficient to achieve a desired therapeutic effect and therefore does not require a cure or complete remission. In embodiments of this application, the therapeutic effect is an improvement in any of the disease indicators, and the therapeutic effect amount is sufficient to cause an improvement in a clinically significant condition / symptom in the treated individual. The expressions “therapeutic effective amount” and “effective amount” are used herein to mean an amount sufficient to reduce, most preferably prevent, a clinically significant deficit in activity, function and response in the treated individual by at least about 15%, preferably at least 50%, more preferably at least 90%, or by at least about 50%, at least about 100%, at least about 200%, more preferably at least about 500%.

[0048] The effective dose may vary depending on factors such as the subject's physique and weight, the type of disease, or the specific drug being administered. For example, the choice of drug can affect what constitutes the "effective dose." A person skilled in the art could study the factors included herein and make a determination regarding the effective dose of the drug in this application without conducting excessive experiments.

[0049] The administration method may affect what constitutes the effective dose. The agents of this application can be administered to a subject either before or after the diagnosis or manifestation of a disease. Furthermore, they can be administered in several divided doses, at staggered times, daily, weekly, monthly, quarterly, sequentially, by continuous infusion, or by bolus injection. In addition, the dosage of one or more agents of this application may be increased or decreased proportionally, as indicated by the urgency of the therapeutic or preventive situation.

[0050] As used herein, the terms “treat,” “administer,” or “procedure” have the meanings commonly understood in medical technology and therefore include any beneficial or desired clinical outcome, without requiring a cure or complete remission. Non-limiting examples of such beneficial or desired clinical outcomes include an extension of survival compared to the expected survival without treatment, and relief of one or more symptoms including: weakness and atrophy of proximal skeletal muscles, inability to sit or walk independently, dysphagia, dyspnea, etc.

[0051] In this specification, “prevention” or “delay” of a disease means preventing the complete onset of the disease.

[0052] The term "biological sample" refers to any tissue, cell, fluid, or other substance derived from an organism (e.g., a human subject). In some embodiments, the biological sample is serum or blood.

[0053] As used herein, the terms “identity” or “homology” refer to the sequence similarity between one oligonucleotide strand (sense strand or antisense strand) of an siRNA and the coding strand or template strand in a region of the target gene. As used herein, “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 differ from the reference sequence. To determine the percentage identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Next, the nucleotides at the corresponding nucleotide positions are compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two arrays is a function of the number of identical positions the arrays share, taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap.

[0054] Sequence comparison and determination of percentage identity between two sequences can be achieved using mathematical algorithms, such as the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program of the GCG software package (www.gcg.com). Percent identity between two nucleotide sequences can be determined using the E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) algorithm incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. It is understood that the molecules described herein may have additional conserved or non-essential nucleic acid substitutions that do not substantially affect their function.

[0055] In embodiments of this application, one target gene is SOD1. A “target sequence” refers to a sequence fragment in which the sense strand or antisense oligonucleotide of an siRNA is homologous or complementary. For example, in some embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a human SOD1 transcript.

[0056] As used herein, the term “non-target” means that the referenced accessory oligonucleotide (ACO) (e.g., siRNA, saRNA, etc.) that binds to the target oligonucleotide is not specifically complementary to the target sequence on which the target oligonucleotide functions, and / or the referenced oligonucleotide (i.e., the ACO) does not share the same target sequence on which the target oligonucleotide (e.g., siRNA, saRNA, etc.) specifically functions. The target oligonucleotides disclosed herein are nucleic acid sequences that are specifically complementary to the target sequence or region. In some embodiments, the term “non-target oligonucleotide” may include any referenced oligonucleotide other than the “target sequence.” In some cases, “specifically complementary” may mean that the complementarity between the target oligonucleotide and the target sequence or region is at least about 95%. Non-target oligonucleotides ("ACOs") do not induce biological activity through known mechanisms, nor do they induce ASO (i.e., "mixer" or "gapmer") activity on a specific subject, subject organ, subject tissue, or complementary nucleic acid sequence (i.e., mRNA) within the subject's cells when administered. Non-target oligonucleotides (i.e., ACOs) facilitate the introduction of the target oligonucleotide (e.g., siRNA, saRNA, etc.) to which they are bound when an oligonucleotide conjugate is administered, into a specific subject, subject organ, subject tissue, subject cell, or subject cell nucleus.

[0057] As used herein, the term “gapmer” refers to a short DNA antisense oligonucleotide (ASO) structure having modified RNA segments on both sides of a central DNA structure. In some embodiments, at least one of the modified RNA segments comprises a lock nucleic acid (LNA) and one or more modified nucleotides selected from 2'-OMe or 2'-F modified nucleotides to increase affinity to a target, increase nuclease resistance, decrease immunogenicity, and / or reduce toxicity. In some embodiments, the gapmer comprises at least one nucleotide modified with a phosphorothioate (PS) group. In some embodiments, the gapmer is designed to hybridize to a target portion of RNA and silence the gene transcript via induction of RNase H cleavage. As an example, the ASO drug “Tofersen” is a gapmer that knocks down SOD1 mRNA for the treatment of ALS. A possible example of a dual-acting oligonucleotide (DAO) with the gapmer ASO disclosed herein may be “siSOD1-Tofersen conjuagte”.

[0058] As used herein, the term “mixer” refers to an antisense oligonucleotide (ASO) characterized as a mixed structure of DNA and a chemically modified nucleic acid analog. Optionally, a mixer consists of a fully modified nucleotide or nucleic acid analog. In some embodiments, a mixer is designed to bind to and mask a complementary RNA sequence to sterically inhibit a protein, factor, or other RNA from interacting with a target RNA. In some embodiments, a mixer is designed to alter premRNA splicing by displacing a spliceosome. In some embodiments, a mixer is designed to bind to and sequester a microRNA (miRNA), and the mixer is referred to by the alternative name “antagomir” or “anti-miR”.

[0059] As used herein, the terms “sense strand” and “sense oligonucleotide strand” are interchangeable. The sense oligonucleotide strand of an siRNA molecule may include, for example, a first nucleic acid strand of the siRNA containing a sequence in the human genome or a fragment of a target gene sequence.

[0060] As used herein, the terms “antisense strand” and “antisense oligonucleotide strand” are interchangeable. The antisense oligonucleotide strand of an siRNA molecule may, for example, include a second nucleic acid strand in the siRNA duplex that is complementary to the sense oligonucleotide strand. The antisense strand of siRNA may be complementary to a contiguous fragment of the target gene sequence and can bind to contiguous fragments having 0, 1, 2, 3, 4, or 5 mismatches without affecting the function of the siRNA.

[0061] As used herein, the term “coding strand” refers to the DNA strand of a target gene that is not transcribed, whose nucleotide sequence is identical to that of the RNA produced by transcription (in RNA, the T in DNA is replaced by U). The coding strand of a double-stranded DNA sequence of a target gene promoter as described herein refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0062] As used herein, the term “template strand” refers to another strand of double-stranded DNA of the target gene that is complementary to the coding strand and can be transcribed as a template to RNA complementary to the transcribed RNA bases (AU, GC). During transcription, RNA polymerase binds to the template strand and moves along the 3'→5' direction of the template strand, catalyzing RNA synthesis in the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter as described herein refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

[0063] As used herein, the term “overhang” refers to an oligonucleotide having a non-base-paired nucleotide (maybe multiple) at the oligonucleotide chain end (5' or 3') resulting from another chain extending beyond one of the chains in the siRNA. A single-stranded region extending beyond the 3' and / or 5' ends of a double helix is ​​called an overhang. In some embodiments, the overhang is 0–6 nucleotides long. An overhang of 0 nucleotides is understood to mean no overhang.

[0064] As used herein, the term “natural overhang” refers to an overhang consisting of one or more nucleotides identical or complementary to the corresponding position on the target sequence. A natural overhang on the sense strand consists of one or more nucleotides identical to the corresponding position on the mRNA target. A natural overhang on the antisense strand consists of one or more nucleotides complementary to the corresponding position on the mRNA target.

[0065] As used herein, the terms “gene silencing,” “knockdown of gene expression,” “gene downregulation,” “reduced gene expression,” and “downregulation of gene expression” are interchangeable and refer to a reduction or downregulation of the transcription, translation, expression, or activity of a particular nucleic acid sequence, determined by measuring the transcription level, mRNA level, protein level, enzyme activity, methylation status, chromatin status or arrangement, translation level, or activity or status of the gene in a cellular or biological system. These activities or states can be determined directly or indirectly. Furthermore, “gene downregulation” 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, gene downregulation may occur at the transcriptional or post-transcriptional level, reducing transcription to RNA, and RNA levels may be translated to proteins at levels lower than baseline, thereby reducing protein expression.

[0066] As used herein, the terms “short interfering RNA,” “siRNA,” and “silencing RNA” are interchangeable and refer to ribonucleic acid molecules that can downregulate, knock down, or silence target gene expression. These may also refer to double-stranded nucleic acid molecules. siRNA primarily binds to target mRNA in the cytoplasm and downregulates gene expression post-transcriptionally via the RNA interference (RNAi) mechanism.

[0067] siRNA can contain either native or chemically modified nucleotides. Modifications can confer increased nuclease stability and / or increased intracellular potency. Examples of chemical modifications include phosphorothioate backbone modifications, 2'-deoxyribonucleotides, 2'-OCH3-containing ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethylribonucleotides, and combinations thereof. siRNA can have varying lengths (e.g., 10-200 bps) and structures (e.g., hairpin, single-stranded / double-stranded, bulge, nick / gap, mismatch) and are processed intracellularly to knock down target mRNA. Double-stranded siRNA may have the same number of nucleotides on each strand (blunt end) or asymmetric ends (overhang). For example, a 1-2 nucleotide overhang may be present on the sense and / or antisense strand, and also on the 5'- and / or 3'-ends of a given strand.

[0068] siRNA molecules typically have lengths of approximately 10–60, 10–50, 15–30, 17–29, 18–28, 19–27, 20–26, 21–25, and 22–24 base pairs, and are typically about 15, 16, 17, 18, 19, 20, 21, 23, 25, 30, 40, or 50 base pairs. Furthermore, the terms “small interfering RNA,” “silencing RNA,” and “siRNA” include non-ribonucleotide nucleic acids, including but not limited to modified nucleotides or analogues.

[0069] The term "equal-length portion" refers to a portion of a sequence that, when compared to a target sequence (e.g., a sequence of oligonucleotides from siRNA), has the same length (equal number of base pairs) as the target sequence.

[0070] As used herein, the term “sequence-specific mode” refers to a mode of joining or hybridizing two nucleic acid fragments according to their nucleotide sequence, such as Watson-Crick base sequences (A to T, A to U, C to G, etc.) or other modes that enable the formation of a double helix (such as Hoogsteen or reverse Hoogsteen base pairing).

[0071] As used herein, the terms “isolated target site,” “target site,” and “isolated polynucleotide” are interchangeable and refer herein to nucleic acid target sites to which siRNA is complementary or hybridizes. For example, an isolated nucleic acid sequence of a target site may include a nucleic acid sequence to which a region of siRNA is complementary or hybridizes.

[0072] As used herein, the term “complementarity” refers to the ability to form base pairs between two oligonucleotide chains. Base pairs are generally formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains can be paired by the Watson-Crick method (AT, AU, CG, etc.) or by other methods that enable the formation of double helices (such as Hoogsteen or reverse Hoogsteen base pairing).

[0073] Complementarity can be categorized into complete and incomplete complementarity. "Complete complementarity" or "100% complementarity" means that each nucleotide in the first oligonucleotide chain can form a hydrogen bond with the corresponding nucleotide in the second oligonucleotide chain within the double-stranded region of the siRNA molecule, and there are no "misspairs" in the base pairs. "Incomplete complementarity" or "mismatch" means that not all nucleotide units in the two chains are bonded to each other by hydrogen bonds. For example, in the case of two oligonucleotide chains, each 20 nucleotides long in the double-stranded region, if only two base pairs can be formed by hydrogen bonds in this double-stranded region, the complementarity of the oligonucleotide chains is 10%. In the same example, if 18 base pairs in this double-stranded region can be formed by hydrogen bonds, the oligonucleotide chains have 90% complementarity. Substantial complementarity refers to at least approximately 75%, 79%, 80%, 85%, 90%, 95%, or 99% complementarity.

[0074] As used herein, “ODV” and “oligonucleotide delivery vehicle” are used interchangeably and refer to oligonucleotide molecules including double-stranded or double-stranded RNA (e.g., siRNA or saRNA) and ACOs covalently bound to the double-stranded RNA via a linker, as described in more detail below.

[0075] As used herein, “covalent linker,” “linker,” and “binding component” are used interchangeably and refer to an organic moiety that links two parts of a compound. For example, one or more single-stranded oligonucleotides (e.g., ACOs) and dsRNAs (e.g., siRNAs), or two dsRNAs, are covalently linked by, for example, a nucleic acid linker, a peptide linker, etc., and include disulfide linkers.

[0076] As used herein, the term “synthesis” refers to the method by which oligonucleotides are synthesized and includes any means by which RNA can be synthesized or chemically modified, such as chemical synthesis, in vitro transcription, and vector expression.

[0077] The terms “oligonucleotide modulator” and “oligonucleotide agent” can be used interchangeably and refer to an oligonucleotide-containing substance comprising at least one or more siRNAs of the present invention, having activity to modulate target gene expression or activity to enhance the effect of siRNA, and may further comprise siRNA(s) conjugates, complexes, or mixed with other oligonucleotide moieties / components (such as ASOs or accessory oligonucleotides (ACOs)) or non-oligonucleotide moieties / components. In certain embodiments, the oligonucleotide modulator comprises RNA (such as siRNAs of the present invention), DNA, BNA, LNA, GNA, or PNA.

[0078] As used herein, the term "LNA" refers to a locked nucleic acid in which a 2'-oxygen atom and a 4'-carbon atom are linked by an extra bridge. As used herein, the term "BNA" refers to a 2'-O and 4'-aminoethylene crosslinked nucleic acid that may contain a 5-membered or 6-membered crosslinked structure with NO bonds. As used herein, the term "PNA" refers to a nucleic acid mimetic having a pseudopeptide backbone consisting of N-(2-aminoethyl)glycine units having nucleic acid bases linked to glycine nitrogen via a carbonylmethylene linker. As used herein, the term "GNA" is also called glycerol nucleic acid and is a nucleic acid similar to DNA or RNA, but with a different sugar-phosphodiester backbone composition, using propylene glycol instead of ribose or deoxyribose.

[0079] In this application, singular forms such as "a" and "this" include multiple subjects unless explicitly specified in the context.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by experts in the technical field to which this application pertains. siRNA

[0081] Embodiments of this application are based in part on the remarkable discovery that oligonucleotide agents (e.g., siRNA, also referred herein as “SOD1 gene siRNA” or “SOD1 siRNA”) can inhibit or downregulate the expression of the intracellular SOD1 gene. The reduction in functional SOD1 gene transcript after administration of the oligonucleotide agents of the present invention can achieve a significant reduction or downregulation of SOD1 mRNA and SOD1 protein levels in cells or mammals.

[0082] In particular, the inventors have found that a functional oligonucleotide agent capable of inhibiting the expression of superoxide dismutase 1 (SOD1) comprises a short interfering RNA (siRNA), where the siRNA comprises a sense strand and an antisense strand forming a double helix, where the antisense strand comprises a nucleotide sequence containing at least 10 consecutive nucleotides having a 0, 1, 2, or 3 mismatch, and has at least 85% nucleotide sequence complementarity or homology to a portion of the nucleotide sequence of SOD1 mRNA.

[0083] As a beneficial result, a target sequence (e.g., an isolated nucleic acid sequence containing 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 siRNA. In one embodiment, SOD1 mRNA is reduced by at least 80%. Based at least in part on these findings, this application features siRNA, compositions, and pharmaceutical compositions for inhibiting / downregulating SOD1 mRNA transcripts by at least 10% compared to baseline levels of SOD1 mRNA. Furthermore, this specification also provides a method for preventing or treating a disease or condition induced in an individual by overexpression of the SOD1 protein, mutation in the SOD1 gene, and / or high or abnormal SOD1 levels, the method comprising administering any of the siRNAs, compositions, and / or pharmaceutical compositions described herein to the individual.

[0084] In certain embodiments, the oligonucleotide sequences disclosed herein have at least 85%, at least 90%, or at least 95% homology or complementarity with nucleotide sequences selected from SEQ ID NOs: 2-269. In certain embodiments, the sense strand of the oligonucleotide sequences disclosed herein has at least 85%, at least 90%, or at least 95% homology with nucleotide sequences selected from SEQ ID NOs: 2-269. In certain embodiments, the antisense strand of the oligonucleotide sequences disclosed herein has at least 85%, at least 90%, or at least 95% complementarity with nucleotide sequences selected from SEQ ID NOs: 2-269.

[0085] Embodiments of this application are also partly based on the finding that SOD1 mRNA inhibitory oligonucleotide agents include siRNA having a sense strand having at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from SEQ ID NOs: 270-537.

[0086] In some other embodiments, the SOD1 mRNA inhibitory oligonucleotide agent comprises an siRNA having an antisense strand having at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from SEQ ID NO: 538-805.

[0087] In some embodiments, the SOD1 mRNA inhibitory oligonucleotide agent comprises an siRNA, where the sense strand and antisense strand of the siRNA independently have nucleotide sequences having at least 85%, at least 90%, or at least 95% homology to nucleotide sequences selected from SEQ ID NO: 808-849, 867, and 868.

[0088] The oligonucleotide siRNAs described herein contain an RNA chain (antisense chain) having a length of 60 nucleotides or less, i.e., 15–40 nucleotides, and generally 19–25 nucleotides, which is substantially complementary to at least a portion of the mRNA transcript of the SOD1 gene. The use of these siRNAs enables targeted degradation of mRNA of genes involved in pathological conditions related to SOD1 expression in mammals. Low-dose SOD1 siRNAs, in particular, can specifically and efficiently mediate RNAi and significantly inhibit the expression of the SOD1 gene. Using cell-based assays, the inventors have demonstrated that SOD1-targeting siRNAs specifically and efficiently mediate RNAi, resulting in significant inhibition of SOD1 gene expression. Therefore, methods and oligonucleotide agents containing these siRNAs are useful for treating pathological processes that can be mediated by SOD1 downregulation, such as diseases that cause elevated SOD1 levels, such as amyotrophic lateral sclerosis (ALS). The following detailed description discloses the preparation and use of oligonucleotide agents, including siRNA, for inhibiting the expression of the SOD1 gene, as well as oligonucleotide agents and methods for treating diseases and disorders caused by the expression of this gene.

[0089] In one embodiment, RNA interference agents include single-stranded RNA that interacts with a target RNA sequence to induce cleavage of the target RNA. While we do not wish to be bound by theory, long double-stranded RNA introduced into plant or invertebrate cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer is an enzyme similar to ribonuclease III that processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Subsequently, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unravel the siRNA double helix, allowing a complementary antisense strand to recognize the target (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Therefore, in one embodiment, the present invention relates to single-stranded RNA that promotes the formation of a RISC complex, resulting in the silencing of a target gene.

[0090] In some embodiments, the sequence oligonucleotide sequence of the siRNA has 5 or fewer nucleotide differences or mismatches with respect to the isolength portion of the SOD1 mRNA, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches. In some embodiments, the sequence oligonucleotide sequence of the sense strand of the siRNA has 3 or fewer nucleotide differences or mismatches with respect to the isolength portion of the SOD1 mRNA, i.e., 3, 2, 1, or 0 nucleotide differences or mismatches. In some embodiments, the sequence oligonucleotide sequence of the antisense strand of the siRNA has 3 or fewer nucleotide differences or mismatches with respect to the isolength portion of the SOD1 mRNA, i.e., 3, 2, 1, or 0 nucleotide differences or mismatches.

[0091] In some embodiments, the SOD1 mRNA disclosed herein does not contain nucleotide mutations. 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 on the targeting site of the siRNA. In some embodiments, the SOD1 mRNA disclosed herein contains at least one nucleotide mutation upstream and / or downstream of the targeting site of the siRNA.

[0092] In some embodiments, the difference or mismatch is located in the middle or 3' end of the oligonucleotide sequence of the siRNA. Methods and principles of siRNA molecular design are well known to those skilled in the art, for example, Place et. al., Molecular Therapy-Nucleic Acids (2012) 1, e15; and Li et. al., PNAS, 2006, vol. 103, no. 46, 17337-17342, which are incorporated herein by reference in their entirety.

[0093] In some embodiments, the siRNA disclosed herein comprises a sense strand and an antisense strand. The sense strand and antisense strand include complementary regions capable of forming a double-stranded nucleic acid structure that reduces intracellular SOD1 transcript levels via an RNAi mechanism. As used herein, the RNAi mechanism (also called RNA interference) refers to a mechanism by which a double-stranded nucleic acid structure sequence-specifically downregulates a target gene at the transcriptional level. The sense strand and antisense strand of the siRNA may reside on two different nucleic acid strands or on a single nucleic acid strand (e.g., a contiguous nucleic acid sequence). If the sense strand and antisense strand reside on two different strands, at least one strand of the siRNA has a 3' overhang of 0–6 nucleotides in length, for example, an overhang of 0, 1, 2, 3, 4, 5, or 6 nucleotides in length, and possibly both strands have a 3' overhang of 2 or 3 nucleotides in length. The nucleotides of the overhang are, in some cases, thymine deoxyribonucleotides (dTs), or, in some cases, native overhangs that are nucleotides selected from the corresponding position on the DNA target or complementary nucleotides. When the sense and antisense strands are located on a single nucleic acid strand, in some cases the siRNA is a hairpin-type single-stranded nucleic acid molecule, where the complementary regions of the sense and antisense strands form a double-stranded nucleic acid structure with respect to each other. In some embodiments of the siRNA disclosed herein, the sense strand has a length in the range of 10–60 nucleotides. For example, in some embodiments, the sense and antisense strands independently consist of lengths of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. In some embodiments, the antisense strand has a length in the range of 10–60 nucleotides. For example, in some embodiments, the sense strand and antisense strand independently consist of lengths of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0094] In some embodiments, the antisense strands disclosed herein are capable of interacting with the target nucleic acid sequence of the SOD1 gene mRNA in a sequence-specific manner, meaning that the antisense strands are capable of hybridizing with the target nucleic acid via hydrogen bonding. In some embodiments, the antisense strand has a nucleotide sequence that, when described in the 5' to 3' direction, constitutes 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 described in the 5' to 3' direction, constitutes the reverse complement of the target portion in the fragment of the SOD1 gene transcript. ACO

[0095] While several previous studies have demonstrated that siRNAs capable of inhibiting SOD1 mRNA and reducing SOD1 protein expression can be used to treat SOD1 protein-related diseases, such as amyotrophic lateral sclerosis (ALS), the inventors have found two unresolved problems. One is the lack of potency of the SOD1 siRNA molecule, and the other is the lack of an efficient delivery method for delivering the siRNA molecule to target organs or tissues, such as cells in the CNS.

[0096] "Delivery to cells," when referring to siRNA, means efficient uptake or absorption by cells, as understood by those skilled in the art. The absorption or uptake of siRNA may occur by unassisted diffusive or active cellular processes, or by auxiliaries or devices. The meaning of this term is not limited to in vitro cells; it is also possible to "introduce" siRNA into cells, where the cells are part of a living organism. In such cases, introduction into cells also includes delivery to a living organism. For example, in vivo introduction may involve injecting siRNA into a tissue site or administering it systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches not known in the art are described below.

[0097] The present invention has found that when an siRNA agent, such as siRNA, is bound to a non-target single-stranded accessory oligonucleotide (ACO) as disclosed, the bioavailability, in vivo distribution, and / or cellular uptake, and in vivo efficacy of isRNA are significantly improved compared to oligonucleotide agents without ACOs. In particular, in several in vivo examples in this application, ACOs in oligonucleotide agents increased the in vivo distribution of siRNA in one, two, or more target tissues compared to oligonucleotide agents without ACOs.

[0098] Accordingly, aspects of this application relate to oligonucleotide agents and ACOs that can inhibit the expression of superoxide dismutase 1 (SOD 1), including small interfering RNA (siRNA).

[0099] In some embodiments, the oligonucleotide agent comprises one or more conjugated ACOs to enhance the in vivo distribution of the oligonucleotide agent in specific tissues and to increase the permeability and membrane-crossing ability of the oligonucleotide agent, such as the blood-brain barrier.

[0100] In some embodiments, ACO is an oligonucleotide containing a 5' end and a 3' end.

[0101] In some embodiments, siRNA and ACO are covalently bonded together, with or without one or more linking components, to form an oligonucleotide agent.

[0102] In some embodiments, the length of the ACO includes nucleotide lengths in the range of 6 to 19 nucleotides, such as 6 nucleotides or more, 7 nucleotides or more, 8 nucleotides or more, 9 nucleotides or more, 10 nucleotides or more, 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, 15 nucleotides or more, 16 nucleotides or more, 17 nucleotides or more, 18 nucleotides or more, 22 nucleotides or more, 20 nucleotides or more, 21 nucleotides or more, and 22 nucleotides or more. In some embodiments, the length of the ACO is 6 to 18 consecutive oligonucleotides.

[0103] In some embodiments, the length of the ACO can modulate the activity and / or biodistribution of an oligonucleotide agent within a target tissue or cell of interest. For example, we have found that shorter ACOs exhibit activity throughout the central nervous system, while longer ACOs exhibit activity only in specific regions of the brain, such as the cerebellum.

[0104] In another aspect of this application, an oligonucleotide agent comprising siRNA and a non-targeted ACO is 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.

[0105] In some embodiments, the oligonucleotide agents of this application comprise one or more ACOs, e.g., 2, 3, 4, 5, 6, 7, 9, or 10 ACOs, covalently linked to an siRNA, with or without one or more linkers between the ACOs and the siRNA. The number of ACOs can vary from 1-4, 2-10, branched, or liner-like, linked to the siRNA via a polyvalent linker, e.g., a polymer linker. In some embodiments, multiple ACOs are covalently linked in one or more agents to two or more siRNAs, e.g., 2, 3, 4, 5, 6, 7, 9, 10, or more siRNAs. PCT application WO2023280190A1, whose entire contents are incorporated herein by reference for all purposes, describes the principles of ACO design and examples of ACOs conjugated to siRNA to improve pharmacokinetic properties, including accessory delivery of double-stranded RNA to cells.

[0106] chemical modification In the siRNAs or ACOs disclosed herein, all nucleotides may be natural nucleotides or unmodified nucleotides, and at least one nucleotide may be a chemically modified nucleotide. Non-limiting examples of chemical modifications include one or more of the following combinations: 1) Modification of phosphodiester bonds of nucleotides in the nucleotide sequence of siRNA or ACO; 2) Modification of the 2'-OH group of ribose in the nucleotide sequence of siRNA or ACO; 3) Modification of bases in nucleotides of siRNA or ACO; 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 nucleotide sequence of ACO is a deoxyribonucleotide (DNA).

[0107] The chemical modifications described herein are well known to those skilled in the art, and phosphodiester bond modifications refer to modifications of oxygen in the phosphodiester bond, including phosphorothioate modifications and boranophosphate modifications. The modifications disclosed herein stabilize the siRNA structure and maintain high specificity and high affinity for base pairing. The modifications disclosed herein also stabilize the ACO structure and maintain delivery support properties, including bioavailability, in vivo distribution, and / or cellular uptake, of oligonucleotide drugs in various tissues, including the prefrontal cortex, cerebellum, spinal cord (e.g., cervical, thoracic, and lumbar), muscle, lung, eye, liver, and kidney.

[0108] In some embodiments, the chemical modification involves substituting phosphodiester bonds on the nucleotide sequence backbone of the oligonucleotide agent disclosed herein with phosphorothioate (PS) bonds. In some embodiments, the oligonucleotide agent disclosed herein comprises at least one PS backbone modification. In some embodiments, the ACO comprises at least one PS backbone modification. In some embodiments, the ACO comprises 6–17 PS backbone modifications.

[0109] In some embodiments, the siRNA or ACO of this application comprises at least one chemically modified nucleotide modified with a 2'-OH group of the pentose of the nucleotide, for example, 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2,4'-dinitrophenol modification, loc nucleic acid (LNA), 2'-amino modification, or 2'-deoxy modification, for example, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide.

[0110] In some embodiments, the siRNA or ACO of this application comprises at least one chemically modified nucleotide modified with a base of the nucleotide, for example, 5'-bromouracil modification, 5'-iodoluracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.

[0111] In some embodiments, the chemical modification of the siRNA or ACO is the addition of an (E)-vinylphosphonate moiety at the 5' end of the sense or antisense sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5'-methylcytosine moiety at the 5' end of the sense or antisense sequence.

[0112] In some embodiments, the siRNA or ACO of this application comprises a nucleotide in which at least one nucleotide in the siRNA / ACO nucleotide sequence consists of a chemically modified nucleic acid, such as loc nucleic acid, abasic nucleic acid, glycerol nucleic acid (GNA), morpholinonucleotide, phosphoramidate, and a non-natural base. In some embodiments, the siRNA disclosed herein comprises "endo-light" modification with a nucleotide containing a 2′-O-methyl modified nucleotide and a nucleotide containing a 5′-phosphorothioate group.

[0113] In some embodiments, the siRNA or ACO of this application is chemically modified to enhance stability or other beneficial properties. Nucleic acids characterized in this application include, for example, conventional methods such as "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., which is incorporated herein by reference. Modifications include, for example, (a) terminal modifications, e.g., 5' terminal modifications (phosphorylation, conjugation, reverse bond, etc.) and 3' terminal modifications (conjugation, DNA nucleotide, reverse bond, etc.), (b) base modifications, e.g., stabilizing bases, destabilizing bases, or substitutions of bases that form base pairs with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) skeletal modifications, including modification or substitution of phosphodiester bonds. Specific examples of siRNA molecules that can be used in this application include, but are not limited to, RNA containing a modified skeleton or RNA that does not contain natural nucleoside bonds. In some embodiments, the RNA having a modified backbone includes, among other things, RNA that does not have a phosphorus atom in its backbone. In some embodiments, the modified RNA that does not have a phosphorus atom in its internucleoside backbone may also be an oligonucleoside. In some embodiments, the modified oligonucleotide will have a phosphorus atom in its internucleoside backbone.

[0114] Modified oligonucleotide skeletons include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramides and aminoalkyl phosphoramidates, thionophosphoramides, thioalkyl phosphonates, thioalkyl phosphotriesters, and boranophosphates having the usual 3'-5' bond, their 2'-5' bonded analogues, and those having reverse polarity where adjacent pairs of nucleoside units are bonded from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0115] In some embodiments, the siRNA or ACO consists of one or more RNA, DNA, BNA, LNA, GNA, or PNA. covalent bond

[0116] Aspects of this application include oligonucleotide agents comprising covalently bonded double-stranded targeted oligonucleotides (i.e., siRNAs) and ACOs.

[0117] In some embodiments, any of the oligonucleotides in the oligonucleotide preparation of this application include a linking component. In some embodiments, the siRNA and ACO are covalently linked by the linking component. In some embodiments, the siRNA and ACO are linked by a covalent linker. Various combinations of strands can be linked, 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.

[0118] 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' and 5' ends 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' and 5' ends of the antisense strand of the siRNA. In some embodiments, one or more ACOs are covalently linked to the siRNA. In some embodiments, 2-10 ACOs are covalently linked to the siRNA. In some embodiments, one or more siRNAs are covalently linked to the ACO. In some embodiments, 2-10 ACOs are covalently linked to the siRNA.

[0119] In some embodiments, the ACO is bound to a linking component. In some embodiments, the 5' or 3' end of the ACO is bound to the linking component. In some embodiments, the siRNA and the ACO are covalently linked by the linking component. In some embodiments, the sense or antisense strand of the siRNA is covalently linked to the ACO by the linking component.

[0120] Linkers are typically direct bonds, or units such as atoms like oxygen or sulfur, NR1, C(O), C(O)O, C(O)NR1, SO, SO2, SO2NH, or substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryl, heteroaryl, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroaryl Rylalkynyl, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkenyl, alkenyl heterocyclylalkynyl, alkylheterocyclylalkynyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenyl heterocyclylalkynyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkenyl heterocyclylalkynyl, alkenyl heterocyclylalkynyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkenylaryl, alkylheteroaryl, alkenyl heteroaryl, alkenyl heteroaryl, alkenyl heteroaryl, where one or more methylene groups are O, S, S(O), SO2, N(R')2, C(O), cleavable linking groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, substituted or unsubstituted heterocyclic groups; Here, R1 is hydrogen, acyl, aliphatic, or substituted aliphatic.

[0121] The subject's combination may include, but is not limited to, various types of linker functionality, including, severable and non-severable linkers, as well as reversible and irreversible linkers.

[0122] In some embodiments, the linker is a cleavable linker. A cleavable linker is one that releases two bound parts, e.g., ACO and dsRNA, depending on processes within the target cell, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes or endosomes, or cleavage by specific intracellular enzymes (e.g., proteases). Thus, a cleavable linker allows the dsRNA to be released in its original form after the conjugate has been internalized and processed within the target cell. Cleavable linkers include, but are not limited to, those whose binding is cleavable by enzymes (e.g., peptide linkers), those whose binding is cleavable under reducing conditions (e.g., disulfide linkers), and those whose binding is cleavable under acidic conditions (e.g., hydrazones and carbonates).

[0123] In some embodiments, the linking component is selected from one or more of ethylene glycol chains, alkyl chains, peptides, RNA, DNA, carbohydrates, thiol linkages, phosphodiesters, phosphorothioates, phosphoramidates, amides, and carbamates. In some embodiments, the linking component includes, but is not limited to, the following: spacer phosphoramidite 18 (phosphoramide acid, N,N-bis(1-methylethyl)-, 19,19-bis(4-methoxyphenyl)-phenyl-3, 6, 9, 12, 15, 18-Hexaoxanonadec--Il di-cyanoethyl ester); Spacer-9(3-[2-[bis(4-methoxyphenyl)-phenylmethoxy]ethoxy]ethoxy-[di(propan-2-yl)amino]phosphanyl]oxypropanenitrile); 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 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-hexaoxahenicosan-CPG). In some embodiments, the linking component includes the compound structures shown in Table 15.

[0124] In some embodiments, the linking component is spacer phosphoramidite 18 (phosphoramidic acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecyl-1-yl 2-cyanoethyl ester). [Table 1]

[0125] In some embodiments, siRNA and ACO are covalently linked by a phosphodiester bond. In some embodiments, siRNA and ACO are covalently linked by a phosphorothioate bond.

[0126] In some embodiments, the siRNA includes a sense strand covalently bound to the ACO. In some embodiments, the siRNA includes an antisense strand covalently bound to the ACO.

[0127] In some embodiments, the siRNA and ACO are covalently linked by one or more nucleotides.

[0128] A non-limiting example of a covalent linker is described in U.S. Patent Application Publication No. 20200332292, which is incorporated herein by reference in its entirety. The covalent linker can be used to link siRNA and ACO.

[0129] In some embodiments, the covalent linker includes RNA and / or DNA and / or peptide linkers. The linker may be single-stranded, double-stranded, partially single-stranded, or partially double-stranded. In some embodiments, the linker includes disulfide bonds. The linker may be cleavable or non-cleavable. In some embodiments, the covalent linker comprises a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is [ka]

[0130] In some embodiments, the covalent linker includes peptide bonds, for example, amino acids. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably consisting of 4-5 amino acids, and optionally X-Gly-Phe-Gly-Y, where X and Y represent any amino acids.

[0131] In some embodiments, the covalent linker includes a hexaethylene glycol linker, HEG. ODV-siRNA oligonucleotide agent

[0132] In some embodiments, the oligonucleotide agent reduces the expression of the SOD1 gene or SOD1 protein. Administration of the oligonucleotide agent to a patient treats or delays the onset of ALS, such as familial ALS, sporadic ALS, or Lou-Georg's disease. In some embodiments, the described oligonucleotide agent reduces the amount of SOD1 protein or full-length SOD1 mRNA, for example, by downregulating SOD1 transcript levels. 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, SOD1 protein is reduced by an amount sufficient to attenuate symptoms associated with ALS. In some embodiments, the 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, the SOD1 protein is reduced by at least 80%.

[0133] In some embodiments, the oligonucleotide agent that reduces the expression of the SOD1 gene or SOD1 protein is an siRNA-ACO conjugate (or ODV-siRNA). The SOD1 siRNA-ACO conjugate reduces or downregulates the expression of the SOD1 gene in cells in which the SOD1 gene is abnormally or overexpressed.

[0134] In a typical embodiment, the first strand of the SOD1 siRNA in the oligonucleotide agent includes a segment having at least 75% sequence identity or complementarity with a 6-60 nucleotide fragment of the selective target region of the SOD1 gene, thereby resulting in inactivation or downregulation of gene expression.

[0135] In some embodiments, the ooligonucleotide 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 siSOD having the nucleotide sequence of SEQ ID NO: 834, which has an antisense chain complementary to the ODV structured sense chain fragment of SEQ ID NO: 850.

[0136] 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 siSOD having the nucleotide sequence of SEQ ID NO: 842, which has an antisense strand complementary to the ODV structured sense strand fragment of SEQ ID NO: 851.

[0137] 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 siCON having the nucleotide sequence of SEQ ID NO: 858, which has an antisense strand complementary to the ODV structured sense strand fragment of SEQ ID NO: 852.

[0138] 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 siCON-AC, which has an antisense strand that is complementary to the ODV structured sense strand fragment of SEQ ID NO: 853.

[0139] 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 siCON having the nucleotide sequence of SEQ ID NO: 860, which has an antisense strand complementary to the ODV structured sense strand fragment of SEQ ID NO: 854.

[0140] 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 siSOD having the nucleotide sequence of SEQ ID NO: 861, in which the antisense strand is complementary to the ODV structured sense strand fragment of SEQ ID NO: 855.

[0141] 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 siSOD having the nucleotide sequence of SEQ ID NO: 848, whose antisense strand is complementary to the ODV structured sense strand fragment of SEQ ID NO: 850.

[0142] 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 siSOD having the nucleotide sequence of SEQ ID NO: 862, whose antisense strand is complementary to the ODV structured sense strand fragment of SEQ ID NO: 856.

[0143] 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 siSOD having the nucleotide sequence of SEQ ID NO: 849, in which the antisense strand is complementary to the ODV structured sense strand fragment of SEQ ID NO: 852.

[0144] 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 siSOD having the nucleotide sequence of SEQ ID NO: 863, in which the antisense strand is complementary to the ODV structured sense strand fragment of SEQ ID NO: 857.

[0145] Furthermore, to facilitate the entry of siRNA into cells, based on the above modifications, chemically conjugated groups other than those disclosed by ACO can be introduced to the terminus of the sense or antisense strand of the siRNA to facilitate its action via the cell membrane, which consists of the nuclear membrane, the intranuclear lipid bilayer, and the mRNA region.

[0146] In some embodiments, the siRNAs disclosed herein are covalently bonded to one or more conjugated groups. In some embodiments, the conjugated groups modify one or more properties of the conjugated oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cell distribution, cell uptake, charge, and clearance. In some embodiments, the conjugated groups confer novel properties to the conjugated oligonucleotide, such as a fluorophore or reporter group that enables the detection of the oligonucleotide.Specific conjugated groups and conjugated moieties include, for example, the cholesterol moiety (Letsinger et al., ProcNatl.Acad.Sci.USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg.Med.Chem.Lett., 1994, 4, 1053-1060), thioethers, for example, hexyl-S-tritylthiol (Manoharan et al., Ann.NYAcad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg.Med.Chem.Lett., 1993, 3, 2765-2770), and thiocholesterol (Oberhauser et al., Nucl.Acids Res., 1992, 20, 533-538), aliphatic chains, e.g., dodecane-diol or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-; Shea et al., Nucl.Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), adamantane acetate palmityl moiety (Mishra et al., Biochim.Biophys.Acta, 1995, 1264, 229-237), octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al, 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc cluster (e.g., WO2014 / 179620).

[0147] In some embodiments, the siRNA of this application relates to a sense or antisense strand of siRNA conjugated to one or more conjugated groups selected from: intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and dyes.

[0148] In some embodiments, the conjugated group is, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiazide, chlorothiazide, diazepine, indomethicine, barbiturates, cephalosporins, sulfonamides, antidiabetic drugs, antibacterial agents or antibiotics.

[0149] In some embodiments, the siRNA of this application is conjugated to one or more conjugated groups selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0150] In some embodiments, the siRNA of this application relates to a sense or antisense chain of siRNA conjugated to one or more conjugated groups selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acid, cholesterol, glucose, and N-acetylgalactosamine.

[0151] In some embodiments, the siRNA conjugated to one or more conjugated groups disclosed in the embodiments is directly contacted, transported, delivered, or administered to cells or subjects. Cells containing siRNA

[0152] After contact with cells, the oligonucleotide agents disclosed herein can effectively inhibit or downregulate the expression of the SOD1 gene in cells, for example, by at least 10% (e.g., compared to baseline levels of SOD1 transcript).

[0153] In some embodiments, this application relates to cells containing the oligonucleotide agents disclosed herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells, and are human cells from various tissues, such as the prefrontal cortex, cerebellum, spinal cord (e.g., cervical, thoracic, lumbar), muscle, liver, and kidney.

[0154] The cells disclosed herein may be in vitro or ex vivo, such as cell lines, and may be present in the body of a mammal, such as the human body. The human body disclosed herein is a subject suffering from a disease or condition caused by a mutation in the SOD1 gene, abnormal SOD1 mRNA levels, and / or overexpression of the SOD1 protein in the CNS.

[0155] In some embodiments, the cells are derived from the CNS tissue of subjects with ALS. In some embodiments, the cells are derived from subjects with ALS. In some embodiments, the cells are derived from subjects with Alzheimer's disease (AD), Parkinson's disease (PD), or Down syndrome (DS). Composition containing siRNA

[0156] Another aspect of this application provides a composition or pharmaceutical composition that can downregulate the level of SOD1 mRNA transcripts by a mechanism of RNA interference (MoA), comprising an oligonucleotide agent disclosed herein, for the treatment or prevention of the onset of SOD1-related diseases (particularly ALS).

[0157] In some embodiments, this application relates to a composition or pharmaceutical composition comprising the siRNA of this application.

[0158] In some embodiments, this application relates to compositions or pharmaceutical compositions comprising siRNA and ACO as described herein. In some embodiments, this application relates to compositions or pharmaceutical compositions comprising siRNA and ACO covalently bonded by a linking component as described herein.

[0159] In one embodiment, a pharmaceutically acceptable carrier comprises one or more of the following: an aqueous carrier, liposomes or LNPs, polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates (e.g., GalNAc), and polypeptides. In one embodiment, the aqueous carrier may be, for example, RNase-free water or an RNase-free buffer. The composition may contain an oligonucleotide agent or nucleic acid encoding the full-length or partial amount of an oligonucleotide agent according to this application in a concentration of 1-150 nM, e.g., 1-100 nM, e.g., 1-50 nM, e.g., 1-20 nM, e.g., 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM, e.g., 50 nM.

[0160] In some embodiments, the composition comprises 1-150 nM of the oligonucleotide agent of the present invention.

[0161] In another embodiment, the present invention provides a pharmaceutical composition or drug comprising the oligonucleotide agent of the present invention and a therapeutically inert carrier, diluent or pharmaceutically acceptable excipient, as well as a method of using the oligonucleotide agent of the present invention to prepare such compositions and drug products.

[0162] Typical formulations are prepared by mixing the drug of this application with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art, 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 R. C, Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. Formulations may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, lubricants, processing aids, colorants, sweeteners, flavorings, diluents, and other known additives to provide a sophisticated presentation of the drug (i.e., the drug of this application or its pharmaceutical composition) or to assist in the manufacture of a pharmaceutical (i.e., a pharmaceutical).

[0163] The compositions of this application are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals.

[0164] The oligonucleotide composition of this application may be delivered optionally by parenteral infusion, including intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intraventricular, intravitreous, or subcutaneous administration; or by oral, intranasal, inhalation, vaginal, or rectal administration.

[0165] In another embodiment, the application provides the use of oligonucleotide agents or compositions according to any one of the embodiments described herein in the manufacture of pharmaceuticals for the treatment of gene or protein-related conditions in an individual. One embodiment may include SOD1-related diseases, including ALS, AD, PD, and / or DS. Use by specific embodiments is also provided, where the individual is a mammal, preferably a human. kit

[0166] In another embodiment, any of the compositions described herein may be provided in one or more kits, which optionally include instructions for use of the composition. That is, the kit may include a description of the use of an oligonucleotide agent or composition or pharmaceutical composition in the method described in any one of the items herein. As used herein, “kit” typically defines a package, assembly, or container (such as an insulated container) that includes one or more components or embodiments of this application and / or other components related to this application, such as those described above. Any embodiment or component of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder, frozen, etc.).

[0167] In some cases, a kit may contain one or more components, which may be in the same container, in two or more receptacles, and / or any combination thereof. Containers may contain liquids, and non-limiting examples include bottles, vials, jars, tubes, flasks, beakers, etc. In some cases, containers may be leak-proof (when closed, liquid will not leak from the container regardless of its orientation).

[0168] Examples of other compositions or components related to the agents, compounds 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, etc., used for specific purposes, modifying, assembling, preserving, packaging, preparing, mixing, diluting, and / or storing the components. In embodiments in which a liquid form of any of the components is used, the liquid form may be concentrated or ready for immediate use.

[0169] In further embodiments, the kit may include instructions in any form or to a website or other source provided for using the kit in relation to the components and / or methods described herein. For example, the instructions may include instructions for the use, modification, mixing, dilution, storage, assembly, preservation, packaging, and / or preparation of the components and / or other components related to the kit. In some cases, the instructions may also include instructions for shipping or storage of the components, for example, at room temperature, below freezing, or cryogenic temperatures. The instructions may be provided in any way and in any form useful to the user of the kit, such as written or oral (e.g., by telephone), digital, optical, visual (e.g., videotape, DVD, etc.), and / or electronic communication (including internet or web-based communication). How to use

[0170] Another aspect of this application relates to an oligonucleotide agent of this application used in therapeutic approaches for treating diseases such as ALS.

[0171] In non-limiting embodiments, the present application provides a method for reducing the transcription level of the SOD1 gene or SOD1 protein, comprising administering a pharmaceutical composition disclosed herein to a subject.

[0172] In some embodiments, this application relates to a method for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising the step of administering a pharmaceutical composition disclosed herein to the subject. In some embodiments, the subject has sporadic ALS (sALS). In some embodiments, the subject has familial ALS (fALS). In some embodiments, the pharmaceutical composition reduces the transcription of the SOD1 gene or the SOD1 protein.

[0173] In some embodiments, ACOs in oligonucleotide agents improve the stability, bioavailability, in vivo distribution, and / or cellular uptake of siRNAs compared to oligonucleotide agents without ACOs.

[0174] In some embodiments, ACOs in oligonucleotide agents increase the in vivo distribution of siRNA within one or more target tissues compared to oligonucleotide agents without ACOs.

[0175] In some embodiments, ACOs in oligonucleotide agents increase the in vivo distribution of siRNA in two or more target tissues compared to oligonucleotide agents without ACOs.

[0176] In some embodiments, one or more target tissues are selected from the prefrontal cortex, cerebellum, cerebrum, spinal cord, muscles, lungs, eyes, liver, and kidneys.

[0177] In some embodiments, the oligonucleotide agents of this application achieve a reduction in full-length SOD1 protein of less than that achieved by administering the same amount of double-stranded oligonucleotides, such as siRNA substances that do not have an ODV structure, used individually, with higher potency, reduced toxicity, or undesirable side effects. In some embodiments, the oligonucleotide agents of this application achieve a reduction in full-length SOD1 protein of less than the additive effect of treatment with the same amount of siRNA used individually.

[0178] Specifically, the oligonucleotide agents of this application 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, when the oligonucleotide agents disclosed in the embodiments are administered, for example, to one or more cells, the SOD1 mRNA transcripts are inhibited / downregulated by at least 50%, 60%, 70%, 77%, 79%, 81%, 84%, 85%, and 88% in in vitro cell lines compared to baseline SOD1 mRNA transcripts in a control group with a 10 nM treatment. In some embodiments, the oligonucleotide agents inhibit or downregulate SOD1 mRNA transcripts by about 80%.

[0179] In some embodiments, SOD1 gene expression is inhibited / downregulated by at least 0.01 nM of the oligonucleotide agent disclosed in the embodiments, for example, 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, the protein encoding the SOD1 gene (SOD1 protein) is inhibited / downregulated, for example, by administering the oligonucleotide agent disclosed in the embodiments to cells or subjects. Knockdown of the SOD1 protein is 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 expression of the SOD1 protein. In some embodiments, oligonucleotide agents inhibit or downregulate SOD1 protein expression by about 80%. In some embodiments, the SOD1 protein is inhibited / downregulated by administering the oligonucleotide agents disclosed in the embodiments to cells at concentrations of at least 0.01 nM, for example, 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.

[0180] In some embodiments, the oligonucleotide agents disclosed in the embodiments have dose-dependent knockdown activity in cells. In some embodiments, the oligonucleotide agents knock down SOD1 mRNA transcripts with IC50 values ​​of less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.8 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, less than 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.

[0181] Another aspect of this application relates to a method for preventing or treating a disorder or condition induced by overexpression of the SOD1 protein, SOD1 gene mutation, and / or high SOD1 mRNA levels in an individual, comprising the steps of: administering an effective amount of an siRNA, oligonucleotide agent, or composition comprising an oligonucleotide agent disclosed herein to the individual. In some embodiments, the effective amount of the siRNA disclosed herein is a concentration in the range of 0.01 nM to 50 nM, e.g., 0.01 nM, 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, the disorder or condition is ALS. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0182] In any of the embodiments provided herein, such cells may be in vitro, such as in a cell line, or they may be present in the body of a mammal, such as a human. In some embodiments, the human is a subject or individual suffering from SOD1 protein-related disease or ALS, AD, PD, DS.

[0183] Another aspect of this application relates to administering an effective amount of an oligonucleotide agent or composition to an individual using the administration routes described herein. In some embodiments, the administration route is selected from one or more of parenteral infusion, oral administration, nasal administration, inhalation administration, vaginal administration, and rectal administration. In some embodiments, the administration route is selected from one or more of intrathecal administration, intramuscular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intravesical administration, intraventricular administration, intravitreous administration, and subcutaneous administration. Administration regimen and route of administration

[0184] Aspects of this application relate to pharmaceutical compositions comprising the oligonucleotide agent of this application. In some embodiments, the application relates to pharmaceutical compositions comprising the oligonucleotide agent of this application and a pharmaceutically acceptable carrier, a therapeutically inactive carrier, a diluent, or a pharmaceutically acceptable excipient. The pharmaceutical compositions disclosed herein are developed as pharmaceuticals for the prevention or treatment of SOD1 protein-related diseases or ALS.

[0185] Aspects of this application also relate to methods of using the oligonucleotide agent of this application to prepare such compositions.

[0186] Another aspect of this application relates to the use of the oligonucleotide agent of this application in the manufacture of the pharmaceutical compositions disclosed herein.

[0187] Another aspect of this application relates to the use of oligonucleotide agents or compositions according to any one of the embodiments described herein in the manufacture of pharmaceuticals for the prevention or treatment of gene or protein-related conditions induced by overexpression of the SOD1 protein, SOD1 gene mutations, and / or high levels of the SOD1 protein in an individual. In the use according to a particular embodiment, the condition may include SOD1 protein mutation-related disorders or conditions that constitute ALS. In the use according to a particular embodiment, the condition induced by overexpression of the abnormal SOD1 protein is ALS. Use according to a particular embodiment is also relevant when the individual is a mammal, such as a human.

[0188] The doses to which the oligonucleotide agents or compositions of this application may be administered may vary over a wide range and be adapted to the individual requirements of each case. In some embodiments, the initial dose of the pharmaceutical composition of this application is administered when the subject is less than one week old, less than one month old, less than three months old, less than six months old, less than one year old, less than two years old, less than fifteen years old, or 15 years old or older.

[0189] The single dose of an oligonucleotide agent can be, for example, in the range of approximately 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 doses described herein may contain two or more of the oligonucleotide agent sequences described herein.

[0190] In some embodiments, the proposed number of doses is approximate. For example, in some embodiments, if the proposed dosing frequency is an administration on day 1 and a second administration on day 29, an ALS patient may receive the second administration 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 days after the first administration. In some embodiments, if the proposed dosing frequency is an administration on day 1 and a second administration on day 15, an ALS patient may receive the second administration 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after the first administration. In some embodiments, if the proposed dosing frequency is an administration on day 1 and a second administration on day 85, an ALS patient may receive the second administration 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days after the first administration.

[0191] In some embodiments, the dose and / or amount of the injection is adjusted based on the subject's age, the subject's weight, and / or other factors that may necessitate adjustments to the injection parameters.

[0192] In some embodiments, the pharmaceutical composition includes a cosolvent system. Some such cosolvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such a cosolvent system is used for hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which consists of 3% w / v benzyl alcohol and 8% w / v nonpolar surfactant polysorbate 80 TM and an absolute ethanol solution containing 65% w / v polyethylene glycol 300. The proportions of such cosolvent systems can be changed considerably without significantly altering solubility or toxic properties. Furthermore, the identity of the cosolvent components can be changed: for example, Polysorbate 80 TMOther surfactants may be used instead; the fractional size of polyethylene glycol may be changed; other biocompatible polymers, such as polyvinylpyrrolidone, may be used instead of polyethylene glycol; and other sugars or polysaccharides may be used instead of dextrose.

[0193] Examples of other compositions or components related to 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, etc., for example, those for using, modifying, assembling, preserving, packaging, preparing, mixing, diluting, and / or storing the components for specific uses. In embodiments in which a liquid form of any of the components is used, the liquid form may be concentrated or ready for immediate use.

[0194] In some embodiments, the lipid moiety used in nucleic acid therapy may be applied in this application for the delivery of oligonucleotide agent molecules disclosed herein. In such a method, nucleic acids (e.g., one or more oligonucleotide agents described herein) are introduced into pre-formed liposomes or lipoplexes consisting of a mixture of cationic and neutral lipids. In certain methods, complexes of oligonucleotide agents with mono- or polycationic lipids are formed without the presence of neutral lipids. In some embodiments, the lipid moiety is selected to increase the distribution of the pharmaceutical agent to specific cells or tissues. In some embodiments, the lipid moiety is selected to increase the distribution of the pharmaceutical agent to adipose tissue. In some embodiments, the lipid moiety is selected to increase the distribution of the pharmaceutical agent to muscle tissue.

[0195] 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 are useful for preparing certain pharmaceutical compositions, including those containing hydrophobic compounds. In some embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0196] In some embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more of the drugs of this application to a specific tissue or cell type. For example, in some embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0197] In some embodiments, oligonucleotide agents may be delivered or administered via a vector. Any vector that can be used for gene delivery may be used. In some embodiments, viral vectors may be used. Non-limiting examples of viral vectors that may be used in this application include, but are not limited to, human immunodeficiency virus;HSV, herpes simplex virus;MMSV, Moloney's mouse sarcoma virus;MSCV, mouse stem cell virus;SFV, Semryki forest virus;SIN, Sindbis virus;VEE, Venezuelan encephalitis virus;VSV, vesicular stomatitis virus;VV, vaccinia virus;AAV, adeno-associated virus; adenovirus; lentivirus; and retrovirus.

[0198] In some embodiments, the vector is a recombinant AAV vector (rAAV). The AAV vector is a relatively small DNA virus that can be incorporated into the genome of an infected cell in a stable and site-specific manner. AAV can infect a variety of cells without affecting cell growth, morphology, or differentiation, and does not appear to be involved in human pathogenesis. The AAV genome has been cloned, sequenced, and characterized. The genome consists of approximately 4,700 base pairs, with approximately 145 base pairs of reverse terminal repeat (ITR) regions at both ends, which serve as the origin of viral replication. The remainder of the genome is divided into two important regions responsible for encapsulation: the left side of the genome, which contains the rep gene involved in viral replication and viral gene expression, and the right side of the genome, which contains the cap gene encoding the viral capsid protein.

[0199] The formulations, pharmaceutical compositions, or medicinal products of this disclosure are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals.

[0200] In the case of the formulations, pharmaceutical compositions, or pharmaceuticals of this disclosure, delivery may be selectively parenteral infusion, including intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intraventricular, intravitreous, or subcutaneous; or by oral, intranasal, inhalation, vaginal, or rectal administration.

[0201] Typical formulations of the oligonucleotide modulators of this disclosure are prepared by mixing the siRNA of this disclosure with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art, 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 R. C, Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, lubricants, processing aids, colorants, sweeteners, flavorings, diluents, and other known additives to provide a sophisticated presentation of a pharmaceutical product (i.e., siRNA or a pharmaceutical composition thereof) or to assist in the manufacture of a pharmaceutical product (i.e., a drug). [Examples]

[0202] The following examples are provided to give a complete disclosure and explanation of the manufacturing method and use of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be the present invention, nor are they intended to represent all or only experiments. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental errors and deviations should be explained. Unless otherwise stated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations may be used. For example, bp is base pair, kb is kilobase, pl is picoliters, s or sec is seconds, min is minutes, h or hr is hours, aa is amino acid, nt is nucleotide, im is intramuscular, ip is intraperitoneal; sc is subcutaneous; icv or icv or ICV is intracerebral, etc. Example 1: Development of siRNA drug candidates to knock down human SOD1

[0203] The sequence of the human SOD1 transcript (NM_000454.5) was searched in the NCBI nucleotide database. It contained a 465 bp open reading frame (ORF) located between base pairs 78 and 542, which was used as a template for siRNA design (Table 1). [Table 2]

[0204] A total of 268 siRNA duplexes were designed and synthesized with a length of 21 nucleotides (nt), no more than four repeating nucleotides per row, and a GC content between 35-65%. Target sites and congeneral siRNA chain sequences are shown in Table 2. The knockdown activity of each siRNA was evaluated in HEK293A cells at concentrations of 0.1 nM and 10 nM using high-throughput RT-qPCR. The data were ranked according to the average knockdown activity of 121 siRNAs, which reduced SOD1 by ≥90% at 10 nM (Figure 1A). As an indicator of potency, 69 and 15 siRNAs reduced SOD1 levels by ≥50% and ≥75%, respectively, at 0.1 nM treatment. Twenty-five running siRNAs underwent additional screening in HEK293A cells at six concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM) to demonstrate dose-dependent activity. In this screening, propidium iodide (PI) was incorporated into sample preparation to monitor changes in nucleic acid content and serve as an indicator of cytotoxicity (Figure 2). Figure 1B shows the data for the top five siRNAs with the most potent knockdown activity (i.e., siSOD1-063, 047, 104, 005, 258), which also demonstrate the absence of apparent cytotoxicity (i.e., a reduction of less than 20% in PI staining).

[0205] Next, dose-response curves were generated for each of the five candidate siRNAs to validate their efficacy in representative model cell lines of neurological diseases, including SK-N-AS cells (Figure 1C) and T98G cells (Figure 3). As summarized in Table 3, the in vitro efficacy of each double-stranded siRNA was in the low picomolar range in both cell lines. Cytotoxicity was also evaluated 72 hours after treatment at concentrations far exceeding 200 times the extrapolated IC50 value. As shown in Figures 4A-B, only siSOD1-047 and 005 did not have a 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 with respect to caspase 3 / 7 activity in T98G cells and were inversely correlated with cell viability. SK-N-AS cells appeared to be more resistant to treatment, but a similar pattern was observed with respect to cell viability.

[0206] Several medicinal chemistry patterns, referred to as M1, M2, M3, and M4, representing different double-strand lengths (20, 21, 22, and 23 nt, respectively), containing phosphorothionate (PS) backbone modifications with 2''-O-methylation (2''Ome) or 2''-fluoro(2''F) substitutions at each nucleotide, were applied to each lead candidate and screened for target mRNA knockdown activity. As shown in Figure 1D, M3 variants (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, and 258M3) generally showed superior knockdown activity compared to other chemically modified siRNAs at a treatment concentration of 0.1 nM in SK-N-AS cells. A nearly identical pattern was observed in T98G cells (Figure 5). To further characterize the efficacy, dose-response curves were generated for M3-modified double helices (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, 258M3, and 270M3) in both SK-N-AS (Figure 1E) and T98G (Figure 6) cell lines. As summarized in Table 3, the in vitro efficacy was largely maintained after chemical modification.

[0207] Coordinate oligonucleotide (ACO) conjugates were developed to confer self-delivery properties similar to ASOs by sharing pharmaceutically acceptable pharmaceutically acceptable properties with conventional siRNAs. Therefore, the sense strands of each M3 variant (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, and 258M3) were covalently bonded to a 14-nucleotide ACO (referred to as AC1) via a short linker (L9, i.e., a spacer-9 linker). Here, the ACO has PS backbone substitution and 2'-O-methoxyethyl (2'MOE) modification at all positions (Figure 7A). As shown in Figure 7B, AC1 alone did not detect SOD1 knockdown in vitro at concentrations of 0.25 and 2.5 nM, but activity was recognized only in the conjugate with siRNA. In additional dose-response analyses, the AC1 conjugate (i.e., siSOD1-005M3-AC1) showed approximately 10-fold reduced siRNA potency compared to the chemically modified double helix (i.e., siSOD1-005M3) alone (Figure 7C). However, modification of the 5' end of the guide chain with 5'-(E)-vinylphosphonic acid (5'VP) restored potency (i.e., siSOD1-005M3-AC15). VP (Figure 7D). Tofersen-like ASO (an antisense oligonucleotide ASO in clinical studies that has shown therapeutic effects on ALS by suppressing mutant SOD1 mRNA levels; T. Miller et al, New England Journal of Medicine 383, 109-119 (2020)) is similar in both sequence and chemistry (i.e., ASO SOD1 SOD1 knockdown by either ASOSOD1 (SEQ ID NO: 864) or siRNA-ACO variants (i.e., siSOD1-005M3-AC1 or siSOD1-005M3-AC1VP) was more potent than ASOSOD1, regardless of the 5'VP modification (Figure 7D).

[0208] Subsequently, the siRNA-ACO conjugate was synthesized using M3 chemistry and 5'VP modification (i.e., siSOD1-063M3-AC1 VP ,047M3-AC1 VP, 104M3-AC1 VP , 005M3-AC1 VP , 258M3-AC1 VP , 270M3-AC1 VP ), downstream screening was performed in vivo. Prior to treatment, dose-response curves were generated in both SK-N-AS and T98G cells, and the activity of each siRNA-ACO candidate in suppressing mutant SOD1 mRNA levels was verified (Figures 8A-8B). As summarized in Table 3, the in vitro potency was generally well conserved compared to the unbound form. Apoptosis and cell viability were also quantified 72 hours after treatment to measure changes in cytotoxicity as a result of chemical modification and ACO conjugation. As shown in Figures 9A-9B, siSOD1-047M3-AC1 VP and 005M3-AC1 VP had no nominal effect on cell integrity and were generally unaffected in both SK-N-AS and T98G cells, but siSOD1-104M3-AC1 VP retained signs of detrimental cytotoxicity, and 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 the unmodified form (Figures 4A-B).

Table 3

[0209] Example 2: Selection of siRNA-ACO drug candidates in vivo hSOD1 G93A Transgenic hemizygous mice express a mutant form of human SOD1 and exhibit a disease phenotype similar to ALS, including progressive motor function decline and shortened lifespan due to neuronal degeneration (P. Weydt et al., Neuroreport 14, 1051-1054 (2003); P. H. Tu, et al., Proc Natl Acad Sci USA 93, 3155-60 (1996)). To test knockdown activity in vivo, hSOD1G93A Mice were given an equimolar dose (i.e., 20 nmole / dose) of siRNA-ACO (i.e., siSOD1-047M3-AC1) VP Or siSOD1-005M3-AC1 VP ) and an unconjugated control (i.e., siSOD1-047M3VP or siSOD1-005M3) VP Compared to administration by ICV injection, the effect of AC1 binding on in vivo knockdown activity was demonstrated. All siRNA-ACOs were formulated in aCSF, where only the treatment was used as the solvent control group to establish baseline expression, and siCON2-AC1 was used. VP SISOD1-005M3-AC1 served as a negative control for siRNA-ACO activity. As shown in Figure 10, both siRNA-ACO double helixes showed higher knockdown activity compared to the unconjugated congener in all tissues of the brain (i.e., prefrontal cortex, cerebellum, and cerebrum) and spinal cord (i.e., cervical, thoracic, and lumbar vertebrae), and showed minimal activity in the periphery (i.e., liver). siSOD1-005M3-AC1 compared to non-5'VP control (i.e., siSOD1-005M3-AC1) VP Its knockdown durability is also ASO SOD1 It was characterized in comparison to [another compound]. In summary, the combination of the AC1 conjugate and the 5'VP modification conferred enhanced activity to our siRNA necessary for a durable response in vivo. Example 3: Single administration of siRNA-ACO by ICV injection is effective against SOD1 G93A It slows disease progression and extends survival time in mice.

[0210] Adult hSOD1 G93A In mice, siRNA-ACO candidates (i.e., siSOD1-047M3-AC1) were introduced. VP Or siSOD1-005M3-AC1 VPThe drug was administered via ICV injection to PND 85 or 60 at doses of 50, 100, 200, or 400 mg / dose, respectively. Drug concentrations and hSOD1 expression levels were quantified in a subset of animals 14 days post-treatment in CNS tissue (i.e., cerebellum, cerebrum, and spinal cord). As shown in Figures 11A-B, siSOD1-047M3-AC1 VP and siSOD1-005M3-AC1 VP Both the activity and tissue accumulation of SOD1 knockdown were dose-dependent, inversely correlated with increased siRNA-ACO concentrations in CNS tissues. Table 4 summarizes the drug concentrations predicted to evoke an ED50 (median effective dose) response in each tissue. [Table 4]

[0211] In the remaining animals, changes in body weight were plotted, and growth rate and disease progression were monitored. As shown in Figure 12A, siSOD1-047M3-AC1 VP Or siSOD1-005M3-AC1 VP All groups treated with either of the above methods continued to gain weight compared to aCSF treatment. Furthermore, disease-related weight loss was dose-dependently delayed, as indicated by the time required for the growth rate to return to the starting weight (dotted line). Disease progression was confirmed when peak weight decreased by 10%. Plotting the data on Kaplan-Meier curves showed when animals in each treatment group progressed to progressive disease (Figure 12B). Data were collected until the animals inevitably succumbed to the disease, and survival curves were also generated (Figure 12C). In summary, siSOD1-047M3-AC1 VP (Table 5) and siSOD1-005M3-AC1 VP Both treatments (Table 6) delayed disease progression and extended the survival time of the animals, with the highest dose (i.e., 400 mg) extending survival by 70 days and 111.5 days, respectively, compared to the solvent control group. [Table 5]

[0212] [Table 6]

[0213] Example 4: Pathogenicity mutations at the target site of siRNA-ACO affect lead selection siSOD1-005M3-AC1 VP In silico analysis of pathogenic single nucleotide polymorphisms (SNPs) located within the target site revealed a total of five SNPs, four of which were in a region complementary to the "seed" sequence (Figure 13A). This mismatch in this region is known to inhibit siRNA activity, and is a major factor in the global spread of ALS. SOD1 It is predicted that approximately 8.9–12.4% of patients will be excluded from the treatment pool (O. Abel et al., Hum Mutat 33, 1345-51 (2012); https: / / alsod.ac.uk / , Amyotrophic Lateral Sclerosis online Database - ALSoD). Conversely, siSOD1-047M3-AC1 VP Only two pathogenic SNPs (i.e., P.E22G and P.F21C) have been reported within the target site, and these are the only ones reported in ALS worldwide. SOD1 This includes approximately 4.04% and 2.70% of the population (Figure 13B). siSOD1-047M3-AC1 VP A luciferase reporter construct (i.e., pLuc) containing either a consensus sequence or pathogenic mutations (i.e., P.E22G and P.F21C) that are fully complementary to the guide chain. SOD1 pLuc P.E22G and pLuc P.F21C ) was co-transfected into HEK293A cells along with siRNA-ACO. Luciferase activity knockdown was performed with siSOD1-047M3-AC1 VP It is specific to and scrambled control (i.e., siCON2-AC1 VP Transfection in ) did not reduce reporter expression (Figure 13C). Dose-response data showed that the P.E22G mutation reduced siSOD1-047M3-AC1 compared to the consensus target site sequence.VP It showed no significant effect on knockdown activity / potency, while P.F21C was partially resilient to treatments that resulted in incomplete knockdown and inferior potency (Figure 13D).

[0214] Example 5. siRNA-ACO was administered by IT injection to hSOD1 G93A This method slows disease progression, extends survival time, and improves motor function in mice. Based on the recognized patient population, siSOD1-047M3-AC1 VP This was selected for further in vivo analysis. 雌雄のhSOD1G93Aマウスを、PND68と100に、75、150、または300μg / 用量のsiSOD1-047M3-AC1VPを2回連続してIT注射した。 Nonspecific siRNA-ACO (i.e., siCON3-AC1) VP ) was used as a negative control for treatment efficacy. Animal body weight was monitored in comparison to wild-type animals (i.e., WT), and all siSOD1-047M3-AC1 VP While the treatment yielded similar benefits in male mice, weight gain in females showed a more pronounced dose-dependent effect (Figure 14A). Both disease progression (i.e., 10% loss of peak body weight) and animal survival were also associated with siSOD1-047M3-AC1. VP The treatment was plotted to delay disease progression and extend survival in both male and female mice (Figure 14B-C). Table 7 summarizes the median days of disease progression and survival after siRNA-ACO treatment with IT injection in male and female populations. Overall, siCON3-AC1 VP It did not provide a therapeutic benefit compared to the solvent control group, but an equivalent dose of siSOD1-047M3-AC1 VP (i.e., 150 mg) extended the survival of animals by 61 days and 29 days in males and females, respectively. [Table 7]

[0215] Neuromuscular function was also evaluated in groups including male and female mice. Distance traveled during open field roaming (Figure 15A), rotarod test (Figure 15B), and grip strength (Figure 15C) were all measured using siSOD1-047M3-AC1. VP The administration demonstrated a significant improvement in motor function in hSOD1G93A mice, which was generally well-maintained until the end of the study. Comparing the rotarod performance of each individual at an early point before measurable weight loss (i.e., PND 90) with their respective final points, siSOD1-047M3-AC1 VP The administration of ASOSOD1 was further shown to maintain or improve neuromuscular function in most animals compared to the control group or ASOSOD1 (Figure 16). The latency of rotarod performance in each animal is summarized in Table 8.

[0216] [Table 8] JPEG2026509175000010.jpg224170

[0217] Motor function was scored for all animals before the open field, rotarod, and / or grip strength tests using the ALS Therapy Development Institute (ALS TDI) neuroscore (NS) system, as shown in Figure 15D, for the aCSF group and siCON3-AC1 group. VP In both the control group and the siSOD1-047M3-AC1 mice, all mice developed abnormal spray (i.e., NS2) by approximately 130 PNDs, and the severity continued to increase over time (i.e., ≥NS3). Conversely, all mice with siSOD1-047M3-AC1 VP The average dose score never exceeded NS2 at any point during the study period, and in particular, the average NS remained almost constant at around NS1 in the highest dose group (i.e., 300 mg). ASO SOD1 The administration was also performed in the solvent control group and siCON3-AC1 VP Compared to the control group, NS showed improvement, but with PND150, ASO SOD1150mg dose resulted in siSOD1-047M3-AC1 VP Despite having approximately three times the molecular excess, NS began to increase at a similar rate to the control group.

[0218] In summary, the siRNA-ACO conjugate conferred pharmacological properties necessary for clinical development to SOD1 siRNA, such as potent and sustained activity in CNS delivery and good histobiological distribution. G93A Local administration to mice via ICV or IT injection delayed disease onset / progression and extended animal survival with superior efficacy compared to ASO compounds similar in sequence and chemistry to Tofersen. These results clearly demonstrate that siRNA targeting and knocking down SOD1 for ALS treatment represents a significant improvement over current clinical therapies (i.e., Tofersen).

[0219] Example 6: Knockdown activity of siRNA against SOD1 mRNA expression in HeLa cells and SK-N-AS cells To evaluate the knockdown activity of siRNAs, the listed siRNAs (i.e., RD-15757, RD-18972, RD-1200, RD-18973, RD-18948, and RD-18949) were directly added at 1500 nM to a culture medium containing HeLa cells for 3 days. Untreated cells served as the mock control, and cells treated with RD-11566 served as the double-strand control. As shown in Figure 17, compared to RD-11566, all siRNAs caused varying degrees of reduction in SOD1 mRNA levels (ranging from 1 to 32%), with RD-12500 causing the greatest reduction (32%).

[0220] To further evaluate the dose-dependent knockdown activity of the siRNAs, 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 for 24 hours at the indicated concentrations (i.e., 0.0002, 0.001, 0.0039, 0.0156, 0.0625, 0.25, 1, and 4 nM). SOD1 mRNA levels quantified by two-step RT-qPCR are shown in Figures 18A and 18B. For each of the tested siRNAs that showed dose-dependent knockdown of SOD1 mRNA, the EC50 value was extrapolated to define potency in relation to maximal activity. EC50 after siRNA treatment in SK-N-AS cells 50 The values ​​are summarized in Table 9. [Table 9]

[0221] Materials and methods High-throughput screening of siRNA targeting human SOD1

[0222] An open reading frame (ORF) of the human SOD1 (hSOD1) cDNA sequence (NM_000454.5) was used as a template. A total of 268 double helices were synthesized with a length of 19 nucleotides (Table 2). Plating and transfection of HEK293A cells were performed in a dual 96-well plate containing 32 siRNAs and 8 quality control treatments at two concentrations (i.e., 0.1 and 10 nM). Cells were cultured for 24 hours, and lysis was automated via a Fluent System 780 liquid handling system (Tecan, Hombrechtikon, The Switzerland) using an optimized formulation including propidium iodide (PI) based on Cell-Lysis (CL) buffer for one-step RT-qPCR, as previously described (K. Shatzkes et al, Sci Rep 4, 4659 (2015)). In sample preparation, PI integration was used to monitor cell number variations (e.g., undesirable cytotoxicity) by staining the total nucleic acid content in the crude lysate. Staining was quantified by optical density (OD) at excitation wavelength 535 nm and emission wavelength 615 nm using an Infinite M200 Pro microplate reader (Tecan). Subsequently, samples were transferred to 384-well plates and analyzed by RT-qPCR 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 reaction mixture preparation was automated using an Echo 525 Acoustic Liquid Handler (Beckman Coulter, Brea, CA, USA). Subsequently, secondary screening was performed on the top 30 siRNAs at six concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM). All samples were amplified in triplicate. siRNA synthesis

[0223] Oligonucleotide sequences were synthesized in-house at Ractigen Therapeutics (Rudong, China) using an HJ-12 synthesizer (Highgene-Tech Automation, Beijing, China) on a solid support, and then purified by RP-HPLC with an acetonitrile gradient on a UniPS column (NanoMicro Technology, Suzhou, China). Each sequence was reconstituted in sterile water by buffer exchange. Equimolar amounts of each chain were annealed to the corresponding double helix by briefly heating the mixed chain and cooling it to room temperature. To confirm double helix formation, a single band of the expected molecular weight was separated by gel electrophoresis. Double helix identity was confirmed using ESI-MS, and the overall purity was analyzed by SEC-HPLC using an XBridge Protein BEH SEC 125 A column (Waters Corporation, Milford, MA, USA). Endotoxin levels in each batch were quantified via proenzyme factor C using the Endpoint Chromogenic Endotoxin Quant Kit (Bioendo, Xiamen, China). All control double helices and chemically modified sequences are shown in Table 10. [Table 10] JPEG2026509175000013.jpg255170JPEG2026509175000014.jpg255170JPEG2026509175000015.jpg255170 Cell culture and processing

[0224] HEK293A cells (Cobioer, Nanjing, China, Cat# CBP60436) and SK-N-AS cells (Procell, Wuhan, China, Cat# CL-0621) were maintained in DMEM medium supplemented with 10% bovine calf serum (Sigma-Aldrich), penicillin (100 U / ml, Gibco), and streptomycin (100 mg / ml, Gibco). T98G cells (Cobioer, Cat# CBP60301) were maintained in MEM medium supplemented with 10% FBS, 1% NEAA, sodium pyruvate (1 mM), penicillin (100 U / ml), and streptomycin (100 ug / ml). Human cervical cancer cells (HeLa(ATCC)) were cultured in modified RPMI1640 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% bovine calf serum and 1% penicillin / streptomycin. All cell lines were cultured at 37°C under a humidified atmosphere of 5% CO2. Transfection was performed using Lipofectamine RNAiMax (ThermoFisher, Waltham, MA, USA) in antibiotic-free growth medium according to the manufacturer's protocol. RT-qPCR One-step reverse transcription-quantitative polymerase chain reaction (one-step RT-qPCR)

[0225] After transfection, the culture medium was discarded, and the cells were washed once per well with 150 μL of PBS. After discarding the PBS, 100 μL of cell lysate was added to each well, and the cells were incubated at room temperature for 5 minutes. 0.5 μL of cell lysate was collected from each well and analyzed by RT-qPCR using a Roche Lightcycler 480 real-time PCR instrument with a One Step TB Green™ PrimeScrip™ RT-PCR kit II (Takara, RR086A). The PCR reaction was prepared using an Echo 525 Acoustic Liquid Handler (Beckman Coulter). Each transfection sample was repeatedly amplified in three wells. The PCR reaction conditions are shown in Table 11.

Table 11

[0226] The reaction conditions were as follows: Reverse transcription reaction (first step): 42°C, 5 minutes, 95°C, 10 seconds; PCR reaction (second step): 95°C, 5 seconds, 59°C 20 seconds, 72°C 10 seconds, amplification for 40 cycles, melting curve (third step). The human SOD1 gene was amplified as the target gene. Human TBP or mouse Tbp was used as the reference gene and was also amplified as an internal control for RNA loading. The primer sequences are shown in Table 12.

Table 12

[0227] Two-step RT-qPCR

[0228] Animal tissues frozen with RNALater (Sigma-Aldrich, St. Louis, MO, USA) were homogenized with Total RNA Isolation Reagent (Biosharp, Hefei, China) using Bioprep-24 Homogenizer (Allsheng, Hangzhou, China). Chloroform was added to the homogenate, the aqueous phase was removed, and it was mixed with isopropanol. Total RNA was extracted from tissue slides using the RNeasy RNA kit (Qiagen) according to the manufacturer's protocol. RNA from cell cultures was extracted using the Auto-Pure 96A (Allsheng) nucleic acid extraction system. Reverse transcription (RT) was performed using 1 μg of total RNA with the PrimeScript RT kit with gDNA Eraser (Takara, Shlga, Japan). The obtained cDNA was triple-amplified on a Roche LightCycler 480 Multiwell Plate 384 (Roche, ref: 4729749001, US) using SYBR Premix Ex Taq II (Takara, Shlga, Japan), a primer set specific to human SOD1 (hSOD1), and an internal control of either human (i.e., TBP) or mouse (i.e., mTbp) sample. Melting curves were performed after amplification to confirm primer specificity. The reaction conditions were as follows: reverse transcription (step 1): 42°C, 5 min, 95°C, 10 sec; PCR reaction (step 2): 95°C, 5 sec, 60°C, 30 sec, 72°C, 10 sec, 40 amplification cycles, melting curve (step 3). The PCR reaction conditions are shown in Tables 13 and 14. The primer sequences are shown in Table 12. [Table 13]

[0229] [Table 14]

[0230] To calculate the expression level (Erel) of SOD1 mRNA in siRNA transfection samples compared to the control treatment (Mock), the mean Ct values ​​of the target gene and internal reference gene were substituted into Equation 1.

number

[0231] Here, CtT m This is the Ct value of the target gene in the mock-treated sample, CtT s This refers to 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 mock-treated sample, and Ct is the Ct value of the internal reference gene in the siRNA-treated sample. Caspase 3 / 7 Activity Assay

[0232] Caspase 3 / 7 activity was quantified 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 using an Infinite M200 Pro microplate reader (Tecan). Relative caspase 3 / 7 activity was calculated by subtracting the blank background signal from the luminescence value of each well and normalizing the data against an untreated (mock) control. Cell viability assay

[0233] In vitro cell viability was measured using the CCK-8 assay (Dojindo, Mashiki-machi, Japan) according to the manufacturer's protocol. Briefly, fresh medium containing WST-8 substrate was added to each well of a tissue culture plate and incubated at 37°C for at least 1 hour. Absorbance was measured at 450 nm using an Infinite M200 Pro microplate reader (Tecan). Relative viability was calculated by subtracting the background absorbance of the blank control group from the OD value of each well and normalizing the data to the untreated (mock) control group. Luciferase reporter constructs and knockdown evaluation

[0234] Target sequences containing P.E22G or P.F21C mutant SNPs were cloned into the multiple cloning site (MCS) of the luciferase reporter vector pmirGLO (Promega) between the NheI and SalI restriction enzyme (RE) sites downstream of the firefly luciferase gene (luc2), respectively, and pLuc P.E22G and pLuc P.F21C A construct was generated. A consensus h that is perfectly complementary to the siSOD1-047M3-AC1 guide chain. SOD1 A control reporter construct containing the sequence (i.e., pLucSOD1) was also prepared. All constructs were subcloned into DH5a bacteria (Tolobio, Shanghai, China), and colonies were selected for DNA sequencing to confirm insertion of the target sequence. Typical colonies were scaled up for plasmid isolation using midiPrep (Qiagen, Hilden, Germany). HEK293A cells were plated in 96-well cell culture plates at 30,000 cells / well in the absence of antibiotics. 100 ng / well of the reporter plasmid (i.e., pLucSOD1) was isolated using 0.3 μL of Lipofectamine 2000 (ThermoFisher). P.E22G pLuc P.F21C , or pLuc SOD1 ) and the indicated concentration of siSOD1-047M3-AC1 VPAlternatively, cells were co-transfected with a scrambled control group. Wells treated in the absence of the test substance (0 nM) were used as the untreated control group. Cells were cultured for 24 hours, and luciferase activity was quantified using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol. Briefly, cells were lysed in 50 μL of Passive lysis buffer (Promega), 20 μL of the lysis solution was mixed with 20 μL of Dual-Glo Luciferase reagent, and incubated at room temperature for 10 minutes. Luciferase activity was then quantified by measuring luminescence using an Infinite 200 Pro microplate reader (Tecan). 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 quantify the lenira activity used to normalize the luciferase reporter results. The data was calculated as the ratio of reporter emission to lenira emission relative to the untreated control group. The knockdown rate (% KD) was calculated as 1 - (Ratio siRN / Ratio non-treated Calculated using *100. RatiosiRNA = siRNA firefly luciferase activity / siRNA reniral luciferase activity, Rationon-treated = untreated group firefly luciferase activity / untreated group reniral luciferase activity. Animal handling and grouping

[0235] Parent transgenic hSOD1 G93AThe mice (Strain ID #004435) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and imported into China via Nantong University (Jiangsu Province, China). The mice were brought to the animal facility at 6 weeks of age and subsequently bred domestically at Nantong University. All animal experiments were approved by the IACUC at Nantong University. Oligonucleotides were dissolved in aCSF to prepare stock solutions, which were then diluted to the desired therapeutic concentration. Animals were randomly assigned to study groups based on body weight and sex. Animals in poor health or with obvious abnormalities were excluded from the experiment. Randomization was analyzed using a standard one-way ANOVA via GraphPad Prism version 8.3.0 Windows (GraphPad Software, San Diego, CA, USA). Female hSOD1 G93A The typical weight of the mice was about 20–25% less than that of male littermates. Intraventricular (ICV) injection

[0236] Aveltin (1.2%) was prepared fresh and sterilized through a 0.2 micron filter. Mice were injected intraperitoneally (IP) at a dose of 0.30–0.35 ml per 10 g of body weight using a stereotactic device to rapidly induce anesthesia for up to 30 minutes. An incision of approximately 11.5 mm was made in the scalp of the animals, and a 25-gauge needle attached to a Hamilton syringe containing the appropriate siRNA preparation was placed at the bregma level. The needle was moved to the appropriate anterior-posterior and medial / lateral coordinates (0.2 mm anterior-posterior, 1 mm medial / lateral). A total of 10 μL was injected into the lateral ventricle at a rate of approximately 1 μl / second. After treatment, the needle was slowly withdrawn and the wound was sutured. Intrathecal injection (IT)

[0237] Anesthesia was performed using 3.0% isoflurane for 10 consecutive minutes in the induction room. The hair around the injection site at the base of the tail was shaved and washed with 75% ethanol. The space between the L5-L6 spinous processes was identified, and a 30-gauge injection needle attached to a microliter syringe containing the appropriate formulation was slowly inserted into the epidural cavity until tail flicker was observed. Subsequently, the position of the needle was fixed, and 10 μL of the solution was injected over 1 minute. Quantification of siRNA-ACO in animal tissues

[0238] Tissue lysates were prepared with lysis buffer (0.5% CA-630, 1 mM EDTA, 150 mM NaCl) using a Bioprep-24 homogenizer (Allsheng). Subsequently, the samples were heated to 95 °C to inactivate the sample proteins. Serial dilutions of the untreated lysate with added siRNA-ACO were made to create an 8-point standard curve. The reverse transcription (RT) reaction was performed using the PrimeScript RT reagent kit (Takara) in combination with a custom stem-loop primer specific to the siRNA guide strand. Each sample was amplified in triplicate on a 480 Real-Time PCR system (Roche) using a primer set specific to the guide strand cDNA and SYBR Premix Ex Taq II (Takara) reaction mix. To confirm primer specificity, a melting curve was performed after amplification. The absolute amount of siRNA was extrapolated by linear regression using an appropriate standard curve. Tissue concentration was calculated as the ratio of the absolute amount of siRNA (ng) to the total weight (g) of the tissue sample pretreated for lysis. Clinical observations and endpoint criteria

[0239] Animals were observed until 4 hours after injection and then daily until the endpoint. Body weight was measured at intervals before and after administration of the test substance. Animals in which the weight loss on the day of administration was 20% or more of the initial weight or those with a neurological score of NS4 met the endpoint criteria. Neurological scoring

[0240] Regarding animals treated with IT injection, hSOD1 G93A The ALS Therapy Development Institute (ALS TDI) Neurological Score (NS) system, developed to provide an unbiased assessment of disease progression based on hindlimb dysfunction common to mice, was used to evaluate mice for signs of motor impairment (T. Hatzipetros, et al., Journal of Visualized Experiments (2015), https: / doi.org / 10.3791 / 53257). The NS was assigned based on the following four-point scale: 0 for no signs of motor dysfunction (i.e., pre-symptom), 1 for obvious hindlimb tremors when suspended by the tail (i.e., initial symptom), 2 for gait abnormalities (i.e., onset of paralysis), 3 for dragging at least one hindlimb (i.e., partial paralysis), and 4 for inability to move back and forth on one's own within 10 seconds (i.e., terminal paralysis). Open field testing

[0241] During the daytime, each mouse was placed in a corner of an open-field enclosure (50cm x 50cm x 50cm) and allowed to roam freely for 15 minutes. An overhead camera recorded each animal's movement path. The video footage was analyzed using automated tracking software Samart 3.0 (Bioseb, Vitrolles, France) to calculate the total distance traveled. Rotarod Analysis

[0242] The animals were trained starting three days before data acquisition. The mice were placed on a non-exercise rotor rod device (XinRuan Information Technology, Shanghai, China) equipped with a 60mm diameter rotating bar. The rotation speed was accelerated from 0 to 30 rpm over 300 seconds. The latency period was recorded as the time it took for each animal to fall from the rotating bar. Each animal underwent three trials, and the longest value represented the latency period. Grip strength test

[0243] Mice were placed on a grid plate and allowed to grasp the grid with their forelegs and hind legs. Their tails were gently pulled, and their maximum grip strength was measured in mass units using an XR501 dynamometer (XinRuan Information Technology) until the animal released its grip. Each animal underwent three tests, and the average value was used as the grip strength. statistical analysis

[0244] Data analysis was performed using GraphPad Prism version 8.3.0 for Windows. Dose-response curves and ICs were analyzed. 50 The values ​​were extrapolated using nonlinear regression with a four-parameter concentration inhibition model. Where specified, Tukey's multiple comparison test was used to compare means to determine statistical differences between different dose-response curves. ED 50 The relationship between knockdown activity, including extrapolation of values, and drug levels in tissues was examined using nonlinear regression with a three-parameter concentration-response model. Temporally stratified data (i.e., peak gravimetric analysis and animal survival) were plotted via Kaplan-Meier graphs, with statistical significance verified using the Mantel-Cox test.

[0245] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the preceding specification, the descriptions and illustrations of specific embodiments herein are not intended to be constrained. Numerous variations, modifications, and substitutions will arise for those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions described herein, depending on various conditions and variables. It should be understood that various alternative forms to the embodiments of the present invention described herein may be employed in carrying out the present invention. Therefore, the present invention is intended to also cover such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are intended to be covered thereby. [Table 15] JPEG2026509175000022.jpg255170JPEG2026509175000023.jpg255170JPEG2026509175000024.jpg255170JPEG2026509175000025.jpg255170 JPEG2026509175000026.jpg255170JPEG2026509175000027.jpg255170JPEG2026509175000028.jpg255170JPEG2026509175000029.jpg255170

[0246] Note: The target sequence is identical to the identified sense sequence, but the nucleotide "U" is replaced with "T", and the two overhanging nucleotides "TT" are removed.

Claims

1. An siRNA that forms a double-stranded structure comprising a sense strand and an antisense strand, wherein at least one of the sense strand and the antisense strand contains a nucleotide sequence having at least 85% nucleotide sequence complementarity or homology with a portion of the nucleotide molecule described in Sequence ID No. 1, and the siRNA inhibits or suppresses the expression of superoxide dismutase 1 (SOD1) transcript in cells.

2. siRNA according to claim 1, wherein at least one of the two strands may have 0, 1, 2, or 3 mismatches with respect to a portion of the nucleotide sequence described in SEQ ID NO: 1, and / or the portion of the nucleotide sequence may include a sequence selected from any of the sequences described in SEQ ID NOs: 2-269, and / or the sense strand may have at least 85% nucleotide sequence homology with respect to a sequence described in any of SEQ ID NOs: 270-537, and / or the antisense strand may have at least 85% nucleotide sequence homology with respect to a sequence described in any of SEQ ID NOs: 538-805, and / or the siRNA is SOD1 An siRNA that can inhibit or suppress intracellular superoxide dismutase 1 (SOD1) transcript expression by at least 10% compared to baseline mRNA levels, and / or the sense strand may contain 10 or more consecutive nucleotides, and / or the antisense strand may contain 10 or more consecutive nucleotides, and / or the sense strand and antisense strand may form a double-stranded structure containing 0, 1, 2, or 3 mismatches.

3. An siRNA according to claim 1, characterized in that the siRNA comprises a sense strand containing the sequence described in sequence number n (any integer between 270 and 537) and an antisense strand containing the sequence described in sequence number n+268.

4. The siRNA according to claim 1, wherein the siRNA may contain at least one chemically modified nucleotide located on the sense strand, the antisense strand, or at least one of both strands, and / or the chemically modified nucleotide may be located at the 5' end, the 3' end, both ends, or inside the strand, and / or at least 50% of the nucleotides in the sense strand, the antisense strand, or both strands may be chemically modified.

5. The siRNA according to claim 4, wherein the chemically modified nucleotide may be one or more selected from the group consisting of the following, which includes modification of the 2' sugar moiety, modification of the base, modification of the phosphorothioate (PS) skeleton, addition of a 5'-phosphate group to the 5' end, or addition of a 5-methylcytosine group.

6. siRNA according to claim 5, wherein the modification of the 2' sugar moiety may be one or more selected from the group consisting of 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, and / or the addition of a phosphate group to the 5' terminus may be one or more selected from the group consisting of the addition of an (E)-vinylphosphonate group to the 5' terminus of the nucleotide sequence.

7. siRNA according to claim 4, wherein the sense strand may have at least 85% nucleotide sequence homology with respect to the sequence described in any of SEQ ID NOs. 808-827 and 867, with or without the addition of an (E)-vinylphosphonate group to the 5′ end, and / or the antisense strand may have at least 85% nucleotide sequence homology with respect to the sequence described in any of SEQ ID NOs. 828-849 and 868, with or without the addition of an (E)-vinylphosphonate group to the 5′ end.

8. The siRNA according to claim 4, wherein the siRNA may include a sense strand containing the sequence described in sequence number m (any integer from 808 to 827) and an antisense strand containing the sequence described in sequence number m+20, or a sense strand containing the sequence described in sequence number 814 and an antisense strand containing the sequence described in sequence number 848, or a sense strand containing the sequence described in sequence number 822 and an antisense strand containing the sequence described in sequence number 849, or a sense strand containing the sequence described in sequence number 814 and an antisense strand containing the sequence described in sequence number 866.

9. An oligonucleotide agent comprising an siRNA according to any one of claims 1 to 8, wherein (a) the siRNA according to any one of claims 1 to 8; and (b) a non-targeting single-stranded oligonucleotide (accessory oligonucleotide, ACO) having a length of about 6 to 22 nucleotides, the siRNA and the ACO are covalently linked via one or more linker components or without linker components.

10. An oligonucleotide agent according to claim 9, wherein the ACO may consist of one or more selected from the group consisting of RNA, DNA, BNA (bridged nucleotide), LNA (locked nucleotide), GNA (glycerol nucleotide), and PNA (peptide nucleotide).

11. An oligonucleotide agent according to claim 9, wherein the ACO may have a length of about 6 to 18 nucleotides.

12. An oligonucleotide agent according to claim 11, wherein the ACO may have a length of about 8 to 16 nucleotides. The oligonucleotide agent according to claim 16, wherein the single-stranded oligonucleotide has a length of 8-11 nucleotides.

13. An oligonucleotide agent according to claim 9, wherein the siRNA may include a sense chain having a length of about 16 to 25 nucleotides and / or an antisense chain having a length of about 19 to 25 nucleotides.

14. An oligonucleotide agent according to claim 9, wherein the oligonucleotide agent is capable of inhibiting or suppressing the transcript of superoxide dismutase 1 (SOD1) in cells by at least 50% compared to baseline SOD1 mRNA levels.

15. An oligonucleotide agent according to claim 9, wherein the ACO has a 5' end and a 3' end, and at least one of the 5' end or 3' end of the ACO may be bound to a linker component.

16. An oligonucleotide agent according to claim 9, wherein the sense strand and / or antisense strand of the siRNA may be covalently linked to the ACO via one or more linker components.

17. An oligonucleotide agent according to claim 9, wherein the linker component may be one or more selected from the group consisting of ethylene glycol chain, alkyl chain, alkenyl chain, alkynyl chain, peptide, RNA, DNA, sugar, thiol bond, phosphodiester bond, phosphorothioate bond, phosphoramidate bond, amide bond, carbamate bond, tetrazole bond, and benzimidazole bond.

18. The oligonucleotide agent according to claim 17, wherein the linker component may be one or more selected from the following: a) Spacer phosphoramidite 18 (phosphoramidic acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecyl-1-yl 2-cyanoethyl ester). b) Spacer-9 (3-[2-[2-[2-[bis(4-methoxyphenyl)phenylmethoxy]ethoxy]ethoxy]ethoxy-[di(propan-2-yl)amino]phosphoranyl]oxypropanitrile) 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-hexaoxahenicosan-CPG.

19. An oligonucleotide agent according to claim 9, wherein the ACO may be covalently linked to the 3′ end, the 5′ end, or both ends of the sense strand of the siRNA, and / or the ACO may be covalently linked to the 3′ end, the 5′ end, or both ends of the antisense strand of the siRNA, and / or the ACO may be covalently linked to one or more nucleotides inside the siRNA.

20. An oligonucleotide agent according to claim 9, wherein one, two or more ACOs may be covalently linked to the siRNA.

21. An oligonucleotide agent according to any one of claims 9-20, wherein at least one nucleotide of the ACO may be chemically modified.

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

23. The oligonucleotide agent according to claim 21, wherein the chemical modification in at least one chemically modified nucleotide may be a modification of a 2′ sugar moiety selected from one or more of the following: 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and 2'-O-(2-methoxyethyl) (2'-O-MOE) modification.

24. An oligonucleotide agent according to claim 21, wherein the chemical modification of the at least one chemically modified nucleotide may be a phosphorothioate (PS) skeleton modification.

25. An oligonucleotide agent according to claim 24, wherein the ACO may contain 6 to 17 phosphorothioate (PS) skeleton modifications.

26. An oligonucleotide agent according to claim 13, wherein the antisense chain may have an (E)-vinylphosphonate group added to the 5′ end of the nucleotide sequence.

27. An oligonucleotide agent according to claim 21, wherein the chemical modification of the at least one chemically modified nucleotide may be the addition of a 5-methylcytosine group to the 5′ end of the nucleotide sequence.

28. An oligonucleotide agent according to claim 10, wherein the ACO and / or the siRNA may be conjugated to one or more conjugation groups.

29. An oligonucleotide agent according to claim 28, wherein the sense strand and / or antisense strand of the siRNA may be bound to one or more conjugation groups.

30. An oligonucleotide agent according to claim 28, wherein the one or more conjugation groups may be one or more selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

31. An oligonucleotide agent according to claim 28, wherein the one or more conjugation groups may be one or more selected from cell-permeable peptides, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

32. An oligonucleotide agent according to any one of claims 10 to 28, wherein the sense strand and antisense strand of the siRNA may each independently contain a nucleotide sequence having at least about 85% sequence homology to any of the following nucleotide sequences: RD-12926 (SEQ ID NO: 867 and SEQ ID NO: 868), siSOD1-063M1 (SEQ ID NOs. 808 and 828), siSOD1-063M2 (SEQ ID NOs. 809 and 829), siSOD1-063M3 (SEQ ID NOs. 810 and 830), siSOD1-063M4 (SEQ ID NOs. 811 and 831), siSOD1-047M1 (SEQ ID NOs. 812 and 832), siSOD1-047M2 (SEQ ID NOs. 813 and 833), siSOD1-047M3 (SEQ ID NOs. 814 and 834), siSOD1-047M4 (SEQ ID NOs. 815 and 835), siSOD1-104M1 (SEQ ID NOs. 816 and 836), siSOD1-104M2 (SEQ ID NOs. 817 and 837), siSOD1-104M3 (SEQ ID NOs. 818 and 838), siSOD1-104M4 (SEQ ID NOs. 819 and 839), siSOD1-005M1 (SEQ ID NOs. 820 and 840), siSOD1-005M2 (SEQ ID NOs. 821 and 841), siSOD1-005M3 (SEQ ID NOs. 822 and 842), siSOD1-005M4 (SEQ ID NOs. 823 and 843), siSOD1-258M1 (SEQ ID NOs. 824 and 844), siSOD1-258M2 (SEQ ID NOs. 825 and 845), siSOD1-258M3 (SEQ ID NOs. 826 and 846), siSOD1-258M4 (SEQ ID NOs. 827 and 847), siSOD1-270M3 (SEQ ID NOs. 814 and 866), siSOD1-047M3VP (SEQ ID NOs. 814 and 848), siSOD1-005M3VP (SEQ ID NOs. 822 and 847), siSOD1-047M3-AC1 (SEQ ID NOs. 850 and 834), siSOD1-005M3-AC1 (SEQ ID NOs. 851 and 842), siCON1-AC1VP (SEQ ID NOs. 852 and 858), siCON2-AC1VP (SEQ ID NOs. 853 and 859), siCON3-AC1VP (SEQ ID NOs. 854 and 860), siSOD1-063M3-AC1VP (SEQ ID NOs. 855 and 861), siSOD1-047M3-AC1VP (SEQ ID NOs. 850 and 848), siSOD1-104M3-AC1VP (SEQ ID NOs. 856 and 862), siSOD1-005M3-AC1VP (SEQ ID NOs. 852 and 847), siSOD1-258M3-AC1VP (SEQ ID NOs. 857 and 863),

33. An oligonucleotide agent according to claim 10, wherein the ACO in the oligonucleotide agent improves the stability, bioavailability, in vivo distribution and / or intracellular uptake of the siRNA compared to an oligonucleotide agent that does not contain ACO.

34. An oligonucleotide agent according to claim 10, wherein the ACO in the oligonucleotide agent increases the in vivo distribution of the siRNA in one or more target tissues compared to an oligonucleotide agent that does not contain ACO.

35. Oligonucleotide agent according to claim 34, wherein the target tissue may be one or more selected from the prefrontal cortex, cerebrum, cerebellum, spinal cord, muscle, lung, eye, liver, and kidney.

36. A vector comprising an siRNA (as described in any of claims 1-9) and / or an oligonucleotide agent as described in any of claims 10-35.

37. A cell comprising siRNA (as described in any of claims 1-9), an oligonucleotide agent as described in any of claims 10-35, and / or a vector as described in claim 36.

38. A cell according to claim 37, wherein the cell may be a mammalian cell.

39. A cell according to claim 37, wherein the cell may be a human cell.

40. A cell according to claim 37, wherein the cell may be a host cell in vitro, in vivo, or ex vivo.

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

42. A pharmaceutical composition according to claim 41, wherein the pharmaceutically acceptable carrier may be one or more selected from aqueous carriers, liposomes or lipid nanoparticles (LNPs), polymers, micelles, colloids, metal nanoparticles, nonmetal nanoparticles, bioconjugates, or polypeptides.

43. A pharmaceutical composition according to claim 41, wherein the pharmaceutical composition reduces the level of the SOD1 gene transcript or the SOD1 protein.

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

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

46. A method for treating or delaying the onset or progression of neurodegenerative diseases or symptoms associated with SOD1 gene mutations, abnormal SOD1 gene expression, or accumulation of abnormal SOD1 protein, comprising the step of administering the pharmaceutical composition according to any one of claims 41 to 43 to a subject who requires it.

47. The method according to claim 46, wherein the neurodegenerative disease or condition may be one selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down syndrome (DS).

48. A method according to claim 46, wherein the pharmaceutical composition may be administered intrathecally or intraventricularly.

49. A method according to claim 47, wherein the ACO in the oligonucleotide agent improves the stability, bioavailability, in vivo distribution and / or intracellular uptake of the siRNA compared to an oligonucleotide agent that does not contain ACO.

50. Use of an siRNA according to any one of claims 1 to 9, an oligonucleotide agent according to any one of claims 10 to 35, a vector according to claim 36, cells according to any one of claims 37 to 40, and / or a pharmaceutical composition according to any one of claims 41 to 43, in the manufacture of a pharmaceutical for the treatment, prevention of onset, or prevention of progression of SOD1-related neurodegenerative disease or its symptoms.

51. The use according to claim 50, wherein the SOD1-related neurodegenerative disease or its symptoms is one selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down syndrome (DS).

52. An siRNA according to any one of claims 1 to 9, an oligonucleotide agent according to any one of claims 10 to 35, a vector according to claim 36, a cell according to any one of claims 37 to 40, and / or a pharmaceutical composition according to any one of claims 41 to 43, which is used for the treatment, prevention of onset or inhibition of progression of SOD1-related neurodegenerative disease or its symptoms.

53. An siRNA, oligonucleotide agent, vector, cell and / or pharmaceutical composition according to claim 52, wherein the neurodegenerative disease or symptom is one selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down syndrome (DS).

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