WO2024175087A1

WO2024175087A1 - Oligonucleotide targeting sod1

Info

Publication number: WO2024175087A1
Application number: PCT/CN2024/078297
Authority: WO
Inventors: Longcheng Li; Moorim KANG
Original assignee: Ractigen Therapeutics
Current assignee: Ractigen Therapeutics
Priority date: 2023-02-24
Filing date: 2024-02-23
Publication date: 2024-08-29
Anticipated expiration: 2025-08-24
Also published as: MX2025010026A; AU2024224435A1; CN120752341A

Abstract

Provided is a siRNA or an oligonucleotide agent for preventing or treating SOD1-associated neurodegenerative diseases or symptoms (such as amyotrophic lateral sclerosis, ALS). The oligonucleotide agent comprises a double-stranded targeting oligonucleotide (siRNA) and a non-targeting single-stranded oligonucleotide (ACO), in which the siRNA targets a mRNA region of the target gene SOD1.

Description

OLIGONUCLEOTIDE TARGETING SOD1

TECHNICAL FIELD

The present application relates to the technical field of nucleic acids, specifically as it relates to an oligonucleotide agent for reducing SOD1 transcript level and pharmaceutical use thereof.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS) is an adult-onset, lethal, paralytic disorder caused by the degeneration of motor neurons. ALS is characterized by progressive, adult-onset degeneration of cranial, brainstem and spinal motor neurons, leading to death by respiratory failure within 3-5 years of diagnosis. ALS presents as a familial or a sporadic form, depending on whether or not there is a family history of the disease, with sporadic ALS (sALS) accounts for 90%of the ALS patients. The most common mutant genes account for ～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%) and fused in sarcoma/translocated in liposarcoma (FUS/TLS; 4%) . Mutations in the C9orf72 and SOD1 genes also account for ～5–8%and ～2–3%of apparently sALS, respectively. To date, there is currently no effective therapy available for ALS and new therapies are needed to treat this disease.

Among the known genes underlining ALS, SOD1 gene remains a major cause of fALS and has been considered to be an important ALS drug target. The first description of the ALS disease dates back to at least 1824 by Charles Bell, however, SOD1 as the first risk gene of ALS was discovered in 1993. When the first human SOD1 transgenic mouse model (hSOD1^G93A) was established in 1994, indicating that the research on ALS entered a new era. SOD1 mutants cause disease most probably via a gain-of-function and reducing its levels is believed beneficial. Therefore, silencing SOD1 transcript level is an important strategy for the treatment of ALS.

SUMMARY OF THE INVENTION

To address the problem, the present application provides an oligonucleotide agent with potent inhibitory effect on the expression of superoxide dismutase 1 (SOD1) . The oligonucleotide agent comprises a small interfering RNA (siRNA) , for treating diseases or conditions caused by the mutation of SOD1 gene such as ALS by targeting SOD1 mRNA and subsequently downregulating SOD1 protein level in a cell or an individual via RNA interference (RNAi) .

In particular, the inventors discovered that an oligonucleotide agent with strong potency for knocking down SOD1 transcript and high CNS delivery efficiency, comprising: (a) a siRNA; and (b) a non-targeting single-stranded accessary oligonucleotide (ACO) , wherein the ACO is 6-22 nucleotides in length, wherein the siRNA and the ACO are covalently linked, with or without one or more linking components, to form the oligonucleotide agent.

In some embodiments, the siRNA comprises a sense strand and an antisense strand forming a duplex structure, wherein the antisense strand comprises a nucleotide sequence comprising at least 10 contiguous nucleotides, with 0, 1, 2 or 3 mismatches, having at least 85%nucleotide sequence complementarity or homology to a portion of the nucleotide sequence of SEQ ID NO: 1 (Table 1) .

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

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

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

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

In some embodiments, an oligonucleotide agent comprising a siRNA is provided, wherein the siRNA comprising 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 the nucleotide sequence selected from SEQ ID NOs: 270-537, 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 the nucleotide sequence selected from SEQ ID NOs: 538-805.

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

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

In some embodiments, the ACO is conjugated to a linking component. In some embodiments, the 5’ end or the 3’ end or one or more of internal nucleotides of the ACO is conjugated to a linking component. In some embodiments, the siRNA and the ACO are covalently linked by a linking component. In some embodiments, the sense strand or the antisense strand of the siRNA are covalently linked to the ACO by a linking component. In some embodiments, the linking component is or comprises one or more selected from the group consisting of ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, RNA, DNA, carbohydrates, thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, a tetrazole linkage, and a benzimidazole linkage. In some embodiments, the linking component is or comprises one or more selected from the group consisting of:

a) Spacer phosphoramidite 18 (Phosphoramidous acid, N, N-bis (1-methylethyl) -, 19, 19-bis (4-methoxyphenyl) -19-phenyl-3, 6, 9, 12, 15, 18-hexaoxanonadec-1-yl 2-cyanoethyl ester) ;

b) Spacer-9 (3- [2- [2- [2- [bis (4-methoxyphenyl) -phenylmethoxy] ethoxy] ethoxy] ethoxy- [di (propan-2-yl) amino] phosphanyl] oxypropanenitrile) ;

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.

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

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%nucleotides of the ACO are chemically modified nucleotides. 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%nucleotides of the sense strand of the siRNA are chemically modified nucleotides. 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%nucleotides of the antisense strand of the siRNA are chemically modified nucleotides. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is a 2’ sugar modification selected from one or more of: 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 the at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification. In some embodiments, the ACO comprises at least one phosphorothioate (PS) backbone modification. In some embodiments, the ACO comprises 6～17 phosphorothioate (PS) backbone modifications. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is an addition of a 5'-phosophate moiety at the 5’ end of the nucleotide sequence. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is an addition of an (E) -vinylphosphonate moiety at the 5’ end of the nucleotide sequence. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is an addition of a 5-methyl cytosine moiety at the 5’ end of the nucleotide sequence.

In some embodiments, the ACO is conjugated to one or more conjugation groups. In some embodiments, the siRNA is conjugated to one or more conjugation groups. In some embodiments, the sense strand or the antisense strand of the siRNA is conjugated to one or more conjugation groups. In some embodiments, the one or more conjugation groups are selected from: a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody. In some embodiments, the one or more conjugation groups are selected from: a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.

Aspects of the present application relate to an oligonucleotide agent comprising a siRNA and a non-targeting ACO, wherein the sense strand has a nucleotide sequence that is at least 85%, at least 90%, or at least 95%homology to the 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 the nucleotide sequence selected from SEQ ID NOs: 828-849 and 861-863.

Aspects of the present application include a vector comprising the oligonucleotide agent disclosed herein.

Another aspect of the present application provides a cell comprising the oligonucleotide agent disclosed herein. In one embodiment, the cell is a mammalian cell, optionally a human cell. In some embodiments, the cell is a host cell. The aforementioned cell may be in vitro, such as a cell line or a cell strain, or may exist or be taken from in a mammalian body, such as a human body.

Yet another aspect of the present application provides a pharmaceutical composition comprising the oligonucleotide agent disclosed herein. In some embodiments, the composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugates, and polypeptide. Also provided by the present application includes a kit comprising the oligonucleotide agent or the pharmaceutical composition disclosed herein.

The present application provides a method of decreasing the transcript of a SOD1 gene or SOD1 protein, comprising administering to a subject a pharmaceutical composition disclosed herein.

The present application also provides a method for treating or delaying the onset or progression of Amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: administering to the subject a pharmaceutical composition disclosed herein. In some embodiments, the subject has sporadic ALS (sALS) . In some embodiments, the subject has familial ALS (fALS) . In some embodiments, the pharmaceutical composition decreases the transcript of the SOD1 gene or SOD1 protein.

In some embodiments, the ACO of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the siRNA as compared to an oligonucleotide agent without the ACO.

In some embodiments, the ACO of the oligonucleotide agent increases the biodistribution of siRNA within one or more target tissues as compared to an oligonucleotide agent without the ACO.

In some embodiments, the ACO of the oligonucleotide agent increases the biodistribution of siRNA within two or more target tissues as compared to an oligonucleotide agent without the ACO.

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

The present application partially relates to a siRNA comprising an oligonucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides, wherein the continuous 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 to an equal length portion of SEQ ID NO: 1, wherein the siRNA inhibits the mRNA transcript of SOD1 gene by at least 80%as compared to the baseline of SOD1 mRNA level.

The inventors also discovered that optimal target sequences/sense strand of an siRNA within the SOD1 gene include sequences having: (1) a GC content between 35%and 65%; (2) less than 5 consecutive identical nucleotides; (3) 3 or less dinucleotide repeats; and (4) 3 or less trinucleotide repeats. As a beneficial consequence, a target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) , upon interacting with the siRNA, can inhibit the SOD1 mRNA transcript level by at least 80%in a cell as compared to a baseline level of SOD1 mRNA. Based at least in part on these discoveries, the present disclosure features siRNA, compositions, and pharmaceutical compositions for inhibiting the SOD1 mRNA transcript level by at least 80%in a cell as compared to baseline levels of SOD1 mRNA. Also provided herein are methods for preventing or treating a disease or symptom induced by SOD1 gene mutation or abnormal level of SOD1 protein in a cell in an individual comprising administering any of the siRNA, compositions, and/or pharmaceutical compositions described herein.

Additional aspects and advantages of the present application will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present application are shown and described. As will be realized, the present application is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENC

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIGs. 1A-1E show the siRNA screen for SOD1 knockdown in vitro. FIG. 1A shows that HEK293A cells were transfected with each siRNA duplex (268 in total) in duplicate at 10 or 0.1 nM concentrations for 24 hours. SOD1 expression levels were quantified via RT-qPCR using gene specific primer sets. TBP was amplified as an internal reference used to normalize expression data. Shown are the expression values of SOD1 mRNA of each experimental replicate relative to Mock treatments (dotted line) . Mock samples were transfected in absence oligonucleotide. FIG. 1B shows the knockdown activity and cell viability of the top performing siRNAs which were quantified in HEK293A cells at 6 escalating concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM) via RT-qPCR and PI staining, respectively. Shown are the results for both SOD1 knockdown and cytotoxicity of the top 5 performing siRNAs (i.e., siSOD1-063, 047, 104, 005, and 258) relative to Mock treatments (dotted line) . FIG. 1C shows that dose response curves were generated in SK-N-AS cells for each of the top 5 siRNAs at 8 treatment concentrations (i.e., 0.00006, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25, and 1 nM) via RT-qPCR. Data represents mean ± SD from 2 experimental replicates. FIG. 1D shows that the SK-N-AS cells were transfected at 0.1 nM with different chemically modified variants of each siRNA or non-specific siRNA control (siCon) for 24 hours. Knockdown activity was assessed via RT-qPCR relative to Mock treatment. FIG. 1E shows that dose response curves were generated in SK-N-AS cells for each of the M3 modified variants (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, and 258M3) via RT-qPCR. Data represents mean ± SD from 3 experimental replicates.

FIGs. 2A-2B show the secondary screening of siRNA potency and untoward cytotoxicity in vitro. Knockdown activity and cell viability in HEK293A cells of the remaining top 25 performing siRNAs identified in the initial screen is shown following transfection at 6 indicated concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4, and 20 nM) . SOD1 knockdown and untoward cytotoxicity were quantified by RT-qPCR in FIG. 2A and PI staining in FIG. 2B relative to Mock treatments, respectively.

FIG. 3 shows the dose-dependent knockdown of the top 5 siRNA candidates in T98G cells. Dose response curves were generated in T98G cells following transfection for 24 hours with each of the top 5 siRNAs at 8 treatment concentrations (i.e., 0.00006, 0.0002, 0.001, 0.004, 0.016, 0.063, 0.25, and 1 nM) . SOD1 expression levels were quantified via RT-qPCR using gene specific primer sets. TBP was amplified as an internal reference used to normalize expression data. Shown are mean values ± SD from 2 experimental replicates relative to controls treated in absence of oligonucleotide.

FIGs. 4A-4B show the untoward cytotoxicity of the top 5 siRNA candidates in vitro. SK-N-AS and T98G cells were transfected with each of the top 5 siRNAs at 4 treatment concentrations (i.e., 1.56, 6.25, 25, and 100nM) in noted excess of their IC₅₀ values for SOD1 knockdown. Treatment with siCon at 100 nM served as a negative control (Neg Con) . Mock samples were transfected in absence oligonucleotide. Untoward cytotoxicity was evaluated 72 hours after treatment by quantifying both caspase 3/7 activity in FIG. 4A and metabolism of WST-8 reagent as a marker for cell viability in FIG. 4B. Data is shown as mean values ± SD from 2 experimental replicates relative to Mock treatments (dotted lines) .

FIG. 5 shows the impact of different chemical modifications patterns on siRNA knockdown activity in T98G cells. T98G cells were transfected at 0.1 nM with 4 different chemically modified variants of each siRNA candidate (i.e., M1, M2, M3, or M4) or a non-specific siRNA control (siCon) for 24 hours. Knockdown activity was assessed via RT-qPCR relative to Mock treatment. Data represents mean ± SD from 2 experimental replicates.

FIG. 6 shows the dose-dependent knockdown of M3-modified siRNAs in T98G cells. Dose response curves were generated in T98G cells following transfection for 24 hours 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 via RT-qPCR using gene specific primer sets. TBP was amplified as an internal reference used to normalize expression data. Shown are mean values ± SD from 3 experimental replicates relative to samples treated in absence of oligonucleotide.

FIGs. 7A-7D show the siRNA-ACO conjugate knockdown activity in vitro. FIG. 7A depicts a visual representation of the siRNA-ACO conjugate structure in which a 14-nt ACO (referred to AC1) is conjugated to the 3’-terminus of the sense strand via short L9 linker (i.e., Spacer-9 linker) . FIG. 7B shows that HEK293A and T98G cells were transfected at 0.25 or 2.5 nM concentrations with exemplary siRNA-ACO (i.e., siSOD1-005M3-AC1) or AC1 only for 24 hours. Mock samples were transfected in absence oligonucleotide. Treatment with siCon served as a negative control for knockdown activity. SOD1 expression levels were quantified via RT-qPCR using gene specific primer sets. TBP was amplified as an internal reference used to normalize expression data. Shown are the expression values of SOD1 mRNA of each experimental replicate relative to Mock treatments (dotted line) . FIG. 7C shows that dose response curves were generated in T98G cells comparing siRNA knockdown activity of siSOD1-005M3 with AC1 (siSOD1-005M3-AC1) or without AC1 conjugation (siSOD1-005M3) 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) via RT-qPCR. FIG. 7D shows that dose response curves were generated comparing knockdown activity of siRNA-ACOs with (siSOD1-005M3-AC1^VP) or without (siSOD1-005M3-AC1) 5’ VP modification as compared to a published ASO sequence cf. T. Miller et al., New England Journal of Medicine 383, 109-119 (2020) ) in both sequence and chemistry (i.e., ASO^SOD1, SEQ ID NO: 864) .

FIGs. 8A-8B show the knockdown activity of siRNA-ACO drug candidates in vitro. Dose response curves were generated in SK-N-AS (FIG. 8A) and T98G (FIG. 8B) cells for each 5’ VP-modified siRNA-ACO (i.e., siSOD1-063M3-AC1^VP, 047M3-AC1^VP, 104M3-AC1^VP, 005M3-AC1^VP, 258M3-AC1^VP, and 270M3-AC1^VP) at gradient treatment concentrations. SOD1 expression levels were quantified via RT-qPCR using gene specific primer sets. TBP was amplified as an internal reference to normalize data. Shown are the expression values of SOD1 mRNA relative to mock transfections. Data represents mean ± SD from 2 experimental replicates.

FIGs. 9A-9B show the untoward cytotoxicity of siRNA-ACO drug candidates in vitro. SK-N-AS and T98G cells were transfected with the lead siRNA-ACO conjugates (i.e., siSOD1-063M3-AC1^VP, 047M3-AC1^VP, 104M3-AC1^VP, 005M3-AC1^VP, 258M3-AC1^VP) at gradient treatment concentrations in excess of their IC₅₀ values (i.e., 1.56, 6.25, 25, and 100 nM) . Treatment with siCon at 100 nM served as a Neg Con. Mock samples were transfected in absence oligonucleotide. Untoward cytotoxicity was evaluated 72 hours after treatment by quantifying both caspase 3/7 activity in FIG. 9A and metabolism of WST-8 reagent as a marker for cell viability in FIG. 9B. Data is shown as mean values ± SD from 2 experimental replicates relative to Mock treatments (dotted lines) .

FIG. 10 shows siRNA-ACO activity in CNS tissue of hSOD1^G93A mice. Adult hSOD1^G93A mice were treated via intracerebroventricular (ICV) injection with each siRNA-ACO drug candidate (i.e., siSOD1-047M3-AC1^VP and 005M3-AC1^VP) at a fixed molecular dose (20 nmole) . Treatment with aCSF alone was used as a vehicle control to establish baseline expression, while a non-specific siRNA-ACO (i.e., siCON2-AC1^VP) served as a negative control. Knockdown activity was quantified in tissues from the brain (i.e., frontal cortex, cerebellum, and cerebrum) , spinal cord (i.e., cervical, thoracic, and lumbar) , and periphery (i.e., liver) via RT-qPCR on day 14 following treatment via ICV injection at a fixed molecular dose (20 nmole) with siRNA-ACO candidates (i.e., siSOD1-047M3-AC1^VP and 005M3-AC1^VP) or their non-conjugated derivatives (i.e., siSOD1-047M3^VP and 005M3^VP) . Mouse Tbp (mTbp or Tbp) was amplified as an internal reference to normalize expression data. Shown are the mean expression values ± SD (n=2-6 mice/group) of human SOD1 (hSOD1 or SOD1) relative to aCSF treatment. The dotted gray line represents 80%knockdown relative to baseline (dashed line) .

FIGs. 11A-11B show the dose-dependent relationship between knockdown activity and tissue accumulation of siRNA-ACO in different CNS tissues. Adult hSOD1^G93A mice were treated with siSOD1-047M3-AC1^VP (FIG. 11A) or siSOD1-005M3-AC1^VP (FIG. 11B) at the indicated doses (i.e., 50, 100, 200, or 400 μg) via ICV injection. Knockdown activity of hSOD1 was quantified via RT-qPCR in select CNS tissues (i.e., cerebellum, cerebrum, and spinal cord) on day 14 following treatment. Animals treated with aCSF alone represent expression levels at baseline and drug quantities detectable in absence of siRNA-ACO treatment (0 μg) . FIGs. 11A-11B show knockdown activity as percent (%) inhibition of SOD1 relative to baseline (0 μg) . Drug concentrations are shown as siRNA-ACO quantities relative to tissue sample mass (μg/g) . Data represents mean ± SD (n=3-4 mice/group) .

FIGs. 12A-12C show the siRNA-ACO delays disease onset and extends animal survival by a single dose ICV injection. Adult hSOD1^G93A mice were treated with siSOD1-047M3-AC1^VP or siSOD1-005M3-AC1^VP at the indicated doses (i.e., 50, 100, 200, or 400 μg) via ICV injection on postnatal day (PND) 85 or PND 60, respectively. Growth rates (i.e., percent change in body weight) relative to first day of treatment (dotted line) were plotted to monitor disease progression in FIG. 12A. Disease onset is plotted as percentage of animals at their peak body weight in each treatment group in FIG. 12B. Animal survival is plotted as percentage of surviving animals in each treatment group in FIG. 12C. Animal numbers (n) are indicated in each graph.

FIGs. 13A-13D show the pathogenic SNPs in target sites of lead siRNA-ACO candidates. FIGs. 13A-13B show the location of pathogenic SNPs within the target sites of siSOD1-005M3-AC1^VP (FIG. 13A) and siSOD1-047M3-AC1^VP (FIG. 13B) . Shown is guide strand sequence including “seed” region (highlighted in grey) complementary to target sites in hSOD1 transcript containing the indicated pathogenic SNPs. Nucleotide mutation is shown in italic bold in which ‘R’ signifies a purine substitution. FIG. 13C shows the luciferase reporter constructs (i.e., pLuc^SOD1, pLuc^P. E22G, pLuc^P. F21C) containing either consensus sequence or one of the pathogenic mutations (i.e., P. E22G and P. F21C) were co-transfected with siSOD1-047M3-AC1^VP or a non-specific scramble control (siCON2-AC1^VP) at 18 nM concentrations in HEK293A cells. Dose response curves for luciferase activity were generated following co-transfection with siSOD1-047M3-AC1^VP at indicated concentrations (i.e., 0.03, 0.07, 0.22, 0.67, 2.0, 6.0, and 18 nM) in FIG. 13D. Data represents mean ± SEM from 2 experimental replicates relative to samples treated in absence of siRNA. Statistical significance (*) was determined using Tukey’s multiple comparison test to compare the mean values at each data point within the three dose response curves.

FIGs. 14A-14C show two-doses administration of the siRNA-ACO delay disease onset and prolong survival. Male and female adult hSOD1^G93A mice were treated twice with siSOD1-047M3-AC1^VP at the indicated doses (i.e., 75, 150, or 300 μg) via intrathecal (IT) injection on PND 68 and PND 100. Non-specific control (i.e., siCON3-AC1^VP) and ASO^SOD1 were dosed at 150 μg/injection. Treatment with aCSF served as a vehicle control. Body weight in grams (g) was plotted comparative to background animals (wild type, WT) to monitor disease progression in FIG. 14A. Disease onset is plotted as percentage of animals at their peak body weight in each treatment group in FIG. 14B. Animal survival is plotted as percentage of surviving animals in each treatment group in FIG. 14C. Animal numbers (n) are indicated in each graph.

FIGs. 15A-15D show the siRNA-ACO treatment improves motor function in hSOD1^G93A mice. Male and female hSOD1^G93A mice treated twice with siSOD1-047M3-AC1^VP at with the indicated doses (i.e., 75, 150, or 300 μg) via IT on PND 68 and PND 100. Male and female hSOD1^G93A mice were subject to open field tests during the daytime. Total distance each animal moved was autonomously recorded in centimeters (cm) over the course of 15 minutes. Data is plotted as the mean ± SD distance traveled for each treatment group in FIG. 15A. Grip strength was assessed in male and female animals organized by treatment group. Grip strength tests were performed in triplicate and the average value was recorded for each animal. Data is plotted as mean ± SD for grip strength for each treatment group in grams (g) in FIG. 15B. Animal fatigue and coordination was assessed by rotarod test for 5 minutes. Experiments were performed in triplicate in which the longest latency time to fall was recorded in seconds (s) for each animal and showed in FIG. 15C. Motor function was scored using the ALS TDI neuroscore (NS) scale for all animals prior to open field, rotarod, and/or grip strength tests. Experiments were performed in triplicate in which the longest latency time to fall was recorded in seconds (s) for each animal in FIG. 15D. Mean NS ± SD is shown at each treatment group at the indicated time points.

FIG. 16 shows the temporal comparison of rotarod performance for each individual animal following siRNA-ACO treatment as indicated in FIG. 14. Latency time to fall in seconds (s) as measured by the rotarod test for each individual animal in their corresponding treatment groups at an early timepoint (i.e., PND 90) in comparison to performance at final timepoint (i.e., the last timepoint before dying, see Table 8) . All tests were performed in triplicate in which the longest latency time was recorded for each animal.

FIG. 17 shows the knockdown activity of siRNA on the expression of SOD1 mRNA in HeLa cells. The indicated siRNAs (i.e., RD-15757, RD-18972, RD-12500, RD-18973, RD-18948 and RD-18949) were directly added into culture medium containing HeLa cell at 1500 nM for 3 days. Cells were transfected in the absence of an oligonucleotide as Mock treatments (not shown) . RD-11566 (dsCon2M3v) served as a non-targeting duplex control. SOD1 mRNA levels were quantified by two-step RT-qPCR using a gene specific primer set in individual PCR reactions. TBP was amplified as an internal reference. The values (y-axis) represent the SOD1 mRNA level relative to Mock treatment after normalized to TBP (mean ± SEM of three replicated transfection wells) .

FIGs. 18A-18B show the knockdown activity of siRNA on the expression of SOD1 mRNA in SK-N-AS cells. The indicated siRNAs (i.e., RD-12926, RD-15757, RD-12500, RD-18947, RD-18948, RD-18949, RD-18946, RD-18972 and RD-18973) were transfected into SK-N-AS cells at indicated concentrations (i.e., 0.0002, 0.001, 0.0039, 0.0156, 0.0625, 0.25, 1 and 4 nM) for 24 hours. Cells were transfected in the absence of an oligonucleotide as Mock treatments (not shown) . RD-11566 (dsCon2M3v) served as a non-targeting duplex control (not shown) . FIGs. 18A-18B showed the SOD1 mRNA level as quantified by two-step RT-qPCR using a gene specific primer set in individual PCR reactions. TBP was amplified as an internal reference. The values (y-axis) represent the SOD1 mRNA level relative to Mock treatment after normalized to TBP (mean ± SEM of three replicated transfection wells) .

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Aspects of the present application include methods of treating Amyotrophic lateral sclerosis (ALS) by administering an effective amount of an oligonucleotide agent comprising a SOD1-targeting siRNA. The oligonucleotide agent interferes with SOD1 mRNA transcript through the RNA silencing mechanism (RNAi) . The present inventors have developed SOD1 siRNAs with potent inhibitory effect, improved delivery, biodistribution, bioavailability, and other pharmacological properties, for use in the treatment of ALS.

The present application is based on investigations related to oligonucleotide agent, compositions and methods that, a targeting oligonucleotide (siRNA etc. ) , in combination with an oligonucleotide-delivery vehicle (ODV) , can downregulate/decrease a gene expression, to improve therapeutic effects for genetic conditions. The term “oligonucleotide-delivery vehicle (ODV) ” refers to a structure by conjugating an “accessory oligonucleotide (ACO) ” to a molecule, e.g., a duplex oligonucleotide, to facilitate the introduction of the molecule into or uptake by a cell, a tissue, or an organ of an individual.

The present inventors found that some siRNA duplex sequences showed better inhibitory potency on SOD1 transcript than the sequences having the same or similar target sequence in the prior art. The present inventors also found that chemical modification to the siRNA can improve siRNA activity in vitro. Surprisingly, by conjugated to a single stranded accessory oligonucleotide (ACO, or ODV) , the oligonucleotide agent (i.e., ODV-siRNA or siRNA-ACO) achieved CNS delivery, desired biodistribution and bioavailability in CNS tissues when administered in either brain or spinal cord tissue via intracerebroventricular (ICV) and/or intrathecal (IT) injections.

DEFINITIONS

In the present application, the related terms are defined as follows:

Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

The transitional terms/phrases (and any grammatical variations thereof) "comprising" , "comprises" , "comprise" , include the phrases "consisting essentially of" , "consists essentially of" , "consisting of" , and "consists of" and can be interchanged throughout the application. The open term "comprise" also includes a closed term "consisting of" as one option. As used herein, the terms "include, " "have" and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

The terms “Amyotrophic lateral sclerosis” or “ALS” include, but are not limited to, familial ALS (fALS) , sporadic ALS (sALS) , Lou Gehrig's disease, diseases associated with mutant genes Chromosome 9 Open Reading Frame 72 gene (C9orf72; 40%) , superoxide dismutase 1 (SOD1; 20%) , transactive response DNA-binding protein 43 (TDP43; 4%) and fused in sarcoma/translocated in liposarcoma (FUS/TLS; 4%) .

The term "target gene" as used herein can refer to nucleic acid sequences in the form of DNA, RNA or DNA/RNA hybrid, transgenes, viral or bacterial sequences, chromosomes or extrachromosomal genes that are naturally present in organisms, and/or can be transiently or stably transfected or incorporated into cells and/or chromatins thereof. The target gene can be a protein-coding gene or a non-protein-coding gene (such as a microRNA gene and a long non-coding RNA gene) , or a transcript of the protein-coding gene, such as, a message RNA (mRNA) or a complementary DNA (cDNA) of the protein-coding gene. "Target sequence" , "target site" or "target" used interchangeably refers to a consecutive oligonucleotide sequence in the sequence of a target gene, such as, the mRNA or cDNA of a target gene, which is homologous or complementary with a sense strand or an antisense strand of a siRNA with or without one or more mismatched based pairs.

As used herein, the terms "SOD1" and "SOD1 gene" can be used interchangeably, and refer to a gene encoding SOD1 protein, preferably a mammalian gene, and more preferably a human gene. As used herein, the term "SOD1 mRNA" refers to a message RNA (mRNA) generated from the expression of SOD1 gene, or the transcription of SOD1 gene. As used herein, the term "SOD1 cDNA" refers to a complementary DNA (cDNA) generated from the reverse-transcription of a SOD1 mRNA. As used herein, the term "SOD1 protein" refers to a protein generated from the expression of SOD1 gene, or translation of the SOD1 mRNA.

As used herein, the term "baseline expression of SOD1 gene" or "baseline level of SOD1 mRNA" used interchangeably refers to the expression of SOD1 gene of a parallel reference (such as a cell or an individual) without or before the treatment of the siRNA.

The term "oligonucleotide agent" or “oligonucleotide” can be used interchangeably, and refers to polymers of nucleotides, and includes, but is not limited to, single-stranded or double-stranded nucleic acid molecules of DNA, RNA, or DNA/RNA hybrid, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl portions and ribosyl portions, as well as modified and naturally or unnaturally existing frameworks for such oligonucleotides. The oligonucleotide for inhibiting mRNA transcript level of target gene described herein is a small inhibiting nucleic acid molecule (siRNA) , an antisense oligonucleotide molecule (ASO) , or an oligonucleotide delivery vehicle (ODV) conjugated siRNA molecule (siRNA-ACO) .

The terms "oligonucleotide strand" , “strand” and "oligonucleotide sequence" as used herein can be used interchangeably, referring to a generic term for short nucleotide sequences having less than 35 bases (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ) . In a non-limiting example, the length of a strand can be any length ranging from 16 to 25 nucleotides.

As used herein, the terms “subject” and “individual” are used interchangeably herein to mean any living organism that may be treated with agents of the present application. The term “patient” means a human subject or individual, including disclosure infants, children, and adults.

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

The effective amount may vary depending on such factors as the size and weight of the subject, the type of illness, or the particular agents of the application. For example, the choice of the agent of the application could affect what constitutes an “effective amount. ” One of the ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the agents of the application without undue experimentation.

The regime of administration may affect what constitutes an effective amount. The agent of the application can be administered to the subject either prior to or after the disease diagnosis or condition. Further, several divided dosages, as well as staggered dosages, can be administered daily, weekly, monthly, quarterly or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the agent (s) of the application could be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

The terms “treat, ” “treated, ” “treating” , or “treatment” as used herein have the meanings commonly understood in the medical arts, and therefore do not require cure or complete remission, and include any beneficial or desired clinical results. Non-limiting examples of such beneficial or desired clinical results are prolonging survival as compared to expected survival without treatment, reduced symptoms including one or more of the followings: weakness and atrophy of proximal skeletal muscles, inability to sit or walk independently, difficulties in swallowing, breathing, etc.

As used herein, “preventing” or “delaying” a disease refers to inhibiting the full development of a disease.

The term “biological sample” refers to any tissue, cell, fluid, or other material derived from an organism (e.g., human subject) . In some embodiments, the biological sample is serum or blood.

The term "identity" or "homology" as used herein means that one oligonucleotide strand (sense or antisense strand) of a siRNA has sequence similarity with a coding strand or template strand in a region of a target gene. As used herein, the "identity" or "homology" may 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, 10 residues that are different from a reference sequence. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, considering the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as using the Needleman and Wunsch ( (1970) J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www. gcg. com) . The percent identity between two nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ( (1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. It is understood that the molecules described herein may have additional conservative or non-essential nucleic acid substitutions, which do not have a substantial effect on their functions.

In embodiments of the present application, one target gene is SOD1. By “target sequence” is meant a sequence fragment to which the sense oligonucleotide strand or antisense oligonucleotide of the siRNA is homologous or complementary. For example, in some embodiments, a SOD1 siRNA is homologous or complementary to a target select sequence within human SOD1 transcript.

As used herein, the term "non-targeting" means that the referenced accessory oligonucleotide (ACO) which conjugates with the targeting oligonucleotide (e.g., siRNA, saRNA, and etc. ) does not specifically complement to the target sequence which the targeting oligonucleotide functions, and/or that the referenced oligonucleotide (i.e., ACO) does not share the same target sequence which the targeting oligonucleotide (e.g., siRNA, saRNA, and etc. ) specifically attends to function to. The targeting oligonucleotide disclosed herein is a nucleic acid sequence that specifically complements to the target sequence or the region thereof. In some embodiments, the term "non-targeting oligonucleotide" may comprise any referenced oligonucleotide except the “targeting sequence” . In some cases, the “specifically complementary” may mean that the complementarity between the targeting oligonucleotide and the target sequence or the region thereof is at least about 95%. The non-targeting oligonucleotide (i.e., ACO) is not to elicit biological activity via any known mechanism, nor intended to elicit activities indicative of ASO (i.e., “mixmer” or “gapmer” ) function onto a complementary nucleic acid sequence (i.e., mRNA) in a certain subject, an organ of the subject, a tissue of the subject, or a cell of the subject, when the oligonucleotide is administered. The non-targeting oligonucleotide (i.e., ACO) is to facilitate the introduction of the targeting oligonucleotide (e.g., siRNA, saRNA, and etc. ) it conjugates into a certain subject, an organ of the subject, a tissue of the subject, a cell of the subject, or a cell nucleus of the subject, when the oligonucleotide conjugate is administered.

As used herein, the term "gapmer" refers to a short DNA antisense oligonucleotide (ASO) structure with modified RNA segments on both sides of the central DNA structure. In some embodiments, at least one of the modified RNA segments comprises one or more of modified nucleotides selected from locked nucleic acids (LNA) , and 2'-OMe or 2'-F modified nucleotides to increase affinity to the target, increase nuclease resistance, reduce immunogenicity, and/or decrease toxicity. In some embodiments, a gapmer comprises at least one nucleotide modified with a phosphorothioate (PS) group. In some embodiments, the gamper is designed to hybridize to a target piece of RNA and silence the gene transcript through the induction of RNase H cleavage. As an example, the ASO drug "Tofersen" is a gapmer that knockdowns SOD1 mRNA for treatment of ALS. A possible example of a dual-action oligonucleotide (DAO) with a gapmer ASO disclosed in the present application could be "siSOD1-Tofersen conjuagte" .

As used herein, the term "mixmer" refers to an antisense oligonucleotide (ASO) characterized as a mixture of DNA and chemically-modified nucleic acid analogs in structure. Optionally, a mixmer is composed of fully modified nucleotides or nucleic acid analogs. In some embodiments, a mixmer is designed to bind and mask complementary RNA sequence to sterically block proteins, factors, or other RNAs from interacting with targeted RNA. In some embodiments, a mixmers is designed to alter pre-mRNA splicing by displacing the spliceosome. In some embodiments, a mixmer is designed to bind and sequester microRNAs (miRNAs) in which it is adopted yet another name called an "antagomir" or an "anti-miR" .

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

As used herein, the terms “antisense strand” and “antisense oligonucleotide strand” are interchangeable. The antisense oligonucleotide strand of a siRNA molecule can include, for example, a second nucleic acid strand in a duplex of siRNA that is complementary to the sense oligonucleotide strand. An antisense strand of a siRNA may be complementary to a consecutive fragment of a target gene sequence and is capable of binding to the consecutive fragment with 0, 1, 2, 3, 4 or 5 mismatches without affecting the function of the siRNA.

As used herein, the term “coding strand” refers to the DNA strand in the target gene that cannot be transcribed, the nucleotide sequence of which is identical to the sequence of the RNA produced by transcription (in RNA the T in DNA is replaced by U) . The coding strand of the double-stranded DNA sequence of the target gene promoter described in the present application refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

As used herein, the term “template strand” refers to another strand of double-stranded DNA of a target gene that is complementary to the coding strand and that can be transcribed as a template into RNA that is complementary to the transcribed RNA base (A-U, G-C) . 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 described in the present application refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

As used herein, the term “overhang” refers to an oligonucleotide strand end (5' or 3 ') with non-base paired nucleotide (s) resulting from another strand extending beyond one of the strands within the siRNA. Single-stranded regions extending beyond the 3' and/or 5' ends of the duplexes are referred to as overhangs. In some embodiments, the overhang is from 0 to 6 nucleotides in length. It is understood that an overhang of 0 nucleotides means that there is no overhang.

The term “natural overhang” as used herein refers to an overhang which consists of one or more nucleotides identical to or complementary to the corresponding position on the target sequence. A natural overhang on a sense strand consists of one or more nucleotides identical to the corresponding position on the mRNA target. A natural overhang on an antisense strand consists of one or more nucleotides complementary to the corresponding position on the mRNA target.

As used herein, the terms “gene silencing” , “knockdown of gene expression” , “gene downregulation” , “decreasing gene expression” and “downregulating gene expression” can be used interchangeably, and means a decrease or downregulation in transcription, translation, expression or activity of a certain nucleic acid sequence as determined by measuring the transcription level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly. In addition, “gene downregulation” or “downregulating 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 occurs at the transcriptional or post-transcriptional level to decrease transcription into RNA and the RNA level is decreased to be translated into lower level of protein than baseline, thereby decreasing the expression of the protein.

As used herein, the terms “short interfering RNA” , “siRNA” and “silencing RNA” can be used interchangeably and refer to a ribonucleic acid molecule that can downregulate, knockdown, or silence target gene expression. It can be a double-stranded nucleic acid molecule. siRNA binds to target mRNA mainly in the cytoplasm to down-regulate gene expression post-transcriptionally via the RNA interference (RNAi) mechanism.

siRNAs may contain natural nucleotides or chemically modified nucleotides. The modifications can impart increased nuclease stability and/or increased cellular potency. Examples of chemical modifications include phosphorothioate backbone modification, 2'-deoxynucleotide, 2'-OCH₃-containing ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethyl ribonucleotides, combinations thereof and the like. The siRNA can have varying lengths (e.g., 10-200 bps) and structures (e.g., hairpins, single/double strands, bulges, nicks/gaps, mismatches) and are processed in cells to knock down target mRNA. A double-stranded siRNA can have the same number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs) . An overhang of 1-2 nucleotides, for example, can be present on the sense and/or the antisense strand, as well as present on the 5'-and/or the 3'-ends of a given strand.

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

The term "equal length portion" refers to a portion of a sequence that is compared with an object sequence (e.g., a continuous oligonucleotide sequence from the siRNA) and has equal length (equal number of bases) to the object sequence.

The term “sequence specific mode” as used herein means a binding or hybridization way of two nucleic acid fragments according to their nucleotide sequence, e.g., a Watson-Crick manner (such as A to T, A to U, and C to G) or any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing) .

As used herein, the terms “isolated target site” , “target site” and “isolated polynucleotide" can be used interchangeably, and herein means a nucleic acid target site to which a siRNA has complementarity or hybridizes to. For example, an isolated nucleic acid sequence of a target site can include a nucleic acid sequence to which a region of siRNAs has complementarity or hybridize to.

As used herein, the term “complementary” refers to the capability of forming base pairs between two oligonucleotide strands. The base pairs are generally formed through hydrogen bonds between nucleotides in the antiparallel oligonucleotide strands. The bases of the complementary oligonucleotide strands can be paired in the Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing) .

Complementarity includes complete complementarity and incomplete complementarity. “Complete complementarity” or “100%complementarity” means that each nucleotide from the first oligonucleotide strand can form a hydrogen bond with a nucleotide at a corresponding position in the second oligonucleotide strand in the double-stranded region of the siRNA molecule, with no base pair being “mispaired” . “Incomplete complementarity” or “mismatch” means that not all the nucleotide units of the two strands are bound with each other by hydrogen bonds. For example, for two oligonucleotide strands each of 20 nucleotides in length in the double-stranded region, if only two base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 10%. In the same example, if 18 base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 90%. Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%or 99%complementarity.

As used herein, “ODV” and “oligonucleotide delivery vehicle” are used interchangeably, which refer to an oligonucleotide molecule comprising a duplex or double-stranded RNA (e.g., siRNA or saRNA) and an ACO which is covalently linked to the duplex RNA via a linker as described in more detail below.

As used herein, “covalent linker” , “linker” and “linking component” are used interchangeably, which refer to an organic moiety that connects two parts of a compound. For example, one or more of a single-stranded oligonucleotide (e.g., ACO) and a dsRNA (e.g., siRNA) , two dsRNAs, etc. are covalently linked by, e.g., a nucleic acid linker, a peptide linker, and the like and, includes disulfide linkers.

As used herein, the term “synthetic” refers to the manner in which oligonucleotides are synthesized, including any means capable of synthesizing or chemically modifying RNA, such as chemical synthesis, in vitro transcription, vector expression, and the like.

The terms “oligonucleotide modulator” and "oligonucleotide agent" can be used interchangeably and refer to an oligonucleotide-containing substance which at least comprises or consists of one or more siRNA of the invention and has the activity of modulating target gene expression or enhance the effect of the siRNA, and may further comprise other oligonucleotide moieties/components (such as ASO, or accessory oligonucleotide (ACO) ) or non-oligonucleotide moieties/components conjugated, combined or mixed with the siRNA (s) . In certain embodiments, the oligonucleotide modulator comprises an RNA (such as the siRNA of the invention) , a DNA, a BNA, an LNA, a GNA or a PNA.

As used herein, the term “LNA” refers to a locked nucleic acid in which the 2’-oxygen and 4’-carbon atoms are joined by an extra bridge. As used herein, the term “BNA” refers to a 2'-O and 4'-aminoethylene bridged nucleic acid that can contain a five-membered or six-membered bridged structure with an N-O linkage. As used herein, the term “PNA” refers to a nucleic acid mimic with a pseudopeptide backbone composed of N- (2-aminoethyl) glycine units with the nucleobases attached to the glycine nitrogen via carbonyl methylene linkers. As used herein, the term “GNA” also referred to as glycerol nucleic acid, is a nucleic acid similar to DNA or RNA but differing in the composition of its sugar-phosphodiester backbone, using propylene glycol in place of ribose or deoxyribose.

In the present application, singular forms, such as “a” and “this” , include plural objects, unless otherwise specified clearly in the context.

Unless otherwise defined, all the technological and scientific terms used therein have the same meanings as those generally understood by those of ordinary skill in the art covering the present application.

siRNA

Embodiments of the present application are based in part on the surprising discovery that an oligonucleotide agent (for example, siRNA, also referred to as “SOD1 gene siRNA” or “SOD1 siRNA” herein) is capable of inhibiting or downregulating the expression of a SOD1 gene in a cell. The decrease in functional SOD1 gene transcript following administration with an oligonucleotide agent of the present application can achieve a significant decrease or downregulation in the levels of SOD1 mRNA and SOD1 protein in a cell or a mammal.

In particular, the inventors discovered that the functional oligonucleotide agents capable of inhibiting expression of superoxide dismutase 1 (SOD1) comprising a small interfering RNA (siRNA) , wherein the siRNA comprises a sense strand and an antisense strand forming a double strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 10 contiguous nucleotides, with 0, 1, 2 or 3 mismatches, having at least 85%nucleotide sequence complementarity or homology to a portion of the nucleotide sequence of SOD1 mRNA.

As a beneficial consequence, a target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) , upon interacting with the siRNA, can inhibit/downregulate the SOD1 mRNA transcript by at least 10%, for example, 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%, as compared to a baseline level of SOD1 mRNA. In one embodiment, SOD1 mRNA is decreased by at least 80%. Based at least in part on these discoveries, the present application features siRNA, compositions, and pharmaceutical compositions for inhibiting/downregulating the SOD1 mRNA transcript by at least 10%as compared to baseline levels of SOD1 mRNA. Also provided herein are methods for preventing or treating a disease or condition induced by over-expression of SOD1 protein, a SOD1 gene mutation, and/or high or abnormal SOD1 level in an individual comprising administering to the individual any of the siRNA, compositions, and/or pharmaceutical compositions described herein.

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

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

In some other embodiments, the SOD1 mRNA inhibitory oligonucleotide agents comprise a siRNA having an antisense strand that is at least 85%, at least 90%, or at least 95%homology to the nucleotide sequence selected from SEQ ID NOs: 538-805.

In some embodiments, the SOD1 mRNA inhibitory oligonucleotide agents comprise a siRNA, wherein the sense strand and the antisense strand of the siRNA have nucleotide sequences that is independently at least 85%, at least 90%, or at least 95%homology to the nucleotide sequence selected from SEQ ID NOs: 808-849, 867 and 868.

The siRNAs of the oligonucleotide agent described herein include an RNA strand (the antisense strand) having a region which is 60 nucleotides or less in length, i.e., 15-40 nucleotides in length, generally 19-25 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a SOD1 gene. The use of these siRNAs enables the targeted degradation of mRNAs of genes that are implicated in pathologies associated with SOD1 expression in mammals. Very low dosages of SOD1 siRNAs in particular can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a SOD1 gene. Using cell-based assays, the present inventors have demonstrated that siRNAs targeting SOD1 can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a SOD1 gene. Thus, methods and oligonucleotide agents including these siRNAs are useful for treating pathological processes that can be mediated by down regulating SOD1, such as in the treatment of a disorder that causes elevated SOD1 levels, e.g., amyotrophic lateral sclerosis (ALS) . The following detailed description discloses how to make and use oligonucleotide agents containing siRNAs to inhibit the expression of a SOD1 gene, as well as oligonucleotide agents and methods for treating diseases and disorders caused by the expression of this gene.

In one aspect, an RNA interference agent includes a single-stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15: 485) . Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3’ overhangs (Bernstein, et al., (2001) Nature 409: 363) . The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107: 309) . Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15: 188) . Thus, in one aspect the invention relates to a single-stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.

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

In some embodiments, the SOD1 mRNA disclosed herein does not contain a nucleotide mutation. In some embodiments, the SOD1 mRNA disclosed herein contains at least one nucleotide mutation. In some embodiments, the SOD1 mRNA disclosed herein contains at least one nucleotide mutation on the targeting site of the siRNA. In some embodiments, the SOD1 mRNA disclosed herein contains at least one nucleotide mutation upper stream and/or downstream the targeting site of the siRNA.

In some embodiments, the differences or mismatches are located in the middle or 3’ terminus of the oligonucleotide sequence of the siRNA. Methods and principles of siRNA molecule design are well known to those skilled in the art and are described in detail in, for example, Place et. al., Molecular Therapy–Nucleic Acids (2012) 1, e15; and Li et. al., PNAS, 2006, vol. 103, no. 46, 17337–17342, which are herein incorporated by reference in their entireties.

In some embodiments, the siRNA disclosed herein comprises a sense strand and an antisense strand. The sense strand and the antisense strand comprise complementary regions capable of forming a double-stranded nucleic acid structure that decreases the SOD1 transcript level in a cell via the RNAi mechanism. The RNAi mechanism (also known as RNA interference) used herein refers to a mechanism that a double-stranded nucleic acid structure is capable of downregulating target genes in a sequence-specific manner at the transcriptional level. The sense strand and the antisense strand of the siRNA can exist either on two different nucleic acid strands or on one nucleic acid strand (e.g., a contiguous nucleic acid sequence) . When the sense strand and the antisense strand are located on two different strands, at least one strand of the siRNA has a 3' overhang of 0 to 6 nucleotides in length, such that the overhangs of 0, 1, 2, 3, 4, 5 or 6 nucleotides in length, and in some cases, both strands have a 3' overhang of 2 or 3 nucleotides in length. The nucleotide of the overhang is, in some cases thymine deoxyribonucleotide (dT) , or in some cases, natural overhangs which are nucleotides selected from or complementary to the corresponding position on the DNA target. When the sense strand and the antisense strand are located on one nucleic acid strand, in some cases, the siRNA is a hairpin single-stranded nucleic acid molecule, where the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure with each other. In the siRNA disclosed herein, in some embodiments, the sense strand has a length ranging from 10 to 60 nucleotides. For example, in some embodiments, the sense strand and the antisense strand, independently comprise a length 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 ranging from 10 to 60 nucleotides. For example, in some embodiments, the sense strand and the antisense strand, independently comprise a length 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 disclosed herein is capable of interacting with a target nucleic acid sequence of a mRNA of a SOD1 gene in a sequence specific manner, meaning that the antisense strand is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. In some embodiments, an antisense strand has a nucleotide sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target portion of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense strand has a nucleotide sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target portion in a fragment of a SOD1 gene transcript.

ACO

Although some prior research proved that siRNAs capable of inhibiting SOD1 mRNA and decreasing SOD1 protein expression can be used to treat SOD1 protein-related diseases, e.g., for amyotrophic lateral sclerosis (ALS) patients, the inventors found there are two unsolved issues, one is lack of potency of SOD1 siRNA molecules and the other is lack of efficient delivery method to deliver the siRNA molecule to cells of a target organ or tissue, e.g., CNS.

“Delivering into a cell, ” when referring to an siRNA, means efficiently uptake or absorption by the cell, as is understood by those skilled in the art. Absorption or uptake of an siRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a siRNA can also be “introduced into a cell, ” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, siRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below which are not known in the art.

When the siRNA agent is conjugated to a non-targeting single-stranded accessory oligonucleotide (ACO) as disclosed, bioavailability, biodistribution, and/or cellular uptake and in vivo potency of the siRNA were significantly improved as compared to an oligonucleotide agent without the ACO. Especially in some in vivo examples in the present application, the ACO of the oligonucleotide agent increased the biodistribution of siRNA within one, or two, or more target tissues as compared to an oligonucleotide agent without the ACO.

Therefore, aspects of the present application further relate to an oligonucleotide agent capable of inhibiting the expression of superoxide dismutase 1 (SOD1) comprising a small interfering RNA (siRNA) , and an ACO.

In some embodiments, the oligonucleotide agent comprising one or more conjugated ACO enhance the biodistribution of the oligonucleotide agent in particular tissues of the oligonucleotide agent, and increase permeability of the oligonucleotide agent and passage through membranes, such as the blood brain barrier.

In some embodiments, the ACO is an oligonucleotide comprising a 5’ end and a 3’ end.

In some embodiments, the siRNA and the ACO are covalently linked, with or without one or more linking components, to form the oligonucleotide agent.

In some embodiments, the length of the ACO comprises a nucleotide length ranging from 6 to 22 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, 19 nucleotides or more, 20 nucleotides or more, 21 nucleotides or more, 22 nucleotides or more. In some embodiments, the length of the ACO is 6 to 18 contiguous oligonucleotides.

In some embodiments, the length of the ACO can modulate the activity and/or biodistribution of the oligonucleotide agent within the target tissue or cell of interest. For example, the present inventors found that shorter ACOs demonstrated activity throughout the central nervous system, while longer ACOs demonstrated activity only in particular regions of the brain, such as the cerebellum.

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

In some embodiments, the oligonucleotide agent of the present application comprises more than one ACO, for example, 2, 3, 4, 5, 6, 7, 9, 10 ACOs, covalently linked to a siRNA, with or without one or more linkers in between the ACOs and siRNA. The number of ACO can vary from 1 to 4, 2 to 10 linked to a siRNA via a multivalent linker, for example, a polymeric linker, in branch or liner form. In some embodiments, multiple ACOs are covalently linked to 2 or more siRNAs, for example, 2, 3, 4, 5, 6, 7, 9, 10 or more siRNAs, in one agent. PCT Application No. WO2023280190A1, the content of which is incorporated herein by reference, in its entirety, for all purposes, describes principles of ACO design and examples of ACO that are conjugated to the siRNAs to improve pharmacokinetics properties including accessory delivering of duplex RNAs to a cell.

Chemical modification

In the siRNAs or ACOs disclosed herein, all nucleotides may be natural or non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide. Non-limiting examples of the chemical modification include one or more of a combination of the following:

1) modification of a phosphodiester bond of nucleotides in the nucleotide sequence of the siRNA or ACO;

2) modification of 2'-OH of the ribose in the nucleotide sequence of the siRNA or ACO;

3) modification of a base in the nucleotide of the siRNA or ACO;

4) at least one nucleotide in the nucleotide sequence of the siRNA or ACO being a BNA, LNA, GNA or PNA, and

5) at least one nucleotide in the nucleotide sequence of the ACO being a deoxyribonucleotide (DNA) .

The chemical modification described herein is well-known to those skilled in the art, and the modification of the phosphodiester bond refers to the modification of oxygen in the phosphodiester bond, including phosphorothioate modification and boranophosphate modification. The modifications disclosed herein stabilize the siRNA structure, maintaining high specificity and high affinity for base pairing. The modifications disclosed herein also stabilize an ACO structure and maintain its delivering accessory properties including bioavailability, biodistribution, and/or cellular uptake of the oligonucleotide agent in various tissues prefrontal cortex, cerebellum, cerebrum, spinal cord (e.g., cervical, thoracic, lumber) , muscle, lung, eye, liver, and kidney.

In some embodiments, the chemical modification is to substitute the phosphodiester bond with phosphorothioate (PS) bond on the backbone of the nucleotide sequence of the oligonucleotide agent disclosed herein. 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.

In some embodiments, the siRNA or ACO of the present application includes at least one chemically modified nucleotide which is modified at 2'-OH in pentose of a nucleotide, i.e., the introduction of certain substituents at the hydroxyl position of the ribose, such as 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2, 4'-dinitrophenol modification, locked nucleic acid (LNA) , 2'-amino modification or 2'-deoxy modification, e.g., a 2’-deoxy-2’-fluoro modified nucleotide, a 2’-deoxy-modified nucleotide.

In some embodiments, the siRNA or ACO of the present application includes at least one chemically modified nucleotide which is modified at the base of the nucleotide, e.g., 5 '-bromouracil modification, 5’-iodouracil modification, N-methyluracil modification, or 2, 6-diaminopurine modification.

In some embodiments, the chemical modification of the siRNA or ACO is an addition of a (E) ‐vinylphosphonate moiety at the 5’ end of the sense or antisense sequence. In some embodiments, the chemical modification of the at least one chemically modified nucleotide is an addition of a 5-methyl cytosine moiety at the 5’ end of the sense or antisense sequence.

In some embodiments, the siRNA or ACO of the present application includes at least one nucleotide in the nucleotide sequence of the siRNA/ACO being a chemically modified nucleic acid, e.g., a locked nucleotide, an abasic nucleotide, a glycerol nucleic acid (GNA) , a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In some embodiments, the siRNA disclosed herein includes an “endo-light” modification with 2’-O-methyl modified nucleotides and nucleotides comprising a 5’-phosphorothioate group.

In some embodiments, the siRNA or ACO of the present application is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the present application may be synthesized and/or modified by conventional methods, such as those described in “Current protocols in nucleic acid chemistry, ” Beaucage, S.L. et al. (Edrs. ) , John Wiley &Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc. ) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc. ) , (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides) , or conjugated bases, (c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of siRNA molecules that can be used in this present application include but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. In some embodiments, RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. In some embodiments, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be oligonucleosides. In some embodiments, the modified oligonucleotide will have a phosphorus atom in its internucleoside backbone.

Modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included.

In some embodiments, the siRNA or the ACO is composed of one or more of RNA, DNA, BNA, LNA, GNA or PNA.

Covalent Linkage

Aspects of the present application include an oligonucleotide agent comprising a double stranded targeting oligonucleotide (i.e., siRNA) and an ACO that are covalently linked.

In some embodiments, any of the oligonucleotides in the oligonucleotide agent of the present application includes a linking component. In some embodiments, a siRNA and a ACO are covalently linked by a linking component. In some embodiments, the siRNA and the ACO are linked with a covalent linker. Various combinations of strands can be linked, e.g., the first and second dsRNA sense strands are covalently linked or, e.g., the first and second dsRNA antisense strands are covalently linked.

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, or 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, or the 5’ end, or both the 3’ and 5’ ends of the antisense strand of the siRNA. In some embodiments, more than one ACO is covalently linked to a siRNA. In some embodiments, 2-10 ACOs are covalently linked to the siRNA. In some embodiments, more than one siRNA is covalently linked to an ACO. In some embodiments, 2-10 siRNAs are covalently linked to an ACO.

In some embodiments, the ACO is conjugated to a linking component. In some embodiments, the 5’ end or the 3’ end of the ACO is conjugated to a linking component. In some embodiments, the siRNA and the ACO are covalently linked by a linking component. In some embodiments, the sense strand or the antisense strand of the siRNA are covalently linked to the ACO by a linking component.

Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR¹, C (O) , C (O) O, C (O) NR¹, SO, SO₂, SO₂NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S (O) , SO₂, N (R') ₂, C (O) , cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R¹ is hydrogen, acyl, aliphatic or substituted aliphatic.

Without limitations, various types of linker functionality can be included in the subject conjugates, including but not limited to cleavable linkers, and non-cleavable linkers, as well as reversible linkers and irreversible linkers.

In some embodiments, the linker is a cleavable linker. Cleavable linkers are those that rely on processes inside a target cell to liberate the two parts the linker is holding together, e.g., the ACO and the dsRNA, as reduction in the cytoplasm, exposure to acidic conditions in a lysosome or endosome, or cleavage by specific enzymes (e.g., proteases) within the cell. As such, cleavable linkers allow the dsRNA to be released in its original form after the conjugate has been internalized and processed inside a target cell. Cleavable linkers include, but are not limited to, those whose bonds can be cleaved by enzymes (e.g., peptide linkers) ; reducing conditions (e.g., disulfide linkers) ; or acidic conditions (e.g., hydrazones and carbonates) .

In some embodiments, the linking component is selected from one or more of ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, carbohydrates, thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, and a carbamate. In some embodiments, the linking component includes, but is not limited to: Spacer phosphoramidite 18 (Phosphoramidous acid, N, N-bis (1-methylethyl) -, 19, 19-bis (4-methoxyphenyl) -19-phenyl-3, 6, 9, 12, 15, 18-hexaoxanonadec-1-yl 2-cyanoethyl ester) ; Spacer-9 (3- [2- [2- [2- [bis (4-methoxyphenyl) -phenylmethoxy] ethoxy] 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 comprises a compound structure shown in Table 15.

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

Table 15. Linkers used in the oligonucleotide agent

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

In some embodiments, the siRNA comprises a sense strand that is covalently linked to the ACO. In some embodiments, the siRNA comprises an antisense strand that is covalently linked to the ACO.

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

Non-limiting examples of covalent linkers can be found in U.S. Patent Application Publication No.: 20200332292, which is hereby incorporated by reference in its entirety. The covalent linker can join the siRNA and the ACO.

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

In some embodiments, the covalent linker includes a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is

In some embodiments, the covalent linker includes a peptide bond, e.g., include amino acids. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably comprising 4-5 amino acids, optionally X-Gly-Phe-Gly-Y wherein X and Y represent any amino acid.

In some embodiments, the covalent linker includes HEG, a hexaethylenglycol linker.

ODV-siRNA oligonucleotide agent

In some embodiments, the oligonucleotide agent decreases the expression of a SOD1 gene or SOD1 protein. Administration of the oligonucleotide agent to a patient treats or delays the onset of ALS, such as familial or sporadic ALS or Leu Lou Gehrig's disease. In some embodiments, the described oligonucleotide agent decreases the amount of SOD1 protein by, for example, downregulating SOD1 transcript level or decrease the amount of full-length SOD1 mRNA. In some embodiments, SOD1 mRNA is decreased 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 decreased by at least 80%. In some embodiments, SOD1 protein is decreased in an amount sufficient to attenuate the symptoms associated with an ALS. In some embodiments, SOD1 protein is decreased by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) . In some embodiments, SOD1 protein is decreased by at least 80%.

In some embodiments, the oligonucleotide agent that decreases the expression of the SOD1 gene or SOD1 protein is a siRNA-ACO conjugate (or ODV-siRNA) . The SOD1 siRNA-ACO conjugate decreases or downregulates the expression of an SOD1 gene in a cell in which the SOD1 gene is abnormally or over expressed.

In typical embodiments, a first strand of the SOD1 siRNA in the oligonucleotide agent comprises a segment that has at least 75%sequence identity or sequence complementarity to a 6-60 nucleotide fragments of a select target region of the SOD1 gene thereby effecting deactivation or downregulation of expression of the gene.

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 sequences of siSOD1-047M3-AC1 whose antisense strand has a nucleotide sequence of SEQ ID NO: 834 that has complementarity with a fragment the of the ODV structured sense strand of SEQ ID NO: 850.

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 sequences of siSOD1-005M3-AC1 whose antisense strand has a nucleotide sequence of SEQ ID NO: 842 that has complementarity with a fragment of the ODV structured sense strand of SEQ ID NO: 851.

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 sequences of siCON1-AC1^VP whose antisense strand has a nucleotide sequence of SEQ ID NO: 858 that has complementarity with a fragment the of the ODV structured sense strand of SEQ ID NO: 852.

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 sequences of siCON2-AC1^VP whose antisense strand has a nucleotide sequence of SEQ ID NO: 859 that has complementarity with a fragment the of the ODV structured sense strand of SEQ ID NO: 853.

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 sequences of siCON3-AC1^VP whose antisense strand has a nucleotide sequence of SEQ ID NO: 860 that has complementarity with a fragment the of the ODV structured sense strand of SEQ ID NO: 854.

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 sequences of siSOD1-063M3-AC1^VP whose antisense strand has a nucleotide sequence of SEQ ID NO: 861 that has complementarity with a fragment of the ODV structured sense strand of SEQ ID NO: 855.

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 sequences of siSOD1-047M3-AC1^VP whose antisense strand has a nucleotide sequence of SEQ ID NO: 848 that has complementarity with a fragment the of the ODV structured sense strand of SEQ ID NO: 850.

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 sequences of siSOD1-104M3-AC1^VP whose antisense strand has a nucleotide sequence of SEQ ID NO: 862 that has complementarity with a fragment the of the ODV structured sense strand of SEQ ID NO: 856.

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 sequences of siSOD1-005M3-AC1^VP whose antisense strand has a nucleotide sequence of SEQ ID NO: 849 that has complementarity with a fragment the of the ODV structured sense strand of SEQ ID NO: 852.

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 sequences of siSOD1-258M3-AC1VP whose antisense strand has a nucleotide sequence of SEQ ID NO: 863 that has complementarity with a fragment the of the ODV structured sense strand of SEQ ID NO: 857.

In addition, to facilitate entry of the siRNA into a cell, chemical conjugation groups other than the ACO disclosure herein may be introduced at the ends of the sense or antisense strands of the siRNA on the basis of the above modifications to facilitate action through a cell membrane composed of lipid bilayers and mRNA regions within the nuclear membrane and nucleus.

In some embodiments, siRNAs disclosed in the present application are covalently attached to one or more conjugate groups. In some embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In some embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide. Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556) , cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060) , a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770) , a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538) , an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54) , a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1, 2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783) , a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &Nucleotides, 1995, 14, 969-973) , or adamantane acetic acid a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237) , an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937) , a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740) , or a GalNAc cluster (e.g., WO2014/179620) .

In some embodiments, the siRNA of the present application relates to the sense strand or the antisense strand of the siRNA that is conjugated to one or more conjugation groups selected from: intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

In some embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S) - (+) -pranoprofen, carprofen, dansylsarcosine, 2, 3, 5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

In some embodiments, the siRNA of the present application is conjugated to one or more conjugation groups selected from: a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody.

In some embodiments, the siRNA of the present application relates to the sense strand or the antisense strand of the siRNA that is conjugated to one or more conjugation groups selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.

In some embodiments, the siRNA conjugated to one or more conjugation groups disclosed in the embodiments is directly contacted, transferred, delivered or administrated to a cell or a subject.

Cell comprising siRNA

After contacting a cell, the oligonucleotide agent disclosed herein can effectively inhibit or downregulate the expression of SOD1 gene in a cell, for example downregulate the expression by at least 10% (e.g., as compared to baseline level of SOD1 transcript) .

In some embodiments, the present application relates to a cell comprising the oligonucleotide agent disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell, such as a human cell in various tissues including prefrontal cortex, cerebellum, spinal cord (e.g., cervical, thoracic, lumber) , muscle, liver, and kidney.

The cell disclosed herein may be in vitro, or ex vivo, such as a cell line or a cell strain, or may exist in a mammalian body, such as a human body. The human body disclosed herein is a subject suffering from a disease or symptom caused by a SOD1 gene mutation, abnormal SOD1 mRNA level, and/or overexpression of SOD1 protein in CNS.

In some embodiments, the cell is from a CNS tissue of a subject suffering from ALS. In some embodiments, the cell is from a subject suffering from ALS. In some embodiments, the cell is from a subject suffering from Alzheimer’s disease (AD) , Parkinson’s disease (PD) , and Down’s syndrome (DS) .

Composition comprising siRNA

Another aspect of the present application provides a composition or pharmaceutical composition capable of downregulated the level of SOD1 mRNA transcript by the mechanism of action (MoA) of RNA interference, comprising the oligonucleotide agent disclosed herein, to treat or prevent onset of a SOD1 related disease (particularly ALS) .

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

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

In one embodiment, the pharmaceutically acceptable carrier includes one or more of an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugates (e.g., GalNAc) , and polypeptide. In one embodiment, the aqueous carrier may be, for example, RNase-free water, or RNase-free buffer. The composition may contain 1-150 nM, for example 1-100 nM, for example 1-50 nM, for example 1-20 nM, for example 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM, for example 50 nM of the oligonucleotide agent or nucleic acid encoding full-length or partial of the oligonucleotide agent according to the present application.

In some embodiments, the composition comprises 1-150 nM of the oligonucleotide agent of the present application.

Another embodiment provides pharmaceutical compositions or medicaments comprising the oligonucleotide agent of the present application and a therapeutically inert carrier, diluent or pharmaceutically acceptable excipient, as well as methods of using the oligonucleotide agent of the present application to prepare such compositions and medicaments.

A typical formulation is prepared by mixing an agent of the present application and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel H.C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams &Wilkins, Philadelphia; Gennaro A.R. 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 formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., an agent of the present application or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament) .

Compositions of the present application are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

For the oligonucleotide agent compositions of the present application, the delivery can be optionally through parenteral infusions including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal or subcutaneous administration; or through oral administration, intranasal administration, inhaled administration, vaginal administration, or rectal administration.

In another aspect, the application provides use of the oligonucleotide agent, according to any one of the embodiments described herein, or a composition according to any one of the embodiments described herein, in the manufacture of a medicament for the treatment of gene or protein-related condition in an individual. The use according to certain embodiments, the condition can include a SOD1 related condition that comprises ALS, AD, PD and/or DS. Also provided is the use according to certain embodiments wherein the individual is a mammal, preferably a human.

Kits

In another aspect, any of the compositions described herein can be provided in one or more kits, optionally including instructions for use of the compositions. That is, the kit can include a description of use of an oligonucleotide agent or composition or pharmaceutical composition in any method described herein. A "kit, " as used herein, typically defines a package, assembly, or container (such as an insulated container) including one or more of the components or embodiments of the application, and/or other components associated with the application, for example, as previously described. Any of the antes or components of the kit may be provided in liquid form (e.g., in solution) , or in solid form (e.g., a dried powder, frozen, etc. ) .

In some cases, the kit includes one or more components, which may be within the same or in two or more receptacles, and/or in any combination thereof. The receptacle is able to contain a liquid, and non-limiting examples include bottles, vials, jars, tubes, flasks, beakers, or the like. In some cases, the receptacle is spill-proof (when closed, liquid cannot exit the receptacle, regardless of orientation of the receptacle) .

Examples of other compositions or components associated with the agents, compounds and methods described herein include, but are not limited to: diluents, salts, buffers, chelating agents, preservatives, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, and the like, for example, for using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the components for a particular use. In embodiments where liquid forms of any of the components are used, the liquid form may be concentrated or ready to use.

In additional embodiments, a kit can include instructions or instructions to a website or other source in any form that are provided for using the kit in connection with the components and/or methods described herein. For instance, the instructions may include instructions for the use, modification, mixing, diluting, preserving, assembly, storage, packaging, and/or preparation of the components and/or other components associated with the kit. In some cases, the instructions may also include instructions for the delivery of the components, for example, for shipping or storage at room temperature, sub-zero temperatures, cryogenic temperatures, etc. The instructions may be provided in any form that is useful to the user of the kit, such as written or oral (e.g., telephonic) , digital, optical, visual (e.g., videotape, DVD, etc. ) and/or electronic communications (including Internet or web-based communications) , provided in any manner.

Method of use

Another aspect of the present application relates to the oligonucleotide agents of the present application being used in therapeutic approaches to treating diseases such as ALS.

By non-limiting embodiments, the present application provides a method of decreasing the transcript level of a SOD1 gene or SOD1 protein, comprising administering to a subject a pharmaceutical composition disclosed herein.

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

In some embodiments, one or more target tissues is selected from: prefrontal cortex, cerebellum, cerebrum, spinal cord, muscle, lung, eye, liver, and kidney.

In some embodiments, the oligonucleotide agent of the present application achieves a decrease in full-length SOD1 protein that is less than the amount achieved by administration of the same amount of double stranded oligonucleotide such as a siRNA substance without an ODV structure used individually, with higher potency, reduced toxicity, or unwanted side effects. In some embodiments, the oligonucleotide agent of the present application achieves a decrease in full-length SOD1 protein that is less than the additive effect of treatment with the same amount of the siRNA used individually.

Specifically, the oligonucleotide agent of the present application inhibits/down-regulates the SOD1 mRNA transcript 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%as compared to baseline SOD1 mRNA transcript) . In some embodiments, upon administering the oligonucleotide agent disclosed in the embodiments, e.g., to a cell or a subject, the SOD1 mRNA transcript is inhibited/downregulated by at least 50%, 60%, 70%, 77%, 79%, 81%, 84%, 85%, and 88%at 10 nM treatment compared to baseline SOD1 mRNA transcript in control group) in an in vitro cell line. In some embodiments, an oligonucleotide agent inhibits or downregulates the SOD1 mRNA transcript by about 80%.

In some embodiments, the expression of SOD1 gene is inhibited/downregulated by administering the oligonucleotide agent disclosed in the embodiments to a cell at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM. In some embodiments, the SOD1 gene coded protein (SOD1 protein) is inhibited/downregulated by administering the oligonucleotide agent disclosed in the embodiments, e.g., to a cell or a subject. The knockdown of the SOD1 protein by at least 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%, as compared to baseline expression of the SOD1 protein) . In some embodiments, an oligonucleotide agent inhibits or downregulates the expression of the SOD1 protein by about 80%. In some embodiments, the SOD1 protein is inhibited/down-regulated by administering the oligonucleotide agent disclosed in the embodiments to a cell at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 4nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM.

In some embodiments, the oligonucleotide agents disclosed in the embodiments have a dose-dependent knockdown activity in cells. In some embodiments, the oligonucleotide agents knockdown the SOD1 mRNA transcript in cells with a IC₅₀ of less than 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.8nM, 0.6 nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM. 0.1nM, 0.08nM, 0.06nM, 0.04nM, 0.02nM, 0.01nM, 0.008nM, or 0.005nM.

Another aspect of the present application relates to a method for preventing or treating a disorder or condition induced by over-expression of SOD1 protein, a SOD1 gene mutation, and/or high SOD1 mRNA levels in an individual comprising: administering an effective amount of the siRNA, the oligonucleotide agent, or the composition comprising the oligonucleotide agent disclosed herein to the individual. In some embodiments, the effective amount of the siRNA disclosed herein can be a concentration ranging from 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.

In any of the embodiments provided herein, such cells may be ex vivo, such as cell lines, and the like, or may be present in mammalian bodies, such as humans. In some embodiments, the human is a subject or individual suffering from a SOD1 protein related condition or ALS, AD, PD, or DS.

Another aspect of the present application relates administering an effective mount of the oligonucleotide agent or the composition to an individual using administration pathway as described herein. In some embodiments, the administration pathway is selected from one or more of: parenteral infusions, oral administration, intranasal administration, inhaled administration, vaginal administration, and rectal administration. In some embodiments, the administration pathway is selected from one or more of: intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal and subcutaneous administrations.

Dose regiments and route of administration

Aspects of the present application relate to a pharmaceutical composition comprising the oligonucleotide agent of the present application. In some embodiments, the pharmaceutical composition comprises the oligonucleotide agent of the present application and a pharmaceutically acceptable carrier, a therapeutically inert carrier, diluent or pharmaceutically acceptable excipient. The pharmaceutical composition disclosed herein is to be developed into a medicament preventing or treating the SOD1 protein related condition or ALS.

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

Another aspect of the present application relates to use of the oligonucleotide agent of the present application in manufacturing the pharmaceutical composition disclosed herein.

Another aspect of the present application relates to use of the oligonucleotide agent, according to any one of the embodiments described herein, or a composition according to any one of the embodiments described herein, in the manufacture of a medicament for the prevention or treatment of gene or protein-related symptom induced by the over-expression of SOD1 protein, a SOD1 gene mutation, and/or high SOD1 protein levels in an individual. For the use according to certain embodiments, the condition can include a SOD1 protein-mutation-related disorder or condition that comprises ALS. For the use according to certain embodiments, the symptom induced by over-expression of abnormal SOD1 protein is ALS. Also related is the use according to certain embodiments wherein the individual is a mammal, for example a human.

The dosage at which the oligonucleotide agents or compositions of the present application can be administered can vary within wide limits and will be fitted to the individual requirements in each case. In some embodiments, a first dose of a pharmaceutical composition according to the present application is administered when the subject is less than one week old, less than one month old, less than 3 months old, less than 6 months old, less than one-year-old, less than 2 years old, less than 15 years old, or older than 15 years old.

The single dose of the oligonucleotide agent can be a single dose ranging from 0.01 mg/kg to 1000 mg/kg for example, about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 40, 50, 75, 100, 120, 150, 200, 250, 300, 400, 500, 750, or 1000 mg/kg. The doses described herein may contain two or more of any of the oligonucleotide agent sequences described herein.

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

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

In some embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3%w/v benzyl alcohol, 8%w/v of the nonpolar surfactant Polysorbate 80^TM and 65%w/v polyethylene glycol 300. The proportions of such co-solvent systems may vary considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80^TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

Examples of other compositions or components associated with the oligonucleotide agent, compositions, pharmaceutical compositions, and methods described herein include, but are not limited to: diluents, salts, buffers, chelating agents, preservatives, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, and the like, for example, for using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the components for a particular use. In embodiments where liquid forms of any of the components are used, the liquid form may be concentrated or ready to use.

In some embodiments, lipid moieties used in nucleic acid therapies can be applied in the present application for delivery of the oligonucleotide agent molecules disclosed herein. In such methods, the nucleic acid (e.g., one or more oligonucleotide agents described herein) is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, oligonucleotide agent complexes with mono-or poly-cationic lipids are formed without the presence of a neutral lipid. In some embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In some embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In some embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In some embodiments, pharmaceutical compositions comprise 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 comprising hydrophobic compounds. In some embodiments, certain organic solvents such as dimethylsulfoxide are used.

In some embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present application to specific tissues or cell types. For example, in some embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

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

In some embodiments, the vector is a recombinant AAV vector (rAAV) . AAV vectors are DNA viruses of relatively small size that can integrate, in a stable and site-specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus.

Preparations, pharmaceutical compositions, or medicaments of the present disclosure are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

For the preparations, pharmaceutical compositions, or medicaments of the present disclosure, the delivery can be optionally through parenteral infusions including intrathecal, intramuscular, intravenous, intra-arterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal or subcutaneous administration; or through oral administration, intranasal administration, inhaled administration, vaginal administration, or rectal administration.

A typical formulation of the oligonucleotide modulator in the present disclosure is prepared by mixing a siRNA of the present disclosure and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel H.C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams &Wilkins, Philadelphia; Gennaro A.R. 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 formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a siRNA of the present disclosure or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament) .

EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; i.c.v. or ICV, intracerebroventricular and the like.

Example 1. Development of siRNA drug candidates for knockdown of human SOD1

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

Table 1. SOD1 cDNA sequence for siRNA design

A total of 268 siRNA duplexes were designed and synthesized at 21 nucleotides (nt) in length with no more than 4 repetitive nucleotides in a row and GC content between 35-65%. Their target sites and cognate siRNA strand sequences are listed in Table 2. Knockdown activity of each siRNA was assessed in HEK293A cells using high throughput RT-qPCR at both 0.1 and 10 nM concentrations. Data was ranked according to mean knockdown activity in which 121 siRNAs reduce SOD1 by more than 90%at 10 nM (Figure 1A) . As an indicator of potency, 69 and 15 siRNAs reduced SOD1 levels at 0.1 nM treatments by over 50%and 75%, respectively. 25 performing siRNAs were subjected to an additional round of screening at 6 concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4 and 20 nM) in HEK293A cells to demonstrate dose dependent activity in which propidium iodide (PI) was integrated into sample preparation to monitor variation in nucleic acid content as an indicator of untoward cytotoxicity (Figure 2) . Shown in Figure 1B is data for the top 5 siRNAs (i.e., siSOD1-063, 047, 104, 005, and 258) with the most potent knockdown activity which also show an absence of overt cytotoxicity (i.e., <20%reduction in PI staining) .

Dose response curves were subsequently generated for each of the 5 siRNA candidates to validate potency in model cell lines representative of neuronal disease including SK-N-AS (Figure 1C) and T98G (Figure 3) cells. As summarized in Table 3, in vitro potency for each duplex was in the low picomolar range for both cell lines. Untoward cytotoxicity was also evaluated 72 hours after treatment at concentrations well above 200X the extrapolated IC₅₀ values. As shown in Figure 4A-B, only siSOD1-047 and 005 had no detectable impact on apoptosis or cell number in either SK-N-AS or T98G cells, whereas the remaining candidates (i.e., siSOD1-063, 104, and 258) had dose-dependent responses with regards to caspase 3/7 activity in T98G cells that inversely correlated with cell viability. While SK-N-AS cells appeared more tolerant to treatment, a similar pattern was observed for cell viability.

Several medicinal chemistry patterns referred to as M1, M2, M3, or M4 representing different duplex lengths (i.e., 20, 21, 22, and 23 nt, respectively) comprised of phosphorothionate (PS) backbone modifications at select positions with 2'-O-methylation (2’Ome) or 2’-fluoro (2’F) substitutions at every 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 had better knockdown activity compared to the other chemically modified siRNAs at 0.1 nM treatment concentrations in SK-N-AS cells. A near identical pattern was also observed in T98G cells (Figure 5) . To further characterize potency, dose response curves were generated for M3-modified duplexes (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, in vitro potency was generally well-retained following chemical modification.

Accessory oligonucleotide (ACO) conjugation was developed to impart self-delivery properties similar to ASOs by sharing medicinal chemistry normally not tolerated by canonical siRNAs. As such, the sense strand of each M3 variant (i.e., siSOD1-063M3, 047M3, 104M3, 005M3, and 258M3) was synthesized covalently linked to a 14-nucleotide ACO (referred to AC1) via a short linker (L9, i.e., Spacer-9 linker) , wherein the ACO that possessed PS backbone substitutions and 2'-O-methoxyethyl (2’MOE) modifications at every position within the ACO (Figure 7A) . As shown in Figure 7B, AC1 treatment alone had no detectable knockdown of SOD1 at 0.25 and 2.5 nM concentrations in vitro, whereas activity was only perceived when conjugated to siRNA. Additional dose response analysis noted a ～10X loss in siRNA potency as a consequence of AC1 conjugation (i.e., siSOD1-005M3-AC1) compared to only chemically modified duplex (i.e., siSOD1-005M3) (Figure 7C) . However, potency was restored upon modification with 5’- (E) -vinylphosphonate (5’VP) at the 5’ terminus of the guide strand (i.e., siSOD1-005M3-AC1^VP) (Figure 7D) . In comparison to an ASO resembling Tofersen (an antisense oligonucleotide ASO under clinical investigation that has shown therapeutic benefit in the treatment of ALS by suppressing mutant SOD1 mRNA levels; cf. T. Miller et al., New England Journal of Medicine 383, 109-119 (2020) ) in both sequence and chemistry (i.e., ASO^SOD1, SEQ ID NO: 864) , SOD1 knockdown with either siRNA-ACO variant (i.e., siSOD1-005M3-AC1 or siSOD1-005M3-AC1^VP) was more potent than ASO^SOD1 regardless of 5’VP modification (Figure 7D) .

siRNA-ACO conjugates were subsequently 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, and 270M3-AC1^VP) for downstream screening in vivo. Prior to treatment, dose response curves were generated in both SK-N-AS and T98G cells to validate activity of each siRNA-ACO candidate in suppressing mutant SOD1 mRNA levels (Figures 8A-8B) . As summarized in Table 3, in vitro potencies were generally well conserved in comparison to their non-conjugate forms. Apoptosis and cell viability were also quantified 72 hours after treatments to measure any changes in cytotoxicity as a consequence of chemical modification and ACO conjugation. As shown in Figures 9A-9B, siSOD1-047M3-AC1^VP and 005M3-AC1^VP remained generally unaffected with nominal impact on cell health in both SK-N-AS and T98G cells, whereas siSOD1-104M3-AC1^VP retained signs of untoward cytotoxicity and the remaining candidates (i.e., siSOD1-063M3-AC1^VP and 258M3-AC1^VP) noted an improvement in in vitro safety (i.e., reduction in caspase 3/7 activity) compared to their non-modified forms (Figure 4A-B) .

Table 3. In vitro potency of drug candidates

*IC₅₀ vales ± SD, the concentration of siRNAs where SOD1 gene is knocked down by half;
“/” , means not tested.

Example 2. In vivo selection of siRNA-ACO drug candidates

Mice hemizygous for hSOD1^G93A transgene express a mutant form of human SOD1 and exhibit disease phenotypes similar to ALS including progressive loss of motor function and abbreviated life span via neuronal degradation (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, hSOD1^G93A mice were treated via ICV injection at equimolar quantities (i.e., 20 nmole/dose) with siRNA-ACOs (i.e., siSOD1-047M3-AC1^VP or siSOD1-005M3-AC1^VP) in comparison to non-conjugate controls (i.e., siSOD1-047M3^VP or siSOD1-005M3^VP) to demonstrate the effect AC1 conjugation imparts on knockdown activity in vivo. All siRNA-ACOs were formulated in aCSF in which treatment alone served as a vehicle control to establish baseline expression, while siCON2-AC1^VP functioned as a negative control for siRNA-ACO activity. As shown in Figure 10, both siRNA-ACO duplexes provided greater knockdown activity comparative to their non-conjugate cognates in all tissues of the brain (i.e., frontal cortex, cerebellum, and cerebrum) and spinal cord (i.e., cervical, thoracic, and lumbar spine) with minimal activity in the periphery (i.e., liver) . Knockdown durability was also characterized for siSOD1-005M3-AC1^VP relative to its non-5’VP control (i.e., siSOD1-005M3-AC1) with comparison to ASO^SOD1. Taken together, the combination of both AC1 conjugation and 5’VP modification provided our siRNAs with enhanced activity needed for a durable response in vivo.

Example 3. Single dose treatment via ICV injection with siRNA-ACO delays disease progression and prolongs survival in hSOD1^G93A mice

Adult hSOD1^G93A mice were treated via ICV injection with siRNA-ACO candidates (i.e., siSOD1-047M3-AC1^VP or siSOD1-005M3-AC1^VP) at 50, 100, 200, or 400 mg/dose on PND 85 or 60, respectively. Drug concentrations and hSOD1 expression levels were quantified in a subset of animals on day 14 after treatment in CNS tissue (i.e., cerebellum, cerebrum, and spinal cord) . As shown in Figure 11A-B, both activity and tissue accumulation of siSOD1-047M3-AC1^VP and siSOD1-005M3-AC1^VP were dose-dependent in which SOD1 knockdown inversely correlated with increasing concentrations of siRNA-ACO within the CNS tissues. Drug concentrations projected to elicit an ED₅₀ (median effective dose) response in each tissue are summarized in Table 4.

Table 4. ED₅₀ and tissue concentrations of siRNA-ACO to trigger median response

*mass (mg) of siRNA-ACO per gram (g) of tissue.
ND: could not be determined

In remaining animals, changes in body weight were plotted to monitor growth rate and disease progression. As shown in Figure 12A, all groups treated with either siSOD1-047M3-AC1^VP or siSOD1-005M3-AC1^VP continued to gain weight in comparison to aCSF treatment. In addition, disease-related weight loss was delayed in a dose-dependent manner as noted by time needed for growth rates to return to starting weight (dotted line) . Disease progression was confirmed in each animal when a 10%loss in peak body weight was recorded. Plotting data via Kaplan-Meier curves indicated when animals in each treatment group transitioned to progressive disease (Figure 12B) . Data was collected until animals inevitably succumbed to their disease in which survival curves were also generated (Figure 12C) . To summarize, both siSOD1-047M3-AC1^VP (Table 5) and siSOD1-005M3-AC1^VP (Table 6) treatment delayed disease progression and extended animal survival in which the highest dose (i.e., 400 mg) prolonged life by 70 and 111.5 days compared to vehicle controls, respectively.

Table 5. Median age for disease onset and animal survival following single dose siSOD1-047M3-AC1^VP on PND 85

*P < 0.001 compared to aCSF group
P < 0.01 compared to aCSF group

Table 6. Median age for disease onset and animal survival following single dose siSOD1-005M3-AC1^VP on PND 60

*P < 0.001 compared to aCSF group

Example 4. Pathogenic mutation in target sites of siRNA-ACO impact lead selection

In silico analysis of pathogenic single nucleotide polymorphisms (SNPs) located within the target site of siSOD1-005M3-AC1^VP revealed 5 total SNPs in which 4 were in the region complementary to its “seed” sequence (Figure 13A) . Mismatches in this region are known to inhibit siRNA activity, which could eliminate a projected ～8.9-12.4%of the global ALS^SOD1 patients from its 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 has only 2 reported pathogenic SNPs within its target site (i.e., P. E22G and P. F21C) comprising approximately 4.04%and 2.70%of the global ALS^SOD1 population, respectively (Figure 13B) . Luciferase reporter constructs (i.e., pLuc^SOD1, pLuc^P. E22G, and pLuc^P. F21C) containing either consensus sequence perfectly complementary to siSOD1-047M3-AC1^VP guide strand or one of pathogenic mutations (i.e., P. E22G and P. F21C) were co-transfected into HEK293A cells along with siRNA-ACO. Knockdown of luciferase activity was specific to siSOD1-047M3-AC1^VP as transfection with a scramble control (i.e., siCON2-AC1^VP) did not reduce reporter expression (Figure 13C) . Dose response data indicated that the P. E22G mutation does not have any significant impact on siSOD1-047M3-AC1^VP knockdown activity/potency compared to consensus target site sequence, while P. F21C was partially resilient to treatment demonstrating incomplete knockdown and inferior potency (Figure 13D) .

Example 5. siRNA-ACO delays disease progression, prolongs survival and improves motor function of hSOD1^G93A mice via IT injection

Based on perceived patient populations, siSOD1-047M3-AC1^VP was selected for further in vivo analysis. Male and female hSOD1^G93A mice were treated via IT injection with two sequential doses of siSOD1-047M3-AC1^VP on PND 68 and 100 at 75, 150, or 300 μg/dose. A non-specific siRNA-ACO (i.e., siCON3-AC1^VP) served as a negative control for therapeutic efficacy. Animal weight was monitored with comparison to wild-type animals (i.e., WT) in which all siSOD1-047M3-AC1^VP treatments provided similar benefit to male mice, whereas weight gain in females appeared more noticeably dose-dependent (Figure 14A) . Both disease progression (i.e., 10%loss in peak body weight) and animal survival were also plotted in which siSOD1-047M3-AC1^VP treatments delayed advance disease and extended survival in both male and female mice (Figure 14B-C) . Table 7 summarizes median days for disease progression and survival following siRNA-ACO treatment via IT injection in male and female populations. Overall, siCON3-AC1^VP provided no therapeutic benefit in comparison to vehicle control, while equivalent doses of siSOD1-047M3-AC1^VP (i.e., 150 mg) extended animal survival in males and females by 61 and 29 days, respectively.

Table 7. Median age of onset and survival in male and female mice following siSOD1-047M3-AC1^VP treatment via IT injection

*P < 0.001 compared to aCSF group
P < 0.01 compared to aCSF group

Neuromuscular performance was also evaluated in groups comprised of both male and female mice. Distance traveled via open field roaming (Figure 15A) , rotarod test, (Figure 15B) , and grip strength (Figure 15C) all showed siSOD1-047M3-AC1^VP treatment greatly improved motor function in hSOD1^G93A mice, which was generally well sustained until the end of study. Comparing rotarod performance of each individual animal at an early timepoint prior to measurable weight loss (i.e., PND 90) to their respective last timepoints further indicates siSOD1-047M3-AC1^VP treatment retained or improved neuromuscular performance in a majority of animals compared to controls or ASO^SOD1 (Figure 16) . The latency of rotarod performance of each individual animal is summarized in Table 8.

Motor function was also scored for all animals prior to open field, rotarod, and/or grip strength tests using the ALS Therapy Development Institute (ALS TDI) neuroscore (NS) system. As shown in Figure 15D, all mice developed abnormal splay (i.e., NS2) by approximately PND 130 in both aCSF and siCON3-AC1^VP control groups that continued to increase in severity over time (i.e., ≥NS3) . Conversely, mean scores for all siSOD1-047M3-AC1^VP doses never surpassed NS2 at any time point during the course of the study particularly for the highest dose group (i.e., 300 mg) in which mean NS remained predominantly flat at around NS1. ASO^SOD1 treatment also demonstrated an improved NS comparative to vehicle and siCON3-AC1^VP controls, however, by PND 150, NS began to increase at a slope similar to controls despite ASO^SOD1 having ～3X molecular excess than that of siSOD1-047M3-AC1^VP at 150 mg dose.

In summary, siRNA-ACO conjugates provided SOD1 siRNAs with pharmacological properties necessary for clinical development including delivery to the CNS and favorable tissue biodistribution with potent and durable activity. Local delivery via ICV or IT injection into hSOD1^G93A mice delayed disease onset /progression and extended animal survival with superior efficacy compared to an ASO compound resembling Tofersen in sequence and chemistry. These results clearly indicate that siRNA represents a vast improvement over current clinical therapeutic modalities (i.e., Tofersen) to target and knockdown SOD1 for treatment of ALS.

Example 6. Knockdown activity of siRNA on the expression of SOD1 mRNA in HeLa and SK-N-AS cells

To assess the knockdown activity of siRNAs, the indicated siRNAs (i.e., RD-15757, RD-18972, RD-12500, RD-18973, RD-18948 and RD-18949) were directly added into culture medium containing HeLa cell at 1500 nM for 3 days. Cells without any treatment served as a mock control and cells treated with RD-11566 served as a duplex control. As shown in FIG. 17, compared to RD-11566, all the siRNA caused varying degree of reduction in SOD1 mRNA level (ranging from 1 to 32%) with RD-12500 causing the greatest reduction (32%) .

To further assess dose-dependent knockdown activity of 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 at indicated concentrations (i.e., 0.0002, 0.001, 0.0039, 0.0156, 0.0625, 0.25, 1 and 4 nM) for 24 hours. SOD1 mRNA levels as quantified by two-step RT-qPCR were shown in FIGs. 18A and 18B. EC₅₀ values were extrapolated to define potency in context to maximal activity for each of the tested siRNAs that demonstrated dose-dependent knockdown of SOD1 mRNA. The resulting EC₅₀ values following siRNA treatment in SK-N-AS cells are summarized in Table 9.

Table 9. EC₅₀ values following siRNA treatment in SK-N-AS cells

MATERIALS AND METHODS

High-throughput screening of siRNAs targeting human SOD1

Open reading frame (ORF) from human SOD1 (hSOD1) cDNA sequence (NM_000454.5) served as the template for siRNA design via in-house algorithm. A total of 268 duplexes were synthesized at 19-nucleotides in length without medicinal chemistry (Table 2) . Plating and transfection of HEK293A cells was performed in 96-well plates each containing 32 siRNAs and 8 quality control treatments at 2 concentrations (i.e., 0.1 and 10 nM) in duplicate. Cells were cultured for 24 hours, and lysis was automated via the Fluent System 780 liquid handling system (Tecan, Hombrechtikon, The Switzerland) using an optimized formula containing propidium iodide (PI) based on the Cell-Lysis (CL) buffer for one-step RT-qPCR as previous described (K. Shatzkes et al, Sci Rep 4, 4659 (2015) ) . Integration of PI in sample preparation served to monitor variation in cell number (e.g., untoward cytotoxicity) by staining total nucleic acid content in crude lysates. Staining was quantified via optical density (OD) at 535 nm excitation and 615 nm emission wavelengths on the Infinite M200 Pro microplate reader (Tecan) . Samples were subsequently transferred to 384-well plates for analysis by RT-qPCR on the 480 Real-Time PCR system (Roche, Basel, Switzerland) using the One-Step TB Green PrimeScript RT-PCR Kit II (Takara, Kyoto, Japan) . Preparation of PCR reactions was automated by the Echo 525 Acoustic Liquid Handler (Beckman Coulter, Brea, CA, USA) . A secondary screen was subsequently performed only on the top 30 performing siRNAs at 6 concentrations (i.e., 0.0064, 0.032, 0.16, 0.8, 4 and 20 nM) . All samples were amplified in triplicate.

siRNA synthesis

Oligonucleotide sequences were synthesized in-house at Ractigen Therapeutics (Rudong, China) on solid-phase support using a HJ-12 synthesizer (Highgene-Tech Automation, Beijing, China) and subsequently purified via RP-HPLC using an acetonitrile gradient over a UniPS column (NanoMicro Technology, Suzhou, China) . Each sequence was reconstituted in sterile water via buffer exchange. Equal molar quantities of each strand were annealed into their corresponding duplexes by briefly heating strand mixtures and cooling to room temperature. Resolution of a single band via gel electrophoresis at predicted molecular weights was used to qualify duplex formation. ESI-MS was used to confirm duplex identity, while overall purity was analyzed via SEC-HPLC using a XBridge Protein BEH SEC 125 A column (Waters Corporation, Milford, MA, USA) . Endotoxin levels in each batch were quantified using the end-point Chromogenic Endotoxin Quant Kit (Bioendo, Xiamen, China) via proenzyme Factor C. All control duplexes and chemically modified sequences are listed in Table 10.

Cell culture and treatment

HEK293A cells (Cobioer, Nanjing, China, Cat#CBP60436) and SK-N-AS (Procell, Wuhan, China, Cat#CL-0621) cells 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 carcinoma cell HeLa (ATCC) cells were cultured in modified RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10%bovine calf serum and 1%penicillin/streptomycin. All cell lines were cultured in a humidified atmosphere of 5%CO₂ at 37℃. Transfections were carried out using Lipofectamine RNAiMax (ThermoFisher, Waltham, MA, USA) in growth media without antibiotics according to the manufacture’s protocol.

RT-qPCR

One-step reverse transcription-quantitative polymerase chain reaction (one-step RT-qPCR)

At the end of transfection, medium was discarded, and cells were washed with 150 μL of PBS once per well. After discarding the PBS, 100 μL of cell lysis was added into each well and incubated at room temperature for 5 min. 0.5 μL of the cell lysis was taken from each well and analyzed by RT-qPCR using One Step TB GreenTM PrimeScripTM RT-PCR kit II (Takara, RR086A) in a Roche Lightcycler 480 real-time PCR machine. PCR reactions were prepared using Echo 525 Acoustic Liquid Handler (Beckman Coulter) . Each transfection sample was amplified in 3 repeat wells. PCR reaction conditions are shown in Table 11.

Table 11. PCR reaction preparation

The reaction conditions were as follows: reverse transcription reaction (stage 1) : 42℃ for 5 min, 95℃ for 10 sec; PCR reaction (stage 2) : 95℃ for 5 sec, 59℃ for 20 sec, 72℃ for 10 sec; 40 cycles of amplification; and melting curve (stage 3) . Human SOD1 gene was amplified as target genes. Human TBP or mouse Tbp was served as reference genes and also amplified as internal controls for RNA loading. Primer sequences are listed in Table 12.

Table 12. Primer sequences for RT-qPCR assay

Two-step RT-qPCR

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

Table 13. RT reaction

Table 14. RT-qPCR reaction

To calculate the expression level (Erel) of SOD1 mRNA in an siRNA-transfected sample relative to control treatment (Mock) , the averaged Ct values of the target gene and the internal reference gene were substituted into Formula 1,

wherein CtT_m was the Ct value of the target gene from the mock-treated sample; CtT_s was the Ct value of the target gene from the siRNA-treated sample; CtR_m was the Ct value of the internal reference gene from the mock-treated sample; CtR_s was the Ct value of the internal reference gene from the siRNA-treated sample.

Caspase 3/7 activity assay

Caspase 3/7 activity was quantified in cell culture by using the Caspase-Glo 3/7 assay system (Promega, Madison, WI, USA) . Briefly, a luminogenic substrate was added directly to culture media and incubated for 20 mins at 37℃. Luminescence was subsequently measured on an Infinite M200 Pro microplate reader (Tecan) . Relative Caspase 3/7 activity was calculated by subtracting background signal of blank from the luminescence values in each well and normalizing data to non-treated (Mock) controls.

Cell viability assay

In vitro cell viability was measured using the CCK-8 assay (Dojindo, Mashiki-machi, Japan) according to the manufacture’s protocol. Briefly, fresh media containing WST-8 substrate was added to each well of the tissue culture plate and incubated for at least 1 hour at 37℃. Absorbance was measured at 450 nm on an Infinite M200 Pro microplate reader (Tecan) . Relative viability is calculated by subtracting background absorbance of the blank control from the OD values in each well and normalizing data to non-treated (Mock) controls.

Luciferase reporter constructs and knockdown assessment

Target sequence containing either P. E22G or P. F21C mutant SNPs were cloned into the multiple cloning site (MCS) of luciferase reporter vector pmirGLO (Promega) between the NheI and SalI restriction enzyme (RE) sites downstream of the firefly luciferase gene (luc2) to generate constructs pLuc^P. E22G and pLuc^P. F21C, respectively. A control reporter construct (i.e., pLuc^SOD1) was also created containing consensus hSOD1 sequence perfectly complementary to siSOD1-047M3-AC1^VP guide strand. All constructs were subcloned in DH5a bacteria (Tolobio, Shanghai, China) and colonies were selected for DNA sequencing to confirm insertion of target sequence. Exemplary colonies were scaled up for plasmid isolation via midiPrep (Qiagen, Hilden, Germany) . HEK293A cells were plated in 96-well cell culture plates at 30,000 cells/well in absence of antibiotics. Cells were co-transfected with one of reporter plasmids (i.e., pLuc^P. E22G, pLuc^P. F21C, or pLuc^SOD1) at 100 ng/well in combination with siSOD1-047M3-AC1^VP or scramble control at the indicated concentrations using 0.3 μL of Lipofectamine 2000 (ThermoFisher) . Wells treated in absence of test article (0 nM) served as non-treated controls. Cells were cultured for 24 hours, and luciferase activity was quantified using the Dual-Glo Luciferase Assay System (Promega) according to the manufacture’s protocol. Briefly, cells were lysed in 50 μL Passive lysis buffer (Promega) in which 20 μL of lysate was mixed with 20 μL of Dual-Glo Luciferase Reagent and incubated for 10 mins at room temperature. Luminescence was subsequently measured on an Infinite 200 Pro microplate reader (Tecan) to quantify luciferase activity. Following measurements, 20 μL of Dual-Glo Stop &Glo Reagent (Promega) was added to each well and incubated for an additional 10 mins at room temperature. Luminescence was again measured to quantify Renilla activity, which served to normalize luciferase reporter results. Data was calculated as the ratio of reporter luminescence to Renilla luminescence relative to the ratio of non-treated controls. Percent knockdown (%KD) was calculated as 1- (Ratio_siRNA/Ratio_non-treated) *100. Ratio_siRNA = Firefly luciferase activity of siRNA /Renilla luciferase activity of siRNA, Ratio_non-treated = Firefly luciferase activity of non-treated group /Renilla luciferase activity of non-treated group.

Animal handling and grouping

Parental transgenic hSOD1^G93A mice (Strain ID #004435) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and imported into China via Nantong University (Nantong City, Jiangsu Province, China) . Mice were delivered to the animal facility at 6 weeks of age and subsequently bred domestically at Nantong University who supplied the animals for this study. All animal procedures were approved by the IACUC at Nantong University. Formulations for animal treatments were prepared fresh prior to use by dissolving allotments of lyophilized oligonucleotide into aCSF to create stock solutions for dilution to the intended treatment concentrations. Animals were randomly allocated into study groups based on body weight and sex. Any animals in poor health or with obvious abnormalities were omitted from the experiments. Randomization was analyzed using Ordinary one-way ANOVA via GraphPad Prism version 8.3.0 Windows (GraphPad Software, San Diego, CA, USA) . Female hSOD1^G93A mice typical weighed approximately 20-25%less than their male liter mates.

Intracerebroventricular (ICV) injection

Avertin (1.2%) was prepared fresh and sterilized via a 0.2-micron filter. Mice were dosed at 0.30-0.35 ml per 10 g body weight via intraperitoneal (IP) injection in a stereotaxic apparatus to rapidly induce anesthesia for up to 30 minutes. An approximate 11.5 mm incision was made in the animal’s scalp and a 25-gauge needle attached to a Hamilton syringe containing the appropriate siRNA formulation was placed at bregma level. The needle was moved to the appropriate anterior/posterior and medial/lateral coordinates (0.2 mm anterior/posterior and 1 mm to the right medial/lateral) . A total of 10 μL was injected into the lateral ventricle at an approximate rate of 1 μl/s. Following treatment, the needle was slowly withdrawn, and the wound sutured close.

Intrathecal (IT) injection

Anesthesia was administered via 3.0%isoflurane in an induction chamber for continuous 10 mins. Hair was shaved around the injection site at the base of the tail and cleaned with 75%ethanol. The space between the L5-L6 spinous processes was identified and a 30-gauge needle attached to a microliter syringe containing the appropriate drug formulations was slowly inserted into the intradural space until a tail flick was observed. The needle position was subsequently secured in which 10 μL total volume of solution was injected over the course of 1 min.

Quantification of siRNA-ACO in animal tissues

Tissue lysate was prepared in lysis buffer (0.5%CA-630, 1mM EDTA, 150 mM NaCl) using a Bioprep-24 Homogenizer (Allsheng) . Samples were subsequently heated to 95℃ to inactivate sample proteins. Serial dilution of non-treated lysate spiked with siRNA-ACO was used to generate 8-point standard curves. Reverse transcription (RT) reactions were performed using the PrimeScript RT reagent kit (Takara) in conjugation with custom stem-loop primers specific to siRNA guide strands. Each sample was amplified in triplicate on the 480 Real-Time PCR system (Roche) using SYBR Premix Ex Taq II (Takara) reaction mix with primer sets specific to guide strand cDNA. Melting curves were performed after amplification to confirm primer specificity. Absolute quantities of siRNA were extrapolated by linear regression using the appropriate standard curves. Tissue concentrations were calculated as the ratio of absolute siRNA mass (ng) relative to the total weight (g) of tissue sample prepped for lysis.

Clinical observation and endpoint criteria

Animals were observed after injection for up to 4 hours and daily thereafter until endpoint. Body weight was determined before test substance administration and at recorded intervals thereafter. Animals with weight loss >20%relative to their initial mass at day of treatment or a neuroscore of NS4 met endpoint criteria.

Neurological scoring

For animals treated via IT injection, mice were evaluated for signs of motor deficit using the ALS Therapy Development Institute (ALS TDI) neuroscore (NS) system, which was developed to provide unbiased assessment of disease progression based on hindlimb dysfunction common to hSOD1^G93A mice (T. Hatzipetros, et al., Journal of Visualized Experiments (2015) , https: /doi. org/10.3791/53257) . NS was assigned based on the following 4-point scale: 0 if no signs of motor dysfunction (i.e., pre-symptomatic) , 1 if hindlimb tremors are evident when suspended by tail (i.e., first symptoms) , 2 if gait abnormalities are present (i.e., onset of paresis) , 3 if dragging at least 1 hindlimb (i.e., partial paralysis) , and 4 if inability to right itself within 10 seconds (i.e., endpoint paralysis) .

Open field test

Each mouse was placed in the corner of an open field apparatus (50 cm length × 50 cm width × 50 cm height) during daylight hours and allowed to freely roam for 15 minutes. An overhead camera recorded the travel path of each animal. Video footage was analyzed by automated tracking software Samart 3.0 (Bioseb, Vitrolles, France) to calculate total distance traveled.

Rotarod analysis

Animals were trained for 3 days prior to data acquisition. Mice were placed on a motionless rotarod apparatus (XinRuan Information Technology, Shanghai, China) with a swivel bar 60 mm in diameter. Rotational speed was accelerated from 0-30 rpm over the course of 300 seconds. Latency time was recorded as the amount of time it took for each animal to fall off the swivel bar. Each animal was tested in triplicate in which the longest value represents latency time.

Grip strength test

Mice were lowered onto a grid plate in which its forepaws and hind paws were allowed to grasp the grid. The tail was gently pulled, and maximal muscle strength was measured on the XR501 grip strength meter (XinRuan Information Technology) in units of mass until the animal relinquished its grasp. Each animal was tested in triplicate in which the mean value represents grip strength.

Statistical analysis

Data analytics were performed using GraphPad Prism version 8.3.0 Windows. Dose response curves and IC₅₀ values were extrapolated using non-linear regression via 4-parameter concentration-inhibition model. Where specified, mean values were compared using Tukey’s multiple comparison test to determine statistical differences between different dose response curves. Drug quantities in tissue in relationship to knockdown activity including extrapolation of ED₅₀ values were performed using non-linear regression via 3-parameter concentration-response model. Time-stratified data (i.e., peak weight analysis and animal survival) was plotted via Kaplan-Meier graphs in which statistical significance was verified using the Mantel-Cox test.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A siRNA comprising a sense strand and an antisense strand forming a duplex structure with the sense strand, wherein at least one of the two strands comprises a nucleotide sequence having at least 85%nucleotide sequence complementarity or homology to a portion of a nucleotide molecule of SEQ ID NO: 1, and wherein the siRNA is capable of inhibiting/down-regulating superoxide dismutase 1 (SOD1) transcript in a cell.
The siRNA of claim 1, wherein said at least one of the two strands has 0, 1, 2, or 3 mismatches to the portion of a nucleotide sequence of SEQ ID NO: 1; and/or

wherein said portion of the nucleotide sequence comprises a sequence selected from any one of SEQ ID NOs: 2-269; and/or

wherein said sense strand has at least 85%nucleotide sequence homology to a sequence selected from any one of SEQ ID NOs: 270-537; and/or

wherein said antisense strand has at least 85%nucleotide sequence homology to a sequence selected form SEQ ID NOs: 538-805; and/or

wherein the siRNA is capable of inhibiting/down-regulating superoxide dismutase 1 (SOD1) transcript in a cell by at least 10%as compared to the baseline of SOD1 mRNA level; and/or

wherein the sense strand comprises at least 10 contiguous nucleotides; and/or

wherein the antisense strand comprises at least 10 contiguous nucleotides; and/or

wherein the sense strand and the antisense strand form a duplex structure which comprises 0, 1, 2, or 3 mismatches.
The siRNA of claim 1, wherein the siRNA comprises a sense strand comprising SEQ ID NO: n, and an antisense strand comprising SEQ ID NO: n+268, n is any integer between 270 to 537.
The siRNA of claim 1, wherein at least one nucleotide of the siRNA is chemically modified nucleotide located in the sense strand, the antisense strand, or both strands; and/or

wherein the chemically modified nucleotide is a nucleotide modified at the 5' end, 3' end, both ends, or in internal part of the strand (s) ; and/or

wherein at least 50%nucleotides in the sense strand, the antisense strand, and/or both strands of the siRNA are chemically modified.
The siRNA of claim 4, wherein the chemically modified nucleotide is one or more selected from a 2’ sugar modification; base modification; a phosphorothioate (PS) backbone modification; an addition of a 5'-phosophate moiety or a 5-methyl cytosine moiety at the 5’ end of the nucleotide sequence.
The siRNA of claim 5, wherein the 2’ sugar modification is one or more selected from: 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

wherein the addition of a 5'-phosophate moiety is one or more selected from an additional of (E) -vinylphosphonate moiety at the 5’ end of the nucleotide sequence.
The siRNA of claim 4, wherein the sense strand has at least 85%nucleotide sequence homology to a sequence selected form SEQ ID NOs: 808-827, 867 with or without the addition of (E) -vinylphosphonate moiety at the 5’ end; and/or

wherein the antisense strand has at least 85%nucleotide sequence homology to a sequence selected form SEQ ID NOs: 828-849, 868 with or without the addition of (E) -vinylphosphonate moiety at the 5’ end.
The siRNA of claim 4, wherein the siRNA comprises a sense strand comprising SEQ ID NO: m, and an antisense strand comprising SEQ ID NO: m+20, m is any integer between 808 to 827; or wherein the siRNA comprises a sense strand comprising SEQ ID NO: 814, and an antisense strand comprising SEQ ID NO: 848; a sense strand comprising SEQ ID NO: 822, and an antisense strand comprising SEQ ID NO: 849; or a sense strand comprising SEQ ID NO: 814, and an antisense strand comprising SEQ ID NO: 866.
An oligonucleotide agent, comprising:

(a) a small interfering RNA (siRNA) according to any one of claims 1-8; and

(b) a non-targeting single-stranded oligonucleotide (accessary oligonucleotide, ACO) ,

wherein the ACO is about 6-22 nucleotides in length, wherein the siRNA and the ACO are covalently linked, without or with one or more linking components, to form the oligonucleotide agent.
The oligonucleotide agent according to claim 9, wherein the ACO is composed of one or more of RNA, DNA, BNA, LNA, GNA and PNA.
The oligonucleotide agent according to claim 9, wherein the ACO is about 6-18 nucleotides in length.
The oligonucleotide agent according to claim 11, wherein the ACO is about 8-16 nucleotides in length.
The oligonucleotide agent according to claim 9, wherein the siRNA comprises a sense strand having a length ranging from about 16-25 nucleotides; and/or

wherein the siRNA comprises an antisense strand having a length ranging from about 19-25 nucleotides.
The oligonucleotide agent according to claim 9, wherein the oligonucleotide agent capable of inhibiting/down-regulating superoxide dismutase 1 (SOD1) transcript in a cell by at least 50%.
The oligonucleotide agent according to claim 9, wherein the ACO comprises a 5’ end and a 3’ end, and wherein the 5’ end or the 3’ end of the ACO is conjugated to a linking component.
The oligonucleotide agent according to claim 9, wherein the sense strand and/or the antisense strand of the siRNA are covalently linked to the ACO by one or more linking components.
The oligonucleotide agent according to claim 9, wherein the linking component is one or more selected from ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, RNA, DNA, carbohydrates, thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, a tetrazole linkage, and a benzimidazole linkage.
The oligonucleotide agent according to claim 17, wherein the linking component is one or more selected from:

a) Spacer phosphoramidite 18 (Phosphoramidous acid, N, N-bis (1-methylethyl) -, 19, 19-bis (4-methoxyphenyl) -19-phenyl-3, 6, 9, 12, 15, 18-hexaoxanonadec-1-yl 2-cyanoethyl ester) ;

b) Spacer-9 (3- [2- [2- [2- [bis (4-methoxyphenyl) -phenylmethoxy] ethoxy] ethoxy] ethoxy- [di (propan-2-yl) amino] phosphanyl] oxypropanenitrile) ;

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.
The oligonucleotide agent according to claim 9, wherein the ACO is covalently linked to the 3’ end, or the 5’ end, or both the 3’ and 5’ ends of the sense strand of the siRNA; and/or

wherein the ACO is covalently linked to the 3’ end, or the 5’ end, or both the 3’ and 5’ ends of the antisense strand of the siRNA; and/or

wherein the ACO is covalently linked to one or more of internal nucleotides of the siRNA.
The oligonucleotide agent according to claim 9, wherein one, two or more ACOs are covalently linked to the siRNA.
The oligonucleotide agent according to any one of claims 9-20, wherein at least one nucleotide of the ACO is chemically modified.
The oligonucleotide agent according to any one of claims 9-20, wherein at least about 50%nucleotides of the ACO are chemically modified nucleotides.
The oligonucleotide agent according to claim 21, wherein the chemical modification of the at least about one chemically modified nucleotide is a 2’ sugar modification selected from one or more of: 2′-fluoro-2′-deoxynucleoside (2′-F) modification, 2′-O-methyl (2′-O-Me) , modification, and 2′-O- (2-methoxyethyl) (2′-O-MOE) modification.
The oligonucleotide agent according to claim 21, wherein the chemical modification of at least one chemically modified nucleotide is a phosphorothioate (PS) backbone modification.
The oligonucleotide agent according to claim 24, wherein the ACO comprises 6～17 phosphorothioate (PS) backbone modifications.
The oligonucleotide agent according to claim 13, wherein the antisense strand comprises an addition of an (E) -vinylphosphonate moiety at the 5’ end of the nucleotide sequence.
The oligonucleotide agent according to claim 21, wherein the chemical modification of the at least about one chemically modified nucleotide is an addition of a 5-methyl cytosine moiety at the 5’ end of the nucleotide sequence.
The oligonucleotide agent according to claim 10, wherein the ACO and/or the siRNA is conjugated to one or more conjugation groups.
The oligonucleotide agent according to claim 38, wherein the sense strand and/or the antisense strand of the siRNA is conjugated to one or more conjugation groups.
The oligonucleotide agent according to claim 38, wherein the one or more conjugation groups is selected from: a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody.
The oligonucleotide agent according to claim 38, wherein the one or more conjugation groups is selected from: a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.
The oligonucleotide agent according to any one of claims 10-38, wherein the sense strand and the antisense strand of the siRNA have nucleotide sequences that is independently at least about 85%homology to the nucleotide sequence selected from:

a) RD-12926 (SEQ ID NO: 867 and SEQ ID NO: 868) ,

b) siSOD1-063M1 (SEQ ID NO: 808 and SEQ ID NO: 828) ,

c) siSOD1-063M2 (SEQ ID NO: 809 and SEQ ID NO: 829) ,

d) siSOD1-063M3 (SEQ ID NO: 810 and SEQ ID NO: 830) ,

e) siSOD1-063M4 (SEQ ID NO: 811 and SEQ ID NO: 831) ,

f) siSOD1-047M1 (SEQ ID NO: 812 and SEQ ID NO: 832) ,

g) siSOD1-047M2 (SEQ ID NO: 813 and SEQ ID NO: 833) ,

h) siSOD1-047M3 (SEQ ID NO: 814 and SEQ ID NO: 834) ,

i) siSOD1-047M4 (SEQ ID NO: 815 and SEQ ID NO: 835) ,

j) siSOD1-104M1 (SEQ ID NO: 816 and SEQ ID NO: 836) ,

k) siSOD1-104M2 (SEQ ID NO: 817 and SEQ ID NO: 837) ,

l) siSOD1-104M3 (SEQ ID NO: 818 and SEQ ID NO: 838) ,

m) siSOD1-104M4 (SEQ ID NO: 819 and SEQ ID NO: 839) ,

n) siSOD1-005M1 (SEQ ID NO: 820 and SEQ ID NO: 840) ,

o) siSOD1-005M2 (SEQ ID NO: 821 and SEQ ID NO: 841) ,

p) siSOD1-005M3 (SEQ ID NO: 822 and SEQ ID NO: 842) ,

q) siSOD1-005M4 (SEQ ID NO: 823 and SEQ ID NO: 843) ,

r) siSOD1-258M1 (SEQ ID NO: 824 and SEQ ID NO: 844) ,

s) siSOD1-258M2 (SEQ ID NO: 825 and SEQ ID NO: 845) ,

t) siSOD1-258M3 (SEQ ID NO: 826 and SEQ ID NO: 846) ,

u) siSOD1-258M4 (SEQ ID NO: 827 and SEQ ID NO: 847) ,

v) siSOD1-270M3 (SEQ ID NO: 814 and SEQ ID NO: 866)

w) siSOD1-047M3^VP (SEQ ID NO: 814 and SEQ ID NO: 848) ,

x) siSOD1-005M3^VP (SEQ ID NO: 822 and SEQ ID NO: 847) ,

y) siSOD1-047M3-AC1 (SEQ ID NO: 850 and SEQ ID NO: 834) ,

z) siSOD1-005M3-AC1 (SEQ ID NO: 851 and SEQ ID NO: 842) ,

aa) siCON1-AC1^VP (SEQ ID NO: 852 and SEQ ID NO: 858) ,

bb) siCON2-AC1^VP (SEQ ID NO: 853 and SEQ ID NO: 859) ,

cc) siCON3-AC1^VP (SEQ ID NO: 854 and SEQ ID NO: 860) ,

dd) siSOD1-063M3-AC1^VP (SEQ ID NO: 855 and SEQ ID NO: 861) ,

ee) siSOD1-047M3-AC1^VP (SEQ ID NO: 850 and SEQ ID NO: 848) ,

ff) siSOD1-104M3-AC1^VP (SEQ ID NO: 856 and SEQ ID NO: 862) ,

gg) siSOD1-005M3-AC1^VP (SEQ ID NO: 852 and SEQ ID NO: 847) ,

hh) siSOD1-258M3-AC1^VP (SEQ ID NO: 857 and SEQ ID NO: 863) , and

ii) siSOD1-270M3-AC1^vp (SEQ ID NO: 850 and SEQ ID NO: 866) .
The oligonucleotide agent according to claim 10, wherein the ACO of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the siRNA as compared to an oligonucleotide agent without the ACO.
The oligonucleotide agent according to claim 10, wherein the ACO of the oligonucleotide agent increases the biodistribution of siRNA within one or two or more target tissues as compared to an oligonucleotide agent without the ACO.
The oligonucleotide agent according to claim 44, wherein the target tissue is selected from: prefrontal cortex, cerebrum, cerebellum, spinal cord, muscle, lung, eye, liver and kidney.
A vector, comprising the siRNA according to any one of claims 1-9 and/or the oligonucleotide agent according to any one of claims 10-35.
A cell, comprising the siRNA according to any one of claims 1-9, the oligonucleotide agent according to any one of claims 10-35 and/or the vector of claim 36.
The cell according to claim 37, wherein the cell is a mammalian cell.
The cell according to claim 37, wherein the cell is a human cell.
The cell according to claim 37, wherein the cell is a host cell in vitro, in vivo or ex vivo.
A pharmaceutical composition, comprising the siRNA according to any one of claims 1-9, the oligonucleotide agent according to any one of claims 10-35, the vector of claim 36, and/or the cell according to any one of claims 37-40; and a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 41, wherein the pharmaceutically acceptable carrier is one or more selected from an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metallic nanoparticle, bioconjugates, and polypeptide.
The pharmaceutical composition according to claim 41, wherein the pharmaceutical composition decreases the transcript level of the SOD1 gene or SOD1 protein.
A kit, comprising the siRNA according to any one of claims 1-9, the oligonucleotide agent according to any one of claims 10-35, the vector of claim 36, the cell according to any one of claims 37-40, and/or the pharmaceutical composition according to any one of claims 41-43.
A method of decreasing the transcript level of a SOD1 gene or SOD1 protein, comprising administering to a subject a pharmaceutical composition according to any one of claims 41-43.
A method for treating or delaying the onset or progression of a neurodegenerative disease or symptom associated with SOD1 gene mutation, abnormal SOD1 gene expression or abnormal SOD1 protein accumulation in a subject in need thereof, the method comprising: administering to the subject a pharmaceutical composition according to any one of claims 41-43.
The method according to claim 46, wherein the neurodegenerative disease or condition is selected from Amyotrophic lateral sclerosis (ALS) , Alzheimer’s disease (AD) , Parkinson’s disease (PD) , and Down’s syndrome (DS) .
The method according to claim 46, wherein the pharmaceutical composition is administered to the subject intrathecally or intracerebroventricularly.
The method according to claim 57, wherein the ACO of the oligonucleotide agent improves the stability, bioavailability, biodistribution, and/or cellular uptake of the siRNA as compared to an oligonucleotide agent without the ACO.
A use of the siRNA according to any one of claims 1-9, the oligonucleotide agent according to any one of claims 10-35, the vector of claim 36, the cell according to any one of claims 37-40, and/or the pharmaceutical composition according to any one of claims 41-43 in manufacturing a medicament for treating or delaying the onset or progression of a SOD1-associated neurodegenerative disease or symptom.
The use according to claim 64, wherein the SOD1-associated neurodegenerative disease or symptom is selected from Amyotrophic lateral sclerosis (ALS) , Alzheimer’s disease (AD) , Parkinson’s disease (PD) , and Down’s syndrome (DS) .
The siRNA according to any one of claims 1-9, the oligonucleotide agent according to any one of claims 10-35, the vector of claim 36, the cell according to any one of claims 37-40, and/or the pharmaceutical composition according to any one of claims 41-43 for use in treating or delaying the onset or progression of a SOD1-associated neurodegenerative disease or symptom.
The siRNA, oligonucleotide agent, vector, cell and/or pharmaceutical composition for use according to claim 52, wherein the neuro-disease or condition is selected from Amyotrophic lateral sclerosis (ALS) , Alzheimer’s disease (AD) , Parkinson’s disease (PD) , and Down’s syndrome (DS).