WO2016077687A1 - Compositions and methods of treating amyotrophic lateral sclerosis (als) - Google Patents

Compositions and methods of treating amyotrophic lateral sclerosis (als) Download PDF

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Publication number
WO2016077687A1
WO2016077687A1 PCT/US2015/060562 US2015060562W WO2016077687A1 WO 2016077687 A1 WO2016077687 A1 WO 2016077687A1 US 2015060562 W US2015060562 W US 2015060562W WO 2016077687 A1 WO2016077687 A1 WO 2016077687A1
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Prior art keywords
sirna
sodl
aav
als
nucleotides
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English (en)
French (fr)
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WO2016077687A4 (en
Inventor
Dinah Wen-Yee Sah
Jinzhao Hou
Mathieu E. NONNENMACHER
Pengcheng ZHOU
Markus Hossbach
Jochen DECKERT
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Voyager Therapeutics Inc
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Voyager Therapeutics Inc
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Priority to BR112017010087-8A priority Critical patent/BR112017010087B1/pt
Priority to US15/526,690 priority patent/US10597660B2/en
Priority to CN202210354987.1A priority patent/CN114717264A/zh
Priority to AU2015346162A priority patent/AU2015346162B2/en
Priority to CN201580072992.1A priority patent/CN107109407A/zh
Priority to SG11201703281RA priority patent/SG11201703281RA/en
Priority to MX2017006216A priority patent/MX2017006216A/es
Priority to RU2017116576A priority patent/RU2716422C2/ru
Priority to KR1020237037989A priority patent/KR20230169197A/ko
Priority to JP2017525847A priority patent/JP2017535266A/ja
Priority to CA2967367A priority patent/CA2967367C/en
Priority to EP15859973.8A priority patent/EP3218484A4/en
Priority to KR1020177012963A priority patent/KR102599909B1/ko
Priority to HK18103755.9A priority patent/HK1244299A1/zh
Priority to IL292999A priority patent/IL292999A/en
Application filed by Voyager Therapeutics Inc filed Critical Voyager Therapeutics Inc
Publication of WO2016077687A1 publication Critical patent/WO2016077687A1/en
Publication of WO2016077687A4 publication Critical patent/WO2016077687A4/en
Priority to IL251911A priority patent/IL251911B/en
Priority to ZA2017/02991A priority patent/ZA201702991B/en
Anticipated expiration legal-status Critical
Priority to US16/774,493 priority patent/US10920227B2/en
Priority to IL278001A priority patent/IL278001B/en
Priority to US17/143,036 priority patent/US11542506B2/en
Priority to JP2022030717A priority patent/JP2022081546A/ja
Priority to AU2022202979A priority patent/AU2022202979A1/en
Priority to US18/085,222 priority patent/US12123002B2/en
Priority to JP2024050822A priority patent/JP2024079794A/ja
Priority to US18/884,711 priority patent/US20250146000A1/en
Ceased legal-status Critical Current

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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • A—HUMAN NECESSITIES
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to compositions, methods and processes for the design, preparation, manufacture, use and/or formulation of modulatory polynucleotides, e.g., small interfering RNA (siRNA) molecules which target the superoxide dismutase 1 (SOD1) gene.
  • a "modulatory polynucleotide” is any nucleic acid sequence(s) which functions to modulate (either increase or decrease) the level or amount of a target gene, e.g., mRNA or protein levels. Targeting of the SOD1 gene may interfere with SOD1 gene expression and SOD1 enzyme production.
  • the nucleic acid sequence encoding the siRNA molecule are inserted into recombinant adeno-associated virus (AAV) vectors.
  • AAV adeno-associated virus
  • ALS Amyotrophic lateral sclerosis
  • MNs motor neurons
  • the loss of motor neurons devastates basic, fundamental movements, such as breathing, and typically causes death to patients within 2 ⁇ 5 years after diagnosis. Progressive deterioration of motor function in patients severely disrupts their breathing ability, requiring some form of breathing aid for survival of the patients. Other symptoms also include muscle weakness in hands, arms, legs or the muscles of swallowing. Some patients (e.g., FTD-ALS) may also develop frontotemporal dementia.
  • ALS According to the ALS Association, approximately 5,600 people in the United States of America are diagnosed with ALS each year. The incidence of ALS is two per 100,000 people, and it is estimated that as many as 30,000 Americans may have the disease at any given time.
  • sALS sporadic ALS
  • fALS familial ALS
  • ROS reactive oxygen species
  • ALS causes of ALS are complicated and heterogeneous.
  • ALS is considered to be a complex genetic disorder in which multiple genes in combination with environmental exposures combine to render a person susceptible. More than a dozen genes associated with ALS have been discovered, including, SOD-1 (Cu 2+ /Zn 2+ superoxide dismutase), TDP-43 (TARDBP, TAR DNA binding protein-43), FUS (Fused in
  • One strategy is based on the neuroprotective and/or regenerative effect of neurotrophic factors, such as Insulin-like growth factor I (IGF-I), Glial cell line- derived neurotrophic factor (GDNF), Vascular endothelial growth factor (VEGF), Colivelin and Activity dependent neurotrophic factor (ADNF) derived peptide, which can promote neuronal survival.
  • neurotrophic factors such as Insulin-like growth factor I (IGF-I), Glial cell line- derived neurotrophic factor (GDNF), Vascular endothelial growth factor (VEGF), Colivelin and Activity dependent neurotrophic factor (ADNF) derived peptide.
  • IGF-I Insulin-like growth factor I
  • GDNF Glial cell line- derived neurotrophic factor
  • VEGF Vascular endothelial growth factor
  • ADNF Activity dependent neurotrophic factor
  • ALS a progressive neurodegenerative disease 2019
  • Stem cells have the potential to generate motor neurons, thereby replacing degenerating motor neurons in the ALS -affected CNS such as primary motor cortex, brainstem and spinal cord.
  • iPSCs induced pluripotent stem cells
  • MSCs mesenchymal stromal cells
  • BMSCs bone marrow mesenchymal stromal cells
  • ASCs adipocyte stem cells
  • neural tissue origin neural stem cells e.g., fetal spinal neural stem cells (NSCs), multipotent neural progenitor cells (NPCs)
  • NPCs multipotent neural progenitor cells
  • mutant SOD 1 -linked fALS toxicity proposes that an aberrant SOD 1 enzyme causes small molecules such as peroxynitrite or hydrogen peroxide to produce damaging free radicals.
  • Other hypotheses for mutant SOD1 neurotoxicity include inhibition of the proteasome activity, mitochondrial damage, disruption of RNA processing and formation of intracellular aggregates.
  • Abnormal accumulation of mutant SOD 1 variants and/or wild-type SOD1 in ALS forms insoluble fibrillar aggregates which are identified as pathological inclusions.
  • Aggregated SOD1 protein can induce mitochondria stress (Vehvilainen P et al, Front Cell Neurosci., 2014, 8, 126) and other toxicity to cells, particularly to motor neurons.
  • RNA interfering (RNAi) mediated gene silencing has drawn researchers' interest in recent years. Small double stranded RNA (small interfering RNA) molecules that target the SODl gene haven been taught in the art for their potential in treating ALS (See, e.g., U.S. Pat. No. 7,632,938 and U.S. Patent Publication No. 20060229268, the contents of which is herein incorporated by reference in its entirety).
  • the present invention develops an RNA interference based approach to inhibit or prevent the expression of SODl in ALS patients for treatment of the disease.
  • the present invention provides novel double stranded RNA (dsRNA) constructs and siRNA constructs and methods of their design.
  • these novel siRNA constructs may be synthetic molecules or be encoded in an expression vector (one or both strands) for delivery into cells.
  • vectors include, but are not limited to adeno-associated viral vectors such as vector genomes of any of the AAV serotypes or other viral delivery vehicles such as lentivirus, etc.
  • the present invention relates to RNA molecule mediated gene specific interference with gene expression and protein production. Methods for treating motor neuron degeneration diseases such as amyotrophic lateral sclerosis are also included in the present invention.
  • the siRNA included in the compositions featured herein encompass a dsRNA having an antisense strand (the antisense strand) having a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of the SODl gene.
  • the present invention provides short double stranded RNA molecules such as small interfering RNA (siRNA) duplexes that target SODl mRNA to interfere with SODl gene expression and/or SODl protein production.
  • siRNA duplexes of the present invention may interfere with both alleles of the SODl gene irrespective of any particular mutation in the SODl gene, and may particularly interact with those found in ALS disease.
  • siRNA molecules, or a single strand of the siRNA molecules are inserted into adeno-associated viral vectors to be introduced into cells, specifically motor neurons and/or other surrounding cells in the central nervous system.
  • the siRNA duplex of the present invention comprises an antisense strand and a sense strand hybridized together forming a duplex structure, wherein the antisense strand is complementary to the nucleic acid sequence of the targeted SOD1 gene, and wherein the sense strand is homologous to the nucleic acid sequence of the targeted SOD1 gene.
  • the 5'end of the antisense strand has a 5' phosphate group and the 3'end of the sense strand contains a 3'hydroxyl group.
  • each strand of the siRNA duplex targeting the SOD1 gene is about 19-25 nucleotides in length, preferably about 19 nucleotides, 20
  • the siRNAs may be unmodified RNA molecules.
  • the siRNAs may contain at least one modified nucleotide, such as base, sugar or backbone modification.
  • an siRNA or dsRNA includes at least two sequences that are complementary to each other.
  • the dsRNA includes a sense strand having a first sequence and an antisense strand having a second sequence.
  • the antisense strand includes a nucleotide sequence that is substantially complementary to at least part of an mRNA encoding SOD1, and the region of complementarity is 30 nucleotides or less, and at least 15 nucleotides in length.
  • the dsRNA is 19 to 24, e.g., 19 to 21 nucleotides in length.
  • the dsRNA is from about 15 to about 25 nucleotides in length, and in other embodiments the dsRNA is from about 25 to about 30 nucleotides in length.
  • the dsRNA either upon contacting with a cell expressing SOD1 or upon transcription within a cell expressing SOD1, inhibits or suppresses the expression of a SOD1 gene by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more, such as when assayed by a method as described herein.
  • AAV vectors comprising the nucleic acids encoding the siRNA duplexes, one strand of the siRNA duplex or the dsRNA targeting
  • the AAV vector serotype may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ8 and/or AAV-DJ, and variants thereof.
  • siRNA duplexes or dsRNA targeting the SODl gene in ALS are selected from the siRNA duplexes listed in Table 3, 11 or 13.
  • the siRNA duplexes or dsRNA targeting SODl gene in ALS are selected from the group consisting of siRNA duplexes: D-2757, D-2806, D-2860, D-2861, D-2875, D-2871, D-2758, D-2759, D-2866, D-2870, D-2823 and D-2858.
  • the present invention also provides pharmaceutical compositions comprising at least one siRNA duplex targeting the SODl gene and a pharmaceutically acceptable carrier.
  • a nucleic acid sequence encodingthe siRNA duplex is inserted into an AAV vector.
  • the present invention provides methods for inhibiting/silencing SODl gene expression in a cell.
  • the siRNA duplexes or dsRNA can be used to substantially inhibit SODl gene expression in a cell, in particular in a motor neuron.
  • the inhibition of SODl gene expression refers to an inhibition by at least about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.
  • the protein product of the targeted gene may be inhibited by at least about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.
  • the SOD 1 gene can be either a wild type gene or a mutated SOD 1 gene with at least one mutation.
  • the SODl protein is either wild type protein or a mutated polypeptide with at least one mutation.
  • the present invention provides methods for treating, or ameliorating amyotrophic lateral sclerosis associated with abnormal SODl gene and/or SODl protein in a subject in need of treatment, the method comprising administering to the subject a pharmaceutically effective amount of at least one siRNA duplex targeting the SODl gene, delivering said siRNA duplex into targeted cells, inhibiting SODl gene expression and protein production, and ameliorating symptoms of ALS in the subject.
  • an AAV vector comprising the nucleic acid sequence encoding at least one siRNA duplex targeting the SODl gene is administered to the subject in need for treating and/or ameliorating ALS.
  • the AAV vector serotype may be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV1 1, AAV12, AAVrh8, AAVrhlO and AAV- DJ, and variants thereof.
  • ALS is familial ALS linked to SODl mutations.
  • ALS is sporadic ALS which is characterized by abnormal aggregation of SOD 1 protein or disruption of SODl protein function or localization, though not necessarily as a result of genetic mutation.
  • the symptoms of ALS ameliorated by the present method may include motor neuron degeneration, muscle weakness, stiffness of muscles, slurred speech and /or difficulty in breathing.
  • the siR A duplexes or dsRNA targeting SOD1 gene or the AAV vectors comprising such siRNA-encoding molecules may be introduced directly into the central nervous system of the subject, for example, by intracranial injection.
  • the pharmaceutical composition of the present invention is used as a solo therapy. In other embodiments, the pharmaceutical composition of the present invention is used in combination therapy.
  • the combination therapy may be in combination with one or more neuroprotective agents such as small molecule compounds, growth factors and hormones which have been tested for their neuroprotective effect on motor neuron degeneration.
  • the present invention provides methods for treating, or ameliorating amyotrophic lateral sclerosis by administering to a subject in need thereof a therapeutically effective amount of a plasmid or AAV vector described herein.
  • the ALS may be familial ALS or sporadic ALS.
  • FIG. 1 is a histogram showing the activity of the constructs encoded in an AAV vector.
  • FIG. 2 is a histogram showing the activity of the guide strand of the modulatory polynucleotides encoded in an AAV vector in HEK293T cells.
  • FIG. 3 is a histogram showing the activity of the passenger strand of the modulatory polynucleotides encoded in an AAV vector in HEK293T cells.
  • FIG. 4 is a histogram showing the activity of the guide strand of the modulatory polynucleotides encoded in an AAV vector in HeLa cells.
  • FIG. 5 is a histogram showing the activity of the passenger strand of the modulatory polynucleotides encoded in an AAV vector in HeLa cells.
  • FIG. 6 is a histogram for the intracellular AAV DNA.
  • FIG. 7 is a histogram showing the activity of the constructs encoded in an AAV vector in human motor neurons.
  • FIG. 8 is a chart showing the dose-dependent silencing of SODl in U251MG cells.
  • FIG. 9 is a chart showing the dose-dependent silencing of SODl in human astrocyte cells.
  • FIG. 10 is a chart showing the time course of the silencing of SODl in U251MG cells.
  • FIG. 1 1 comprises Fig. 1 1A, 1 IB and 11C which are charts showing the dose- dependent effects of a construct.
  • Fig. 1 1A shows the relative SODl expression.
  • Fig. 1 IB shows the percent of guide strand.
  • Fig. 1 1C shows the percent of the passenger strand.
  • FIG. 12 is a diagram showing the location of the modulatory polynucleotide (MP) in relation to the ITRs, the intron (I) and the polyA (P).
  • MP modulatory polynucleotide
  • the present invention relates to modulatory polynucleotides, e.g., RNA or DNA molecules as therapeutic agents.
  • RNA interfering mediated gene silencing can specifically inhibit targeted gene expression.
  • the present invention then provides small double stranded RNA (dsRNA) molecules (small interfering RNA, siRNA) targeting the SODl gene, pharmaceutical compositions comprising such siRNAs, as well as processes of their design.
  • dsRNA small double stranded RNA
  • siRNA small interfering RNA
  • the present invention also provides methods of their use for inhibiting SOD l gene
  • ALS amyotrophic lateral sclerosis
  • the present invention provides small interfering RNA (siRNA) duplexes (and modulatory polynucleotides encoding them) that target SODl mRNA to interfere with SODl gene expression and/or SODl protein production.
  • siRNA duplexes of the present invention may interfere with both alleles of the SOD l gene irrespective of any particular mutation in the SOD 1 gene, and may particularly interact with those found in ALS disease.
  • a nucleic acid sequence encoding such siRNA molecules, or a single strand of the siRNA molecules is inserted into adeno-associated viral vectors and introduced into cells, specifically motor neurons and/or other surrounding cells in the central nervous system.
  • the encoded siRNA duplex of the present invention contains an antisense strand and a sense strand hybridized together forming a duplex structure, wherein the antisense strand is complementary to the nucleic acid sequence of the targeted SODl gene, and wherein the sense strand is homologous to the nucleic acid sequence of the targeted SODl gene.
  • the 5 'end of the antisense strand has a 5' phosphate group and the 3 'end of the sense strand contains a 3 'hydroxyl group.
  • each strand of the siRNA duplex targeting the SODl gene is about 19 to 25, 19 to 24 or 19 to 21 nucleotides in length, preferably about 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length.
  • the siRNAs may be unmodified RNA molecules.
  • the siRNAs may contain at least one modified nucleotide, such as base, sugar or backbone modification.
  • an siRNA or dsRNA includes at least two sequences that are complementary to each other.
  • the dsRNA includes a sense strand having a first sequence and an antisense strand having a second sequence.
  • the antisense strand includes a nucleotide sequence that is substantially complementary to at least part of an mRNA encoding SODl, and the region of complementarity is 30 nucleotides or less, and at least 15 nucleotides in length.
  • the dsRNA is 19 to 25, 19 to 24 or 19 to 21 nucleotides in length. In some embodiments the dsRNA is from about 15 to about 25 nucleotides in length, and in other embodiments the dsRNA is from about 25 to about 30 nucleotides in length.
  • the dsRNA whether directly administered or encoded in an expression vector upon contacting with a cell expressing SODl, inhibits the expression of SODl by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more, such as when assayed by a method as described herein.
  • the siRNA molecules included in the compositions featured herein comprise a dsRNA having an antisense strand (the antisense strand) having a region that is 30 nucleotides or less, generally 19 to 25, 19 to 24 or 19 to 21 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of the SOD l gene.
  • AAV vectors comprising the nucleic acids of the siRNA duplexes, one strand of the siRNA duplex or the dsRNA targeting SODl gene are produced
  • the AAV vector serotypes may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV1 1, AAV12, AAVrh8,
  • AAVrhlO, AAV-DJ8 and AAV-DJ and variants thereof.
  • siRNA duplexes or dsRNA targeting the SODl gene in ALS is selected from the siRNA duplexes listed in Table 3.
  • the siRNA duplexes or dsRNA targeting the SODl gene in ALS is selected from the group consisting of siRNA duplexes: D-2757, D-2806, D-2860, D-2861, D-2875, D-2871, D-2758, D-2759, D-2866, D-2870, D-2823 and D-2858.
  • the present invention also provides pharmaceutical compositions comprising at least one siRNA duplex targeting the SOD1 gene and a pharmaceutically acceptable carrier.
  • the siRNA duplex is encoded by an AAV vector.
  • the present invention provides methods for inhibiting/silencing SOD1 gene expression in a cell. Accordingly, the siRNA duplexes or encoded dsRNA can be used to substantially inhibit SOD1 gene expression in a cell, in particular in a motor neuron.
  • the inhibition of SOD 1 gene expression refers to an inhibition by at least about 20%, such as by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30- 40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40- 70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50- 100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80- 90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the protein product of the targeted gene may be inhibited by at least about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20- 60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30- 80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40- 100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60- 95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90- 100% or 95-100%.
  • the SOD1 gene can be either a wild type gene or a mutated SOD
  • the siRNA duplexes or encoded dsRNA may be used to reduce the expression of SOD 1 protein by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50- 95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the siRNA duplexes or encoded dsRNA may be used to reduce the expression of SOD 1 mRNA by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50- 95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the siRNA duplexes or encodedsRNA may be
  • the siRNA duplexes or encoded dsRNA may be used to reduce the expression of SODl protein and/or mRNA in at least one region of the CNS such as, but not limited to the spinal cord, the forebrain, the midbrain or the hindbrain.
  • SODl protein and/or mRNA is reduced by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20- 95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40- 50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50- 90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70- 95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in at least one region of the CNS.
  • the present invention provides methods for treating, or ameliorating amyotrophic lateral sclerosis associated with abnormal SODl gene and/or
  • SODl protein in a subject in need of treatment comprising administering to the subject a pharmaceutically effective amount of at least one siRNA duplex or a nucleic acid encoding an siRNA duplex targeting the SODl gene, delivering said siRNA duplex (or encoded duplex) into targeted cells, inhibiting SODl gene expression and protein production, and ameliorating symptoms of ALS in the subject.
  • an AAV vector comprising the nucleic acid sequence of at least one siRNA duplex targeting the SODl gene is administered to the subject in need for treating and/or ameliorating ALS.
  • the AAV vector serotype may be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
  • AAVDJ8 and AAV-DJ AAV-DJ (AAVDJ), and variants thereof.
  • the AAV vector serotype is AAV2.
  • the AAV vector is AAVDJ.
  • the AAV vector serotype is AAVDJ8.
  • the serotype which may be useful in the present invention may be AAV-DJ8.
  • the amino acid sequence of AAV-DJ8 may comprise two or more mutations in order to remove the heparin binding domain (HBD).
  • HBD heparin binding domain
  • the AAV-DJ sequence described as SEQ ID NO: 1 in US Patent No. 7,588,772, the contents of which are herein incorporated by reference in their entirety may comprise two mutations: (1) R587Q where arginine (R; arg) at amino acid 587 is changed to glutamine (Q; Gin) and (2) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
  • K406R where lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg)
  • R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gin)
  • R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
  • ALS is familial ALS linked to SOD1 mutations.
  • ALS is sporadic ALS which is characterized by abnormal aggregation of SOD 1 protein or abberations in SOD1 protein funciton and localization.
  • the symptoms of ALS ameliorated by the present method may include, but are not limited to, motor neuron degeneration, muscle weakness, stiffness of muscles, slurred speech and/or difficulty in breathing.
  • the siRNA duplexes or encoded dsRNA targeting the SOD1 gene or the AAV vectors comprising such siRNA molecules may be introduced directly into the central nervous system of the subject, for example, by intracranial injection.
  • the pharmaceutical composition of the present invention is used as a solo therapy. In other embodiments, the pharmaceutical composition of the present invention is used in combination therapy.
  • the combination therapy may be in combination with one or more neuroprotective agents such as small molecule compounds, growth factors and hormones which have been tested for their neuroprotective effect on motor neuron degeneration.
  • the present invention provides methods for treating, or ameliorating amyotrophic lateral sclerosis by administering to a subject in need thereof a therapeutically effective amount of a plasmid or AAV vector described herein.
  • the ALS may be familial ALS or sporadic ALS.
  • ALS Amyotrophic lateral sclerosis
  • ALS an adult-onset neurodegenerative disorder
  • the incidence of ALS is about 1.9 per 100,000.
  • Patients diagnosed with ALS develop a progressive muscle phenotype characterized by spasticity, hyperreflexia or hyporeflexia, fasciculations, muscle atrophy and paralysis. These motor impairments are caused by the denervation of muscles due to the loss of motor neurons.
  • ALS The major pathological features of ALS include degeneration of the corticospinal tracts and extensive loss of lower motor neurons (LMNs) or anterior horn cells (Ghatak et al, J Neuropathol Exp Neurol, 1986, 45, 385-395), degeneration and loss of Betz cells and other pyramidal cells in the primary motor cortex (Udaka et al, Acta Neuropathol, 1986, 70, 289- 295; Maekawa et al, Brain, 2004, 127, 1237-1251) and reactive gliosis in the motor cortex and spinal cord (Kawamata et al., Am J Pathol, 1992, 140,691-707; and Schiffer et al, J Neurol Set, 1996, 139, 27-33). ALS is usually fatal within 3 to 5 years after the diagnosis due to respiratory defects and/or inflammation (Rowland LP and Shneibder NA, N Engl. J. Med., 2001, 344, 1688-1700).
  • a cellular hallmark of ALS is the presence of proteinaceous, ubiquitinated, cytoplasmic inclusions in degenerating motor neurons and surrounding cells (e.g., astrocytes).
  • Ubiquitinated inclusions i.e., Lewy body-like inclusions or Skein-like inclusions
  • Ubiquitinated inclusions are the most common and specific type of inclusion in ALS and are found in LMNs of the spinal cord and brainstem, and in corticospinal upper motor neurons (UMNs) (Matsumoto et al., J Neurol Sci., 1993, 115, 208-213; and Sasak and Maruyama, Acta Neuropathol, 1994, 87, 578-585).
  • HCIs hyaline conglomerate inclusions
  • SCIs axonal 'spheroids' in spinal cord motor neurons in ALS.
  • Other types and less specific inclusions include Bunina bodies (cystatin C-containing inclusions) and Crescent shaped inclusions (SCIs) in upper layers of the cortex.
  • Other neuropathological features seen in ALS include fragmentation of the Golgi apparatus, mitochondrial vacuolization and ultrastructural abnormalities of synaptic terminals (Fujita et al, Acta Neuropathol. 2002, 103, 243-247).
  • FTD-ALS frontotemporal dementia ALS
  • ALS is a complex and multifactorial disease and multiple mechanisms hypothesized as responsible for ALS pathogenesis include, but are not limited to, dysfunction of protein degradation, glutamate excitotoxicity, mitochondrial dysfunction, apoptosis, oxidative stress, inflammation, protein misfolding and aggregation, aberrant R A metabolism, and altered gene expression.
  • ALS familial ALS
  • sALS sporadic ALS
  • familial (or inherited) ALS is inherited as autosomal dominant disease, but pedigrees with autosomal recessive and X-linked inheritance and incomplete penetrance exist. Sporadic and familial forms are clinically indistinguishable suggesting a common pathogenesis.
  • the precise cause of the selective death of motor neurons in ALS remains elusive. Progress in understanding the genetic factors in fALS may shed light on both forms of the disease.
  • GGGGCC hexanucleotide repeat expansion
  • SOD1 is one of the three human superoxide dismutases identified and characterized in mammals: copper-zinc superoxide dismutase (Cu/ZnSOD or SOD1), manganese superoxide dismutase (MnSOD or SOD2), and extracellular superoxide dismutase (ECSOD or SOD3).
  • SOD1 is a 32 kDa homodimer of a 153-residue polypeptide with one copper- and one zinc-binding site per subunit, which is encoded by the SOD1 gene (GeneBank access No.: NM_000454.4) on human chromosome 21 (see Table 2).
  • SOD1 catalyzes the reaction of superoxide anion (0 2 ⁇ ) into molecular oxygen (O2) and hydrogen peroxide (H2O2) at a bound copper ion.
  • the intracellular concentration of SODl is high (ranging from 10 to
  • the protein is localized not only in the cytoplasm but also in the nucleus, lysosomes, peroxisomes, and mitochondrial intermembrane spaces in eukaryotic cells (Lindenau J et al, Glia, 2000, 29, 25-34).
  • A4V Ala4Val
  • SODl gene mutations 40-50% of all Americans with ALS caused by SODl gene mutations have a particular mutation Ala4Val (or A4V).
  • the A4V mutation is typically associated with more severe signs and symptoms and the survival period is typically 2-3 years.
  • the II 13T mutation is by far the most common mutation in the United Kingdom. The most prevalent mutation in Europe is D90A substitute and the survival period is usually greater than 10 years.
  • ALS mouse models include S0D1 G93A , SODl A4V , S0D1 G37R , S0D1 G85R , SOD1 D90A , S0D1 L84V , SODl I113T , S0D 1 H36 H48Q , S0D1 G127X , S0D1 L126X and SODl L126delTT .
  • transgenic rat models carrying two different human SOD1 mutations There are two transgenic rat models carrying two different human SOD1 mutations: S0D1 H46R and S0D1 G93R .
  • These rodent ALS models can develop muscle weakness similar to human ALS patients and other pathogenic features that reflect several characteristics of the human disease, in particular, the selective death of spinal motor neurons, aggregation of protein inclusions in motor neurons and microglial activation. It is well known in the art that the transgenic rodents are good models of human SOD 1 -associated ALS disease and provide models for studying disease pathogenesis and developing disease treatment.
  • SOD1 pathogenic variants cause ALS by gain of function. That is to say, the superoxide dismutase enzyme gains new but harmful properties when altered by SOD1 mutations.
  • some SOD1 mutated variants in ALS increase oxidative stress (e.g., increased accumulation of toxic superoxide radicals) by disrupting the redox cycle.
  • Other studies also indicate that some SOD1 mutated variants in ALS might acquire toxic properties that are independent of its normal
  • mutant SOD 1 is unstable and through aberrant chemistry interacts with nonconventional substrates causing overproduction of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • protein toxicity model unstable, misfolded SOD1 aggregates into cytoplasmic inclusion bodies, sequestering proteins crucial for cellular processes.
  • the aggregated mutant SOD1 protein may also induce mitochondrial dysfunction (Vehvilainen P et al, Front Cell Neurosci., 2014, 8, 126), impairment of axonal transport, aberrant RNA metabolism, glial cell pathology and glutamate excitotoxicity.
  • mitochondrial dysfunction Vehvilainen P et al, Front Cell Neurosci., 2014, 8, 126
  • misfolded wild-type SOD1 protein is found in diseased motor neurons which forms a "toxic conformation" that is similar to that which is seen with familial ALS- linked SOD1 variants (Rotunno MS and Bosco DA, Front Cell Neurosci., 2013, 16, 7, 253).
  • ALS is a protein folding diseases analogous to other
  • neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.
  • arimoclomol a hydroxylamine derivative
  • heat shock proteins which are cellular defense mechanisms against these aggregates.
  • treatment with arimoclomol improved muscle function in SOD 1 mouse models.
  • Other drugs that target one or more cellular defects in ALS may include AMPA antagonists such as talampanel, beta-lactam antibiotics, which may reduce glutamate- induced excitotoxicity to motor neurons; Bromocriptine that may inhibit oxidative induced motor neuron death (e.g. U.S. Patent publication No. 201 10105517; the content of which is incorporated herein by reference in its entirety); 1,3-diphenylurea derivative or multikinase inhibitor which may reduce SOD1 gene expression (e.g., U.S. Patent Publication
  • RNA therapeutic agents that target the SOD1 gene and modulate SOD 1 expression in ALS are taught in the art.
  • RNA based agents include antisense oligonucleotides and double stranded small interfering RNAs. See, e.g., Wang H et al, J Biol. Chem., 2008, 283(23), 15845-15852); U.S. Pat. Nos. 7,498,316; 7,632,938; 7,678,895; 7,951,784; 7,977,314; 8, 183,219; 8,309,533 and 8, 586, 554; and U.S. Patent publication Nos.
  • the present invention provides modulatory polynucleotides, e.g., siRNA molecules targeting the SODl gene and methods for their design and manufacture.
  • the present invention employs viral vectors such as adeno-associated viral (AAV) vectors comprising the nucleic acid sequence encoding the siRNA molecules of the present invention.
  • AAV vectors comprising the nucleic acid sequence encoding the siRNA molecules of the present invention may increase the delivery of active agents into motor neurons.
  • the siRNA duplexes or encoding dsRNA targeting the SODl gene may be able to inhibit SODl gene expression (e.g., mRNA level) significantly inside cells; therefore, ameliorating SODl expression induced stress inside the cells such as aggregation of protein and formation of inclusions, increased free radicals, mitochondrial dysfunction and RNA metabolism.
  • SODl gene expression e.g., mRNA level
  • Such siRNA mediated SODl expression inhibition may be used for treating ALS.
  • methods for treating and/or ameliorating ALS in a patient comprises administering to the patient an effective amount of AAV vector comprising a nucleic acid sequence encoding the siRNA molecules of the present invention into cells.
  • the administration of the AAV vector comprising such a nucleic acid sequence will encode the siRNA molecules which cause the inhibition/silence of SODl gene expression.
  • the vectors e.g., AAV encoding the modulatory
  • mutant SODl reduces the expression of mutant SODl in a subject.
  • the reduction of mutant SODl can also reduce the formation of toxic aggregates which can cause mechanisms of toxicity such as, but not limited to, oxidative stress, mitochondrial dysfunction, impaired axonal transport, aberrant RNA metabolism, glial cell pathology and/or glutamate
  • the vector e.g., AAV vectors, reduces the amount of SOD 1 in a subject in need thereof and thus provides a therapeutic benefit as described herein.
  • compositions of the invention are provided.
  • the present invention relates to RNA interference (RNAi) induced inhibition of gene expression for treating neurodegenerative disorders.
  • RNAi RNA interference
  • siRNAi siRNA interference
  • siRNA molecules duplexes or encoded dsRNA that target the SODl gene
  • siRNA molecules can reduce or silence SODl gene expression in cells, for example, motor neurons, thereby, ameliorating symptoms of ALS such as, but not limited to, motor neuron death and muscle atrophy.
  • RNAi also known as post-transcriptional gene silencing (PTGS), quelling, or co- suppression
  • PTGS post-transcriptional gene silencing
  • the active components of RNAi are short/small double stranded RNAs (dsR As), called small interfering R As (siRNAs), that typically contain 15-30 nucleotides (e.g., 19 to 25, 19 to 24 or 19-21 nucleotides) and 2 nucleotide 3 ' overhangs and that match the nucleic acid sequence of the target gene.
  • dsR As small interfering R As
  • siRNAs small interfering R As
  • These short RNA species may be naturally produced in vivo by Dicer-mediated cleavage of larger dsRNAs and they are functional in mammalian cells.
  • miRNAs Naturally expressed small RNA molecules, named microRNAs (miRNAs), elicit gene silencing by regulating the expression of mRNAs.
  • miRNA mediated down regulation of gene expression may be caused by cleavage of the target mRNAs, translational inhibition of the target mRNAs, or mRNA decay.
  • miRNA targeting sequences are usually located in the 3 '- UTR of the target mRNAs.
  • a single miRNA may target more than 100 transcripts from various genes, and one mRNA may be targeted by different miRNAs.
  • siRNA duplexes or dsRNA targeting a specific mRNA may be designed and synthesized in vitro and introduced into cells for activating RNAi processes.
  • Elbashir et al. demonstrated that 21 -nucleotide siRNA duplexes (termed small interfering RNAs) were capable of effecting potent and specific gene knockdown without inducing immune response in mammalian cells (Elbashir SM et al, Nature, 2001, 411, 494-498). Since this initial report, post-transcriptional gene silencing by siRNAs quickly emerged as a powerful tool for genetic analysis in mammalian cells and has the potential to produce novel therapeutics.
  • siRNA molecules may be introduced into cells in order to activate RNAi.
  • An exogenous siRNA duplex when it is introduced into cells, similar to the endogenous dsRNAs, can be assembled to form the RNA Induced Silencing Complex (RISC), a multiunit complex that facilitates searching through the genome for RNA sequences that are complementary to one of the two strands of the siRNA duplex (i.e., the antisense strand).
  • RISC RNA Induced Silencing Complex
  • the sense strand (or passenger strand) of the siRNA is lost from the complex, while the antisense strand (or guide strand) of the siRNA is matched with its complementary RNA.
  • the targets of siRNA containing RISC complex are mRNAs presenting a perfect sequence complementarity. Then, siRNA mediated gene silencing occurs, cleaving, releasing and degrading the target.
  • the siRNA duplex comprised of a sense strand homologous to the target mRNA and an antisense strand that is complementary to the target mRNA offers much more advantage in terms of efficiency for target RNA destruction compared to the use of the single strand (ss)-siRNAs (e.g. antisense strand RNA or antisense oligonucleotides). In many cases it requires higher concentration of the ss-siRNA to achieve the effective gene silencing potency of the corresponding duplex.
  • Any of the foregoing molecules may be encoded by an AAV vector or vector genome.
  • siRNA sequence preference include, but are not limited to, (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nucleotides in length.
  • highly effective siRNA molecules essential for suppressing mammalian target gene expression may be readily designed.
  • siRNA molecules e.g., siRNA duplexes or encoded dsRNA
  • siRNA molecules can specifically, suppress SODl gene expression and protein production.
  • the siRNA molecules are designed and used to selectively "knock out" SODl gene variants in cells, i.e., mutated SODl transcripts that are identified in patients with ALS disease (e.g., mutations in Tablel).
  • the siRNA molecules are designed and used to selectively "knock down" SODl gene variants in cells.
  • the siRNA molecules are able to inhibit or suppress both wild type and mutated alleles of SODl gene irrelevant of any particular mutations in the SOD 1 gene.
  • an siRNA molecule of the present invention comprises a sense strand and a complementary antisense strand in which both strands are hybridized together to form a duplex structure.
  • the antisense strand has sufficient complementarity to the SODl mRNA sequence to direct target-specific RNAi, i.e., the siRNA molecule has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • the antisense strand and target mRNA sequences are 100% complementary.
  • the antisense strand may be complementary to any part of the target mRNA sequence.
  • the antisense strand and target mRNA sequences comprise at least one mismatch.
  • the antisense strand and the target mRNA sequence are at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30- 50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%, 40-70%, 40- 80%, 40-90%, 40-95%, 40-99%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 60- 70%, 60-80%, 60-90%, 60
  • the siRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprising 10-50 nucleotides (or nucleotide analogs).
  • the siRNA molecule has a length from about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target region.
  • the siRNA molecule has a length from about 19 to 25, 19 to 24 or 19 to 21 nucleotides.
  • the siRNA molecules of the present invention can be synthetic RNA duplexes comprising about 19 nucleotides to about 25 nucleotides, and two overhanging nucleotides at the 3 '-end.
  • the siRNA molecules may be unmodified RNA molecules.
  • the siRNA molecules may contain at least one modified nucleotide, such as base, sugar or backbone modifications.
  • the siRNA molecules of the present invention can be encoded in plasmid vectors, viral vectors (e.g., AAV vectors), genome or other nucleic acid expression vectors for delivery to a cell.
  • viral vectors e.g., AAV vectors
  • genome or other nucleic acid expression vectors for delivery to a cell.
  • DNA expression plasmids can be used to stably express the siRNA duplexes or dsRNA of the present invention in cells and achieve long-term inhibition of the target gene.
  • the sense and antisense strands of a siRNA duplex are typically linked by a short spacer sequence leading to the expression of a stem-loop structure termed short hairpin RNA (shRNA).
  • shRNA short hairpin RNA
  • the hairpin is recognized and cleaved by Dicer, thus generating mature siRNA molecules.
  • AAV vectors comprising the nucleic acids encoding the siRNA molecules targeting SOD 1 mRNA are produced
  • the AAV vector serotypes may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ8 and AAV-DJ, and variants thereof.
  • the siRNA duplexes or encoded dsRNA of the present invention suppress (or degrade) target mRNA (i.e. SODl). Accordingly, the siRNA duplexes or encoded dsRNA can be used to substantially inhibit SODl gene expression in a cell, for example a motor neuron.
  • SODl target mRNA
  • the inhibition of SODl gene expression refers to an inhibition by at least about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20- 80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30- 95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95- 100%.
  • the protein product of the targeted gene may be inhibited by at least about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20- 95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the SODl gene can be either a wild type gene or a mutated SODl
  • the protein is either wild type protein or a mutated polypeptide with at least one mutation.
  • siRNA duplexes or encoded dsRNA targeting human SOD 1 gene were designed and tested for their ability in reducing SOD 1 mRNA levels in cultured cells.
  • Such siRNA duplexes include those listed in Table 3.
  • the siRNA duplexes may be siRNA duplex IDs: D-2757, D-2806, D-2860, D-2861, D-2875, D-2871, D-2758, D-2759, D-2866, D-2870, D-2823 and D-2858.
  • the 3 ' stem arm of the siRNA duplexes or encoded dsRNA targeting the human SODl gene may have 1 1 nucleotides downstream of the 3 ' end of the guide strand which have complementarity to the 1 1 of the 13 nucleotides upstream of the 5' end of the passenger strand in the 5' stem arm.
  • the siRNA duplexes or encoded dsRNA targeting human SODl gene may have a cysteine which is 6 nucleotides downstream of the 3' end of the 3' stem arm of the modulatory polynucleotide.
  • the siRNA duplexes or encoded dsRNA targeting human SODl gene comprise a miRNA seed match for the guide strand. In another embodiment, the siRNA duplexes or encoded dsRNA targeting human SODl gene comprise a miRNA seed match for the passenger strand. In yet another embodiment, the siRNA duplexes or encoded dsRNA targeting human SODl gene do not comprise a seed match for the guide or passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting human SODl gene may have almost no significant full-length off targets for the guide strand. In another embodiment, the siRNA duplexes or encoded dsRNA targeting human SODl gene may have almost no significant full-length off targets for the passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting human SODl gene may have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1-5%, 2-6%, 3-7%, 4-8%, 5-9%, 5-10% 6-10% full-length off targets for the passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting human SODl gene may have almost no significant full-length off targets for the guide strand or the passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting human SODl gene may have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1-5%, 2-6%, 3-7%, 4-8%, 5-9%, 5- 10% 6-10% full-length off targets for the guide or passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting human SODl gene may have high activity in vitro. In another embodiment, the siRNA duplexes or encoded dsRNA targeting the human SODl gene may have low activity in vitro. In yet another embodiment, the siRNA duplexes or dsRNA targeting the human SODl gene may have high guide strand activity and low passenger strand activity in vitro.
  • the siRNA duplexes or encoded dsRNA targeting the human SODl gene have a high guide strand activity and low passenger strand activity in vitro.
  • the target knock-down (KD) by the guide strand may be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or 100%.
  • the target knock-down by the guide strand may be 60-65%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-99.5%, 60-100%, 65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 65-99%, 65-99.5%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%, 70-99.5%, 70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-99.5%, 75-100%, 80-85%, 80-90%, 80-95%, 80-99%, 80-99.5%, 80-100%, 85-90%, 85-95%, 85-99%, 85-99.5%, 85-100%, 90-95%, 90-99%, 90-99.5%, 90-100%, 95-99%, 95- 99.5%, 95-100%, 99-99.5%, 99-100% or 99.5-100%.
  • the ICso of the passenger strand for the nearest off target is greater than 100 multiplied by the ICsoof the guide strand for the target.
  • the siRNA duplexes or encoded dsRNA targeting the human SOD1 gene is said to have high guide strand activity and a low passenger strand activity in vitro.
  • the 5' processing of the guide strand has a correct start (n) at the 5' end at least 75%, 80%, 85%, 90%, 95%, 99% or 100% of the time in vitro or in vivo.
  • the 5' processing of the guide strand is precise and has a correct start (n) at the 5' end at least 99% of the time in vitro.
  • the 5' processing of the guide strand is precise and has a correct start (n) at the 5' end at least 99% of the time in vivo.
  • the guide-to-passenger (G:P) strand ratio expressed is 1 :99, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15, 90: 10, 95:5, or 99: 1 in vitro or in vivo.
  • the guide-to- passenger strand ratio is 80:20 in vitro.
  • the guide-to-passenger strand ratio is 80:20 in vivo.
  • the integrity of the vector genome encoding the dsRNA is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% of the full length of the construct.
  • the siRNA molecules of the present invention when not delivered as a precursor or DNA, may be chemically modified to modulate some features of RNA molecules, such as, but not limited to, increasing the stability of siRNAs in vivo.
  • the chemically modified siRNA molecules can be used in human therapeutic applications, and are improved without compromising the RNAi activity of the siRNA molecules.
  • the siRNA molecules modified at both the 3' and the 5' end of both the sense strand and the antisense strand may be chemically modified to modulate some features of RNA molecules, such as, but not limited to, increasing the stability of siRNAs in vivo.
  • the chemically modified siRNA molecules can be used in human therapeutic applications, and are improved without compromising the RNAi activity of the siRNA molecules.
  • the siRNA molecules modified at both the 3' and the 5' end of both the sense strand and the antisense strand may be chemically modified to modulate some features of RNA molecules, such as, but not limited to, increasing the stability of siRNAs in vivo.
  • the siRNA duplexes of the present invention may contain one or more modified nucleotides such as, but not limited to, sugar modified nucleotides,
  • the siRNA molecule may contain combined modifications, for example, combined nucleobase and backbone modifications.
  • the modified nucleotide may be a sugar-modified nucleotide.
  • Sugar modified nucleotides include, but are not limited to 2'-fluoro, 2'-amino and 2'-thio modified ribonucleotides, e.g. 2'-fluoro modified ribonucleotides.
  • Modified nucleotides may be modified on the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl.
  • the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.
  • the modified nucleotide may be a nucleobase-modified nucleotide.
  • the modified nucleotide may be a backbone-modified nucleotide.
  • the siRNA duplexes of the present invention may further comprise other modifications on the backbone.
  • a normal "backbone”, as used herein, refers to the repeatingly alternating sugar-phosphate sequences in a DNA or R A molecule.
  • the deoxyribose/ribose sugars are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester" bonds/linker (PO linkage).
  • PO backbones may be modified as "phosphorothioate backbone (PS linkage).
  • the natural phosphodiester bonds may be replaced by amide bonds but the four atoms between two sugar units are kept.
  • Such amide modifications can facilitate the solid phase synthesis of oligonucleotides and increase the thermodynamic stability of a duplex formed with siRNA complement. See e.g. Mesmaeker et al, Pure & Appl. Chem., 1997 ' , 3, 437-440; the content of which is incorporated herein by reference in its entirety.
  • Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
  • nucleobase moieties include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination.
  • More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6- methylguanine, ⁇ , ⁇ ,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methy linos ine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5- halouridine, 4-acetylcytidine, 1 -methyladenosine, 2-methyladenosine, 3-methylcytidine, 6- methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azo
  • the modified nucleotides may be on just the sense strand.
  • the modified nucleotides may be on just the antisense strand.
  • the modified nucleotides may be in both the sense and antisense strands.
  • the chemically modified nucleotide does not affect the ability of the antisense strand to pair with the target mRNA sequence, such as the SOD l mRNA sequence.
  • the siRNA molecules described herein can be encoded by vectors such as plasmids or viral vectors.
  • the siRNA molecules are encoded by viral vectors.
  • Viral vectors may be, but are not limited to, Herpesvirus (HSV) vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and the like.
  • the viral vectors are AAV vectors. Retroviral vectors
  • the siRNA duplex targeting SODl gene may be encoded by a retroviral vector (See, e.g., U.S. Pat. Nos. 5,399,346; 5, 124,263; 4,650,764 and 4,980,289; the content of each of which is incorporated herein by reference in their entirety).
  • Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid to a variety of cell types in vivo, and have been used extensively in gene therapy protocols, including for targeting genes to neural cells.
  • Various replication defective adenovirus and minimum adenovirus vectors have been described for nucleic acid therapeutics (See, e.g., PCT Patent Publication Nos. W0199426914, WO 199502697, W0199428152, W0199412649, WO 199502697 and W0199622378; the content of each of which is incorporated by reference in their entirety).
  • Such adenoviral vectors may also be used to deliver siRNA molecules of the present invention to cells.
  • AAV adeno-associated virus
  • parvoviruses which is a single stranded non-enveloped DNA virus having a genome of about 5000 nucleotides in length and which contains two open reading frames encoding the proteins responsible for replication (Rep) and the structural protein of the capsid (Cap).
  • the open reading frames are flanked by two Inverted Terminal Repeat (ITR) sequences, which serve as the origin of replication of the viral genome.
  • ITR Inverted Terminal Repeat
  • the AAV genome contains a packaging sequence, allowing packaging of the viral genome into an AAV capsid.
  • the AAV vector requires a co-helper (e.g., adenovirus) to undergo productive infection in infected cells. In the absence of such helper functions, the AAV virions essentially enter host cells and integrate into the cells' genome.
  • AAV vectors have been investigated for siRNA delivery because of several unique features.
  • Non-limiting examples of the features include (i) the ability to infect both dividing and non-dividing cells; (ii) a broad host range for infectivity, including human cells; (iii) wild-type AAV has not been associated with any disease and has not been shown to replicate in infected cells; (iv) the lack of cell-mediated immune response against the vector and (v) the non-integrative nature in a host chromosome thereby reducing potential for long-term expression.
  • infection with AAV vectors has minimal influence on changing the pattern of cellular gene expression (Stilwell and Samulski et al., Biotechniques, 2003, 34, 148).
  • AAV vectors for siRNA delivery may be recombinant viral vectors which are replication defective as they lack sequences encoding functional Rep and Cap proteins within the viral genome.
  • the defective AAV vectors may lack most or all coding sequences and essentially only contains one or two AAV ITR sequences and a packaging sequence.
  • AAV vectors may also comprise self-complementary AAV vectors (scAAVs).
  • scAAV vectors contain both DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.
  • the AAV vector used in the present invention is a scAAV.
  • the AAV vector used in the present invention is an ssAAV.
  • AAV vectors comprising the nucleic acid sequence for the siRNA molecules may be prepared or derived from various serotypes of AAVs, including, but not limited to, AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAVl 1, AAV 12, AAVrh8, AAVrhlO, AAV-DJ8 and AAV-DJ.
  • different serotypes of AAVs may be mixed together or with other types of viruses to produce chimeric AAV vectors.
  • the AAV vectors comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be introduced into mammalian cells.
  • AAV vectors may be modified to enhance the efficiency of delivery.
  • modified AAV vectors comprising the nucleic acid sequence encoding the siRNA molecules of the present invention can be packaged efficiently and can be used to successfully infect the target cells at high frequency and with minimal toxicity.
  • the AAV vector comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be a human serotype AAV vector.
  • Such human AAV vector may be derived from any known serotype, e.g., from any one of serotypes AAVl -AAVl 1.
  • AAV vectors may be vectors comprising an AAVl-derived genome in an AAVl-derived capsid; vectors comprising an AAV2-derived genome in an AAV2-derived genome; vectors comprising an AAV4-derived genome in an AAV4 derived capsid; vectors comprising an AAV6-derived genome in an AAV6 derived capsid or vectors comprising an AAV9-derived genome in an AAV9 derived capsid.
  • the AAV vector comprising a nucleic acid sequence for encoding siRNA molecules of the present invention may be a pseudotyped hybrid or chimeric AAV vector which contains sequences and/or components originating from at least two different AAV serotypes.
  • Pseudotyped AAV vectors may be vectors comprising an AAV genome derived from one AAV serotype and a capsid protein derived at least in part from a different AAV serotype.
  • such pseudotyped AAV vectors may be vectors comprising an AAV2-derived genome in an AAVl-derived capsid; or vectors comprising an AAV2-derived genome in an AAV6-derived capsid; or vectors comprising an AAV2-derived genome in an AAV4-derived capsid; or an AAV2-derived genome in an AAV9-derived capsid.
  • the present invention contemplates any hybrid or chimeric AAV vector.
  • AAV vectors comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be used to deliver siRNA molecules to the central nervous system (e.g., U.S. Pat. No. 6, 180,613; the contents of which is herein
  • the AAV vectors comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may further comprise a modified capsid including peptides from non-viral origin.
  • the AAV vector may contain a CNS specific chimeric capsid to facilitate the delivery of encoded siRNA duplexes into the brain and the spinal cord.
  • an alignment of cap nucleotide sequences from AAV variants exhibiting CNS tropism may be constructed to identify variable region (VR) sequence and structure.
  • the AAV vector comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may encode siRNA molecules which are polycistronic molecules.
  • the siRNA molecules may additionally comprise one or more linkers between regions of the siRNA molecules.
  • the encoded siRNA molecule may be located downstream of a promoter in an expression vector such as, but not limited to, CMV, U6, CBA or a CBA promoter with a SV40 intron. Further, the encoded siRNA molecule may also be located upstream of the polyadenylation sequence in an expression vector. As a non-limiting example, the encoded siRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides downstream from the promoter and/or upstream of the polyadenylation sequence in an expression vector.
  • the encoded siRNA molecule may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream from the promoter and/or upstream of the
  • the encoded siRNA molecule may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides downstream from the promoter and/or upstream of the polyadenylation sequence in an expression vector.
  • the encoded siRNA molecule may be located with the first 1-5%, 1-10%, 1-15%, 1- 20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20- 25% downstream from the promoter and/or upstream of the polyadenylation sequence in an expression vector.
  • the encoded siRNA molecule may be located upstream of the polyadenylation sequence in an expression vector.
  • the encoded siRNA molecule may be located downstream of a promoter such as, but not limited to, CMV, U6, CBA or a CBA promoter with a SV40 intron in an expression vector.
  • the encoded siRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides downstream from the promoter and/or upstream of the polyadenylation sequence in an expression vector.
  • the encoded siRNA molecule may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream from the promoter and/or upstream of the
  • the encoded siRNA molecule may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides downstream from the promoter and/or upstream of the polyadenylation sequence in an expression vector.
  • the encoded siRNA molecule may be located with the first 1-5%, 1-10%, 1-15%, 1- 20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20- 25% downstream from the promoter and/or upstream of the polyadenylation sequence in an expression vector.
  • the encoded siRNA molecule may be located in a scAAV.
  • the encoded siRNA molecule may be located in an ssAAV.
  • the encoded siRNA molecule may be located near the 5' end of the flip ITR in an expression vector. In another embodiment, the encoded siRNA molecule may be located near the 3' end of the flip ITR in an expression vector. In yet another embodiment, the encoded siRNA molecule may be located near the 5' end of the flop ITR in an expression vector. In yet another embodiment, the encoded siRNA molecule may be located near the 3 ' end of the flop ITR in an expression vector. In one embodiment, the encoded siRNA molecule may be located between the 5' end of the flip ITR and the 3' end of the flop ITR in an expression vector.
  • the encoded siRNA molecule may be located between (e.g., half-way between the 5' end of the flip ITR and 3' end of the flop ITR or the 3' end of the flop ITR and the 5' end of the flip ITR), the 3 ' end of the flip ITR and the 5' end of the flip ITR in an expression vector.
  • the encoded siRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides downstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in an expression vector.
  • the encoded siRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides upstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in an expression vector.
  • an ITR e.g., Flip or Flop ITR
  • the encoded siRNA molecule may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in an expression vector.
  • an ITR e.g., Flip or Flop ITR
  • the encoded siRNA molecule may be located within 1-5, 1-10, 1-15, 1-20, 1- 25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 upstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in an expression vector.
  • an ITR e.g., Flip or Flop ITR
  • the encoded siRNA molecule may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides upstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in an expression vector.
  • an ITR e.g., Flip or Flop ITR
  • the encoded siRNA molecule may be located with the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10- 25%, 15-20%, 15-25%, or 20-25% downstream from the 5 Or 3' end of an ITR (e.g., Flip or Flop ITR) in an expression vector.
  • an ITR e.g., Flip or Flop ITR
  • an expression vector (e.g., AAV vector) may comprise at least one of the modulatory polynucleotides comprising at least one of the expression vectors described herein.
  • an expression vector may comprise, from ITR to ITR recited 5' to 3 ', an ITR, a promoter, an intron, a modulatory polynucleotide, a polyA sequence and an ITR.
  • the vector genome which comprises a nucleic acid sequence encoding the modulatory polynucleotides described herein may be single stranded or double stranded vector genome.
  • the size of the vector genome may be small, medium, large or the maximum size.
  • the vector genome may comprise a promoter and a polyA tail.
  • the vector genome which comprises a nucleic acid sequence encoding the modulatory polynucleotides described herein may be a small single stranded vector genome.
  • a small single stranded vector genome may be 2.7 to 3.5 kb in size such as about 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5 kb in size.
  • the small single stranded vector genome may be 3.2 kb in size.
  • the vector genome may comprise a promoter and a polyA tail.
  • the vector genome which comprises a nucleic acid sequence encoding the modulatory polynucleotides described herein may be a small double stranded vector genome.
  • a small double stranded vector genome may be 1.3 to 1.7 kb in size such as about 1.3, 1.4, 1.5, 1.6, and 1.7 kb in size.
  • the small double stranded vector genome may be 1.6 kb in size.
  • the vector genome may comprise a promoter and a polyA tail.
  • the vector genome which comprises a nucleic acid sequence encoding the modulatory polynucleotides described herein e.g., siRNA or dsR A may be a medium single stranded vector genome.
  • a medium single stranded vector genome may be 3.6 to 4.3 kb in size such as about 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2 and 4.3 kb in size.
  • the medium single stranded vector genome may be 4.0 kb in size.
  • the vector genome may comprise a promoter and a polyA tail.
  • the vector genome which comprises a nucleic acid sequence encoding the modulatory polynucleotides described herein may be a medium double stranded vector genome.
  • a medium double stranded vector genome may be 1.8 to 2.1 kb in size such as about 1.8, 1.9, 2.0, and 2.1 kb in size.
  • the medium double stranded vector genome may be 2.0 kb in size.
  • the vector genome may comprise a promoter and a polyA tail.
  • the vector genome which comprises a nucleic acid sequence encoding the modulatory polynucleotides described herein may be a large single stranded vector genome.
  • a large single stranded vector genome may be 4.4 to 6.0 kb in size such as about 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0 kb in size.
  • the large single stranded vector genome may be 4.7 kb in size.
  • the large single stranded vector genome may be 4.8 kb in size.
  • the large single stranded vector genome may be 6.0 kb in size.
  • the vector genome may comprise a promoter and a polyA tail.
  • the vector genome which comprises a nucleic acid sequence encoding the modulatory polynucleotides described herein may be a large double stranded vector genome.
  • a large double stranded vector genome may be 2.2 to 3.0 kb in size such as about 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 kb in size.
  • the large double stranded vector genome may be 2.4 kb in size.
  • the vector genome may comprise a promoter and a polyA tail.
  • Promoters [00161] A person skilled in the art may recognize that a target cell may require a specific promoter including but not limited to a promoter that is species specific, inducible, tissue- specific, or cell cycle-specific Parr et al, Nat. Med.3: l 145-9 (1997); the contents of which are herein incorporated by reference in its entirety).
  • the promoter is a promoter deemed to be efficient to drive the expression of the modulatory polynucleotide.
  • the promoter is a promoter having a tropism for the cell being targeted.
  • the promoter is a weak promoter which provides expression of a payload e.g., a modulatory polynucleotide, e.g., siRNA or dsRNA, for a period of time in targeted tissues such as, but not limited to, nervous system tissues.
  • a payload e.g., a modulatory polynucleotide, e.g., siRNA or dsRNA
  • Expression may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years,
  • Expression may be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years or 5-10 years.
  • the promoter is a weak promoter for sustained expression of a payload in nervous tissues.
  • the promoter may be a promoter which is less than 1 kb.
  • the promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800.
  • the promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300- 600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600- 700, 600-800 or 700-800.
  • the promoter may be a combination of two or more components such as, but not limited to, CMV and CBA.
  • Each component may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800.
  • Each component may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300- 500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500- 800, 600-700, 600-800 or 700-800.
  • the promoter is a combination of a 382 nucleotide CMV-enhancer sequence and a 260 nucleotide CBA-promoter sequence.
  • the vector genome comprises at least one element to enhance the target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in its entirety).
  • elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.
  • the vector genome comprises at least one element to enhance the target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in its entirety) such as promoters.
  • Promoters for which promote expression in most tissues include, but are not limited to, human elongation factor la-subunit (EF la), immediate-early cytomegalovirus (CMV), chicken ⁇ -actin (CBA) and its derivative CAG, the ⁇ glucuronidase (GUSB), or ubiquitin C (UBC).
  • Tissue-specific expression elements can be used to restrict expression to certain cell types such as, but not limited to, nervous system promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.
  • tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF- ⁇ ), the synapsin (Syn), the methyl-CpG binding protein 2 (MeCP2), CaMKII, mGluR2, NFL, NFH, ⁇ 2, PPE, Enk and EAAT2 promoters.
  • a non-limiting example of a tissue-specific expression elements for astrocytes include the glial fibrillary acidic protein (GFAP) and EAAT2 promoters.
  • a non-limiting example of a tissue-specific expression element for oligodendrocytes include the myelin basic protein (MBP) promoter.
  • the vector genome comprises a ubiquitous promoter.
  • ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-la, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3). Yu et al.
  • NSE 1.8 kb
  • EF EF
  • NSE 0.3 kb
  • GFAP GFAP
  • CMV CMV
  • hENK PPE
  • NFL NFH
  • NFH 920 nucleotide promoter which are both absent in the liver but NFH is abundant in the sensory proprioceptive neurons, brain and spinal cord and NFH is present in the heart.
  • Scn8a is a 470 nucleotide promoter which expresses throughout the DRG, spinal cord and brain with particularly high expression seen in the hippocampal neurons and cerebellar Purkinje cells, cortex, thalmus and hypothalamus (See e.g., Drews et al. 2007 and Raymond et al. 2004; the contents of each of which are herein incorporated by reference in their entireties).
  • the vector genome comprises an UBC promoter.
  • the UBC promoter may have a size of 300-350 nucleotides.
  • the UBC promoter is 332 nucleotides.
  • the vector genome comprises a GUSB promoter.
  • the GUSB promoter may have a size of 350-400 nucleotides.
  • the GUSB promoter is 378 nucleotides.
  • the construct may be AAV-promoter- CMV/globin intron-hFXN-RBG, where the AAV may be self-complementary and the AAV may be the DJ serotype.
  • the vector genome comprises a NFL promoter.
  • the NFL promoter may have a size of 600-700 nucleotides.
  • the NFL promoter is 650 nucleotides.
  • the construct may be AAV-promoter-CMV/globin intron-hFXN-RBG, where the AAV may be self-complementary and the AAV may be the DJ serotype.
  • the vector genome comprises a NFH promoter.
  • the NFH promoter may have a size of 900-950 nucleotides.
  • the NFH promoter is 920 nucleotides.
  • the construct may be AAV-promoter-CMV/globin intron-hFXN-RBG, where the AAV may be self-complementary and the AAV may be the DJ serotype.
  • the vector genome comprises a scn8a promoter.
  • the scn8a promoter may have a size of 450-500 nucleotides.
  • the scn8a promoter is 470 nucleotides.
  • the construct may be AAV-promoter- CMV/globin intron-hFXN-RBG, where the AAV may be self-complementary and the AAV may be the DJ serotype.
  • the vector genome comprises a FXN promoter.
  • the vector genome comprises a PGK promoter.
  • the vector genome comprises a CBA promoter.
  • the vector genome comprises a CMV promoter.
  • the vector genome comprises a liver or a skeletal muscle promoter.
  • liver promoters include hAAT and TBG.
  • skeletal muscle promoters include Desmin, MCK and C5-12.
  • the AAV vector comprises an enhancer element, a promoter and/or a 5'UTR intron.
  • the enhancer may be, but is not limited to, a CMV enhancer
  • the promoter may be, but is not limited to, a CMV, CBA, UBC, GUSB, NSE, Sunapsin, MeCP2, and GFAP promoter
  • the 5 'UTR intron may be, but is not limited to, SV40, and CBA-MVM.
  • the enhancer, promoter and/or intron used in combination may be: (1) CMV enhancer, CMV promoter, SV40 5'UTR intron; (2) CMV enhancer, CBA promoter, SV 40 5'UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5'UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) Synapsin promoter; (8) MeCP2 promoter and (9) GFAP promoter.
  • the AAV vector has an engineered promoter.
  • the vector genome comprises at least one element to enhance the transgene target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in its entirety) such as an intron.
  • introns include, MVM (67-97 bps), F.IX truncated intron 1 (300 bps), ⁇ - globin SD/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice
  • donor/immunoglobin splice acceptor 500 bps
  • SV40 late splice donor/splice acceptor (19S/16S) (180 bps)
  • hybrid adenovirus splice donor/IgG splice acceptor 230 bps
  • the intron may be 100-500 nucleotides in length.
  • the intron may have a length of 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500.
  • the promoter may have a length between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400- 500.
  • the AAV vector genome may comprise a promoter such as, but not limited to, CMV or U6.
  • a promoter such as, but not limited to, CMV or U6.
  • the promoter for the AAV may comprise a promoter such as, but not limited to, CMV or U6.
  • the promoter for the AAV comprising the nucleic acid sequence for the siRNA molecules of the present invention is a U6 promoter.
  • the AAV vector may comprise a CMV and a U6 promoter.
  • the AAV vector may comprise a CBA promoter.
  • the cells may include, but are not limited to, cells of mammalian origin, cells of human origins, embryonic stem cells, induced pluripotent stem cells, neural stem cells, and neural progenitor cells.
  • Nucleic acids including siRNA, carry a net negative charge on the sugar-phosphate backbone under normal physiological conditions.
  • a siRNA molecule In order to enter the cell, a siRNA molecule must come into contact with a lipid bilayer of the cell membrane, whose head groups are also negatively charged.
  • the siRNA duplexes can be complexed with a carrier that allows them to traverse cell membranes such as package particles to facilitate cellular uptake of the siRNA.
  • the package particles may include, but are not limited to, liposomes, nanoparticles, cationic lipids, polyethylenimine derivatives, dendrimers, carbon nanotubes and the combination of carbon-made nanoparticles with dendrimers.
  • Lipids may be cationic lipids and/or neutral lipids.
  • siRNA molecules can be conjugated to a hydrophobic moiety, such as cholesterol (e.g., U.S. Patent Publication No.
  • siRNA molecules of the present invention may also be conjugated to certain cationic cell-penetrating peptides (CPPs), such as MPG, transportan or penetratin covalently or non-covalently (e.g., U.S. Patent Publication No. 20110086425; the content of which is herein incorporated by reference in its entirety).
  • CPPs cationic cell-penetrating peptides
  • siRNA molecules e.g., siRNA duplexes
  • the siRNA molecules may be introduced into cells using any of a variety of approaches such as, but not limited to, viral vectors (e.g., AAV vectors). These viral vectors are engineered and optimized to facilitate the entry of siRNA molecule into cells that are not readily amendable to transfection. Also, some synthetic viral vectors possess an ability to integrate the shRNA into the cell genome, thereby leading to stable siRNA expression and long-term knockdown of a target gene. In this manner, viral vectors are engineered as vehicles for specific delivery while lacking the deleterious replication and/or integration features found in wild-type virus.
  • viral vectors e.g., AAV vectors
  • the siRNA molecules of the present invention are introduced into a cell by contacting the cell with a composition comprising a lipophilic carrier and a vector, e.g., an AAV vector, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention.
  • a composition comprising a lipophilic carrier and a vector, e.g., an AAV vector, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention.
  • the siRNA molecule is introduced into a cell by transfecting or infecting the cell with a vector, e.g., an AAV vector, comprising nucleic acid sequences capable of producing the siRNA molecule when transcribed in the cell.
  • the siRNA molecule is introduced into a cell by injecting into the cell a vector, e.g., an AAV vector, comprising a nucleic acid sequence capable of producing the siRNA molecule when transcribed in the cell.
  • a vector e.g., an AAV vector
  • a nucleic acid sequence encoding the siRNA molecules of the present invention may be transfected into cells.
  • the vectors e.g., AAV vectors, comprising the nucleic acid sequence encoding the siRNA molecules of the present invention may be delivered into cells by electroporation (e.g. U.S. Patent Publication No. 20050014264; the content of which is herein incorporated by reference in its entirety).
  • vectors comprising the nucleic acid sequence for the siRNA molecules described herein may include photochemical internalization as described in U. S. Patent publication No. 20120264807; the content of which is herein incorporated by reference in its entirety.
  • the formulations described herein may contain at least one vector, e.g., AAV vectors, comprising the nucleic acid sequence encoding the siRNA molecules described herein.
  • the siRNA molecules may target the SOD 1 gene at one target site.
  • the formulation comprises a plurality of vectors, e.g., AAV vectors, each vector comprising a nucleic acid sequence encoding a siRNA molecule targeting the SOD1 gene at a different target site.
  • the SOD1 may be targeted at 2, 3, 4, 5 or more than 5 sites.
  • the vectors e.g., AAV vectors, from any relevant species, such as, but not limited to, human, dog, mouse, rat or monkey may be introduced into cells.
  • the vectors may be introduced into cells which are relevant to the disease to be treated.
  • the disease is ALS and the target cells are motor neurons and astrocytes.
  • the vectors e.g., AAV vectors
  • the vectors may be introduced into cells which have a high level of endogenous expression of the target sequence.
  • the vectors e.g., AAV vectors
  • the vectors may be introduced into cells which have a low level of endogenous expression of the target sequence.
  • the cells may be those which have a high efficiency of AAV transduction.
  • compositions which are suitable for administration to humans
  • pharmaceutical compositions which are suitable for administration to humans
  • such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals.
  • Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals.
  • Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which
  • compositions include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • compositions are administered to humans, human patients or subjects.
  • active ingredient generally refers either to the synthetic siRNA duplexes, the vector, e.g., AAV vector, encoding the siR A duplexes, or to the siRNA molecule delivered by a vector as described herein.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the vectors e.g., AAV vectors, comprising the nucleic acid sequence encoding the siRNA molecules of the present invention can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; or (4) alter the biodistribution (e.g., target the viral vector to specific tissues or cell types such as brain and motor neurons).
  • Formulations of the present invention can include, without limitation, saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics and combinations thereof. Further, the viral vectors of the present invention may be formulated using self-assembled nucleic acid nanoparticles.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
  • the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
  • a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use for humans and for veterinary use.
  • an excipient may be approved by United States Food and Drug
  • an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the
  • Excipients which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).
  • any conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • the formulations may comprise at least one inactive ingredient.
  • inactive ingredient refers to one or more inactive agents included in formulations.
  • all, none or some of the inactive ingredients which may be used in the formulations of the present invention may be approved by the US Food and Drug Administration (FDA).
  • FDA US Food and Drug Administration
  • Formulations of vectors comprising the nucleic acid sequence for the siRNA molecules of the present invention may include cations or anions.
  • the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ and combinations thereof.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid).
  • suitable organic acid examples include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • pharmaceutically acceptable salts of the present disclosure include the conventional nontoxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • solvate means a compound of the invention wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered.
  • solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof.
  • Suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N- methylpyrrolidinone ( ⁇ ), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), l,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl- 3,4,5, 6-tetrahydro-2-(lH)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like.
  • water for example, mono-, di-, and tri-hydrates
  • DMSO dimethyl sulfoxide
  • DMF N,N'-dimethylformamide
  • DMAC N,N'-dimethylacetamide
  • DMEU 1,3-dimethyl-2-imidazolidin
  • the vector e.g., AAV vector
  • the nucleic acid sequence for the siR A molecules of the present invention may be formulated for CNS delivery.
  • Agents that cross the brain blood barrier may be used.
  • some cell penetrating peptides that can target siRNA molecules to the brain blood barrier endothelium may be used to formulate the siRNA duplexes targeting the SOD1 gene (e.g., Mathupala, Expert Opin Ther Pat., 2009, 19, 137-140; the content of which is incorporated herein by reference in its entirety).
  • the vector e.g., AAV vector, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be administered by any route which results in a therapeutically effective outcome.
  • routes include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneal,
  • compositions of vector e.g., AAV vector, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be administered in a way which facilitates the vectors or siRNA molecule to enter the central nervous system and penetrate into motor neurons.
  • the vector e.g., AAV vector, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be administered by muscular injection. Rizvanov et al. demonstrated for the first time that siRNA molecules, targeting mutant human SOD 1 mRNA, is taken up by the sciatic nerve, retrogradely transported to the perikarya of motor neurons, and inhibits mutant SOD 1 mRNA in
  • AAV vectors that express siRNA duplexes of the present invention may be administered to a subject by peripheral injections and/or intranasal delivery. It was disclosed in the art that the peripheral administration of AAV vectors for siRNA duplexes can be transported to the central nervous system, for example, to the motor neurons (e.g., U. S. Patent Publication Nos. 20100240739; and 20100130594; the content of each of which is incorporated herein by reference in their entirety).
  • compositions comprising at least one vector, e.g., AAV vector, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be administered to a subject by intracranial delivery (See, e.g., U. S. Pat. No. 8,1 19,611 ; the content of which is incorporated herein by reference in its entirety).
  • the vector e.g., AAV vector, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution.
  • the siRNA duplexes may be formulated with any appropriate and pharmaceutically acceptable excipient.
  • the vector e.g., AAV vector, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be administered in a "therapeutically effective" amount, i.e., an amount that is sufficient to alleviate and/or prevent at least one symptom associated with the disease, or provide improvement in the condition of the subject.
  • the vector e.g., AAV vector
  • the vector may be administered to the CNS in a therapeutically effective amount to improve function and/or survival for a subject with ALS.
  • the vector may be administered intrathecally.
  • the vector e.g., AAV vector
  • the vector may be administered to a subject (e.g., to the CNS of a subject via intrathecal administration) in a therapeutically effective amount for the siRNA duplexes or dsRNA to target the motor neurons and astrocytes in the spinal cord and/or brain steam.
  • the siRNA duplexes or dsRNA may reduce the expression of SOD 1 protein or mRNA.
  • the siRNA duplexes or dsRNA can suppress SOD1 and reduce SOD1 mediated toxicity. The reduction of SOD 1 protein and/or mRNA as well as SOD1 mediated toxicity may be accomplished with almost no enhanced inflammation.
  • the vector e.g., AAV vector
  • a subject e.g., to the CNS of a subject
  • a therapeutically effective amount to slow the functional decline of a subject (e.g., determined using a known evaluation method such as the ALS functional rating scale (ALSFRS)) and/or prolong ventilator- independent survival of subjects (e.g., decreased mortality or need for ventilation support).
  • the vector may be administered intrathecally.
  • the vector e.g., AAV vector
  • the vector may be administered to the cisterna magna in a therapeutically effective amount to transduce spinal cord motor neurons and/or astrocytes.
  • the vector may be administered intrathecally.
  • the vector e.g., AAV vector
  • the vector may be administered using intrathecal infusion in a therapeutically effective amount to transduce spinal cord motor neurons and/or astrocytes.
  • the vector may be administered intrathecally.
  • the vector e.g., AAV vector, comprising a modulatory polynucleotide may be formulated.
  • the baricity and/or osmolality of the formulation may be optimized to ensure optimal drug distribution in the central nervous system or a region or component of the central nervous system.
  • the vector e.g., AAV vector, comprising a modulatory polynucleotide may be delivered to a subject via a single route administration.
  • the vector e.g., AAV vector, comprising a modulatory polynucleotide may be delivered to a subject via a multi-site route of administration.
  • a subject may be administered the vector, e.g., AAV vector, comprising a modulatory polynucleotide at 2, 3, 4, 5 or more than 5 sites.
  • a subject may be administered the vector, e.g., AAV vector, comprising a modulatory polynucleotide described herein using a bolus infusion.
  • the vector e.g., AAV vector
  • a subject may be administered the vector, e.g., AAV vector, comprising a modulatory polynucleotide described herein using sustained delivery over a period of minutes, hours or days.
  • the infusion rate may be changed depending on the subject, distribution, formulation or another delivery parameter.
  • the catheter may be located at more than one site in the spine for multi-site delivery.
  • the vector e.g., AAV vector, comprising a modulatory polynucleotide may be delivered in a continuous and/or bolus infusion.
  • Each site of delivery may be a different dosing regimen or the same dosing regimen may be used for each site of delivery.
  • the sites of delivery may be in the cervical and the lumbar region.
  • the sites of delivery may be in the cervical region.
  • the sites of delivery may be in the lumbar region.
  • a subject may be analyzed for spinal anatomy and pathology prior to delivery of the vector, e.g., AAV vector, comprising a modulatory polynucleotide described herein.
  • the vector e.g., AAV vector
  • a subject with scoliosis may have a different dosing regimen and/or catheter location compared to a subject without scoliosis.
  • the orientation of the spine of the subject during delivery of the vector e.g., AAV vector, comprising a modulatory polynucleotide may be vertical to the ground.
  • the orientation of the spine of the subject during delivery of the vector e.g., AAV vector, comprising a modulatory polynucleotide may be horizontal to the ground.
  • the spine of the subject may be at an angle as compared to the ground during the delivery of the vector, e.g., AAV vector, comprising a modulatory
  • the angle of the spine of the subject as compared to the ground may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 180 degrees.
  • the delivery method and duration is chosen to provide broad transduction in the spinal cord.
  • intrathecal delivery is used to provide broad transduction along the rostral-caudal length of the spinal cord.
  • multi-site infusions provide a more uniform transduction along the rostral-caudal length of the spinal cord.
  • prolonged infusions provide a more uniform transduction along the rostral-caudal length of the spinal cord.
  • compositions of the present invention may be administered to a subject using any amount effective for reducing, preventing and/or treating a SOD1
  • associated disorder e.g., ALS
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • compositions of the present invention are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutic effectiveness for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the siRNA duplexes employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the age and sex of a subject may be used to determine the dose of the compositions of the present invention.
  • a subject who is older may receive a larger dose (e.g., 5-10%, 10-20%, 15-30% ,20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more) of the composition as compared to a younger subject.
  • a larger dose e.g., 5-10%, 10-20%, 15-30% ,20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more
  • a subject who is younger may receive a larger dose (e.g., 5-10%, 10-20%, 15-30% ,20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more) of the composition as compared to an older subject.
  • a larger dose e.g., 5-10%, 10-20%, 15-30% ,20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more
  • a subject who is female may receive a larger dose (e.g., 5- 10%, 10-20%, 15-30% ,20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more) of the composition as compared to a male subject.
  • a larger dose e.g., 5- 10%, 10-20%, 15-30% ,20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more
  • a subject who is male may receive a larger dose (e.g., 5-10%, 10-20%, 15-30% ,20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more) of the composition as compared to a female subject
  • a larger dose e.g., 5-10%, 10-20%, 15-30% ,20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more
  • the doses of AAV vectors for delivering siRNA duplexes of the present invention may be adapted dependent on the disease condition, the subject and the treatment strategy.
  • delivery of compositions in accordance with the present invention to cells may comprise a total concentration per subject between about lxlO 6 VG and about lxlO 16 VG.
  • delivery may comprise a composition concentration of about lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 , 5xl0 6 , 6xl0 6 , 7xl0 6 , 8xl0 6 , 9xl0 6 , lxlO 7 , 2xl0 7 , 3xl0 7 , 4xl0 7 , 5xl0 7 , 6xl0 7 , 7xl0 7 , 8xl0 7 , 9xl0 7 , lxlO 8 , 2xl0 8 , 3xl0 8 , 4xl0 8 , 5xl0 8 , 6xl0 8 , 7xl0 8 , 8xl0 8 , 9xl0 8 , 2xl0 8 , 3x
  • delivery of compositions in accordance with the present invention to cells may comprise a total concentration per subject between about lxlO 6 VG/kg and about lxlO 16 VG/kg.
  • delivery may comprise a composition concentration of about lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 , 5xl0 6 , 6xl0 6 , 7xl0 6 , 8xl0 6 , 9xl0 6 , lxlO 7 , 2xl0 7 , 3xl0 7 , 4xl0 7 , 5xl0 7 , 6xl0 7 , 7xl0 7 , 8xl0 7 , 9xl0 7 , lxlO 8 , 2xl0 8 , 3xl0 8 , 4xl0 8 , 5xl0 8 , 6xl0 8 , 7xl0 8 , 8xl0 8 , 9
  • about 10 5 to 10 6 viral genome (unit) may be administered per dose.
  • delivery of the compositions in accordance with the present invention to cells may comprise a total concentration between about lxlO 6 VG/mL and about lxlO 16 VG/mL.
  • delivery may comprise a composition concentration of about lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 , 5xl0 6 , 6xl0 6 , 7xl0 6 , 8xl0 6 , 9xl0 6 , lxlO 7 , 2xl0 7 , 3xl0 7 , 4xl0 7 , 5xl0 7 , 6xl0 7 , 7xl0 7 , 8xl0 7 , 9xl0 7 , lxlO 8 , 2xl0 8 , 3xl0 8 , 4xl0 8 , 5xl0 8 , 6xl0 8 , 7xl0 8 , 8xl0 8 , 9
  • the desired siRNA duplex dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • multiple administrations e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations.
  • split dosing regimens such as those described herein may be used.
  • a "split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose.
  • a “single unit dose” is a dose of any modulatory polynucleotide therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • a “total daily dose” is an amount given or prescribed in 24 hour period. It may be administered as a single unit dose.
  • the viral vectors comprising the modulatory polynucleotides of the present invention are administered to a subject in split doses. They may be formulated in buffer only or in a formulation described herein.
  • the present invention are methods for introducing the vectors, e.g., AAV vectors, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention into cells, the method comprising introducing into said cells any of the vectors in an amount sufficient for degradation of target SOD 1 mRNA to occur, thereby activating target-specific RNAi in the cells.
  • the cells may be stem cells, neurons such as motor neurons, muscle cells and glial cells such as astrocytes.
  • the method optionally comprises administering to the subject a therapeutically effective amount of a composition comprising at least vectors, e.g., AAV vectors, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention.
  • a composition comprising at least vectors, e.g., AAV vectors, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention.
  • the siRNA molecules can silence SOD1 gene expression, inhibit SOD1 protein production, and reduce one or more symptoms of ALS in the subject such that ALS is therapeutically treated.
  • the composition comprising the vectors, e.g., AAV vectors, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention is administered to the central nervous system of the subject. In other embodiments, the composition comprising the vectors, e.g., AAV vectors, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention is administered to the muscles of the subject
  • the vectors comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be delivered into specific types of targeted cells, including motor neurons; glial cells including oligodendrocyte, astrocyte and microglia; and/or other cells surrounding neurons such as T cells.
  • motor neurons including motor neurons; glial cells including oligodendrocyte, astrocyte and microglia; and/or other cells surrounding neurons such as T cells.
  • the vectors e.g., AAV vectors, comprising a nucleic acid sequence encoding the siRNA molecules of the present invention may be used as a therapy for ALS.
  • the present composition is administered as a solo therapeutics or combination therapeutics for the treatment of ALS.
  • the vectors e.g., AAV vectors, encoding siRNA duplexes targeting the SOD1 gene may be used in combination with one or more other therapeutic agents.
  • AAV vectors encoding siRNA duplexes targeting the SOD1 gene
  • Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • Therapeutic agents that may be used in combination with the vectors e.g., AAV vectors, encoding the nucleic acid sequence for the siRNA molecules of the present invention can be small molecule compounds which are antioxidants, anti-inflammatory agents, anti- apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, and compounds involved in metal ion regulation.
  • Compounds tested for treating ALS which may be used in combination with the vectors described herein include, but are not limited to, antiglutamatergic agents: Riluzole, Topiramate, Talampanel, Lamotrigine, Dextromethorphan, Gabapentin and AMPA antagonist; Anti-apoptosis agents: Minocycline, Sodium phenylbutyrate and Arimoclomol; Anti-inflammatory agent: ganglioside, Celecoxib, Cyclosporine, Azathioprine,
  • Cyclophosphamide Plasmaphoresis, Glatiramer acetate and thalidomide; Ceftriaxone (Berry et al, Plos One, 2013, 8(4)); Beat-lactam antibiotics; Pramipexole (a dopamine agonist) (Wang et al , Amyotrophic Lateral Scler., 2008, 9(1), 50-58); Nimesulide in U.S. Patent Publication No. 20060074991 ; Diazoxide disclosed in U.S. Patent Publication No.
  • Therapeutic agents that may be used in combination therapy with the vectors e.g., AAV vectors, encoding the nucleic acid sequence for the siRNA molecules of the present invention may be hormones or variants that can protect neuronal loss, such as
  • adrenocorticotropic hormone or fragments thereof (e.g., U.S. Patent Publication No. 20130259875); Estrogen (e.g., U.S. Pat. Nos. 6,334,998 and 6,592,845); the content of each of which is incorporated herein by reference in their entirety.
  • Neurotrophic factors may be used in combination therapy with the vectors, e.g., AAV vectors, encoding the nucleic acid sequence for the siRNA molecules of the present invention for treating ALS.
  • a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and /or maturation of a neuron, or stimulates increased activity of a neuron.
  • the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment.
  • Trophic factors may include, but are not limited to, IGF -I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variants thereof.
  • the vector e.g., AAV vector
  • encoding the nucleic acid sequence for the at least one siRNA duplex targeting the SOD 1 gene may be co-administered with AAV vectors expressing neurotrophic factors such as AAV-IGF-I (Vincent et al, Neuromolecular medicine, 2004, 6, 79-85; the content of which is incorporated herein by reference in its entirety) and AAV-GDNF (Wang et al, JNeurosci., 2002, 22, 6920-6928; the content of which is incorporated herein by reference in its entirety).
  • AAV-IGF-I Vincent et al, Neuromolecular medicine, 2004, 6, 79-85; the content of which is incorporated herein by reference in its entirety
  • AAV-GDNF Wang et al, JNeurosci., 2002, 22, 6920-6928; the content of which is incorporated herein by reference in its entirety
  • the composition of the present invention for treating ALS is administered to the subject in need intravenously, intramuscularly, subcutaneously, intraperitoneally, intrathecally and/or intraventricularly, allowing the siRNA molecules or vectors comprising the siRNA molecules to pass through one or both the blood-brain barrier and the blood spinal cord barrier.
  • the method includes administering (e.g., intraventricularly administering and/or intrathecally administering) directly to the central nervous system (CNS) of a subject (using, e.g., an infusion pump and/or a delivery scaffold) a therapeutically effective amount of a composition comprising vectors, e.g., AAV vectors, encoding the nucleic acid sequence for the siRNA molecules of the present invention.
  • the vectors may be used to silence or suppress SODl gene expression, and/or reducing one or more symptoms of ALS in the subject such that ALS is therapeutically treated.
  • the symptoms of ALS include, but are not limited to, motor neuron degeneration, muscle weakness, muscle atrophy, the stiffness of muscle, difficulty in breathing, slurred speech, fasciculation development, frontotemporal dementia and/or premature death are improved in the subject treated.
  • the composition of the present invention is applied to one or both of the brain and the spinal cord.
  • one or both of muscle coordination and muscle function are improved.
  • the survival of the subject is prolonged.
  • administration of the vectors, e.g., AAV vectors encoding a siRNA of the invention, to a subject may lower mutant SODl in the CNS of a subject.
  • administration of the vectors, e.g., AAV vectors, to a subject may lower wild-type SODl in the CNS of a subject.
  • administration of the vectors, e.g., AAV vectors, to a subject may lower both mutant SODl and wild-type SODl in the CNS of a subject.
  • the mutant and/or wild-type SODl may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80- 100%, 90-95%, 90-100% or 95-100% in the CNS, a region of the CNS, or a specific cell of the CNS of a subject.
  • the vectors may lower the expression of wild-type SODl by at least 50% in the motor neurons (e.g., ventral horn motor neurons) and/or astrocytes.
  • the vectors, e.g., AAV vectors may lower the expression of mutant SODl by at least 50% in the motor neurons (e.g., ventral horn motor neurons) and/or astrocytes.
  • the vectors, e.g., AAV vectors may lower the expression of wild-type SODl and mutant SODl by at least 50% in the motor neurons (e.g., ventral horn motor neurons) and/or astrocytes.
  • administering will reduce the expression of mutant and/or wild-type SODl in the spinal cord and the reduction of expression of the mutant and/or wild-type SODl will reduce the effects of ALS in a subject.
  • vectors e.g., AAV vectors
  • the vectors may be administered to a subject who is in the early stages of ALS.
  • Early stage symptoms include, but are not limited to, muscles which are weak and soft or stiff, tight and spastic, cramping and twitching (fasciculations) of muscles, loss of muscle bulk (atrophy), fatigue, poor balance, slurred words, weak grip, and/or tripping when walking.
  • the symptoms may be limited to a single body region or a mild symptom may affect more than one region.
  • administration of the vectors e.g., AAV vectors, may reduce the severity and/or occurrence of the symptoms of ALS.
  • the vectors may be administered to a subject who is in the middle stages of ALS.
  • the middle stage of ALS includes, but is not limited to, more widespread muscle symptoms as compared to the early stage, some muscles are paralyzed while others are weakened or unaffected, continued muscle twitchings (fasciculations), unused muscles may cause contractures where the joints become rigid, painful and sometimes deformed, weakness in swallowing muscles may cause choking and greater difficulty eating and managing saliva, weakness in breathing muscles can cause respiratory insufficiency which can be prominent when lying down, and/or a subject may have bouts of uncontrolled and inappropriate laughing or crying (pseudobulbar affect).
  • administration of the vectors e.g., AAV vectors
  • the vectors may be administered to a subject who is in the late stages of ALS.
  • the late stage of ALS includes, but is not limited to, voluntary muscles which are mostly paralyzed, the muscles that help move air in and out of the lungs are severely compromised, mobility is extremely limited, poor respiration may cause fatigue, fuzzy thinking, headaches and susceptibility to infection or diseases (e.g., pneumonia), speech is difficult and eating or drinking by mouth may not be possible.
  • the vectors may be used to treat a subject with ALS who has a C9orf72 mutation.
  • the vectors e.g., AAV vectors
  • the vectors e.g., AAV vectors
  • nucleic acid refers to any nucleic acid polymers composed of either polydeoxyribonucleotides (containing 2-deoxy-D-ribose), or polyribonucleotides (containing D-ribose), or any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases.
  • polynucleotide refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single stranded RNA.
  • RNA or "RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides
  • DNA or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides.
  • DNA and RNA can be synthesized naturally, e.g., by DNA replication and transcription of DNA, respectively; or be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively).
  • mRNA or “messenger RNA”, as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.
  • RNA interfering refers to a sequence specific regulatory mechanism mediated by RNA molecules which results in the inhibition or interfering or “silencing" of the expression of a corresponding protein-coding gene.
  • RNAi has been observed in many types of organisms, including plants, animals and fungi. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences.
  • RNAi is controlled by the RNA-induced silencing complex (RISC) and is initiated by short/small dsRNA molecules in cell cytoplasm, where they interact with the catalytic RISC component argonaute.
  • RISC RNA-induced silencing complex
  • the dsRNA molecules can be introduced into cells exogenously. Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer, which binds and cleaves dsRNAs to produce double-stranded fragments of 21-25 base pairs with a few unpaired overhang bases on each end. These short double stranded fragments are called small interfering RNAs (siRNAs).
  • siRNAs small interfering RNAs
  • short interfering RNA refers to an RNA molecule (or RNA analog) comprising between about 5-60 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNAi.
  • a siRNA molecule comprises between about 15-30 nucleotides or nucleotide analogs, such as between about 16-25 nucleotides (or nucleotide analogs), between about 18- 23 nucleotides (or nucleotide analogs), between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs), between about 19-25 nucleotides (or nucleotide analogs), and between about 19-24 nucleotides (or nucleotide analogs).
  • nucleotides or nucleotide analogs such as between about 16-25 nucleotides (or nucleotide analogs), between about 18- 23 nucleotides (or nucleotide analogs), between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs), between about 19-25 nu
  • short siRNA refers to a siRNA comprising 5-23 nucleotides, preferably 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides.
  • long siRNA refers to a siRNA comprising 24-60 nucleotides, preferably about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides.
  • Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, or as few as 5 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi.
  • siRNAs may, in some instances, include more than 26 nucleotides, e.g., 27, 28, 29, 30, 35, 40, 45, 50, 55, or even 60 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi or translational repression absent further processing, e.g., enzymatic processing, to a short siRNA.
  • siRNAs can be single stranded RNA molecules (ss-siRNAs) or double stranded RNA molecules (ds-siRNAs) comprising a sense strand and an antisense strand which hybridized to form a duplex structure called siRNA duplex.
  • the term "the antisense strand” or “the first strand” or “the guide strand” of a siRNA molecule refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of the gene targeted for silencing.
  • the antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target- specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process.
  • the term "the sense strand” or “the second strand” or “the passenger strand” of a siRNA molecule refers to a strand that is complementary to the antisense strand or first strand.
  • the antisense and sense strands of a siRNA molecule are hybridized to form a duplex structure.
  • a "siRNA duplex" includes a siRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a siRNA strand having sufficient complementarity to form a duplex with the siRNA strand.
  • Complementary refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands.
  • polynucleotide strands can form base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes.
  • Watson-Crick manner e.g., A to T, A to U, C to G
  • uracil rather than thymine is the base that is considered to be complementary to adenosine.
  • U is denoted in the context of the present invention, the ability to substitute a T is implied, unless otherwise stated.
  • Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can form hydrogen bond with a nucleotide unit of a second polynucleotide strand.
  • Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can form hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can form hydrogen bond with each other, the polynucleotide strands exhibit 10%
  • the term "substantially complementary” means that the siRNA has a sequence (e.g., in the antisense strand) which is sufficient to bind the desired target mRNA, and to trigger the RNA silencing of the target mRNA.
  • targeting means the process of design and selection of nucleic acid sequence that will hybridize to a target nucleic acid and induce a desired effect.
  • gene expression refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide.
  • measurements may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of the amino acid product of translation, e.g., polypeptides or peptides. Methods of measuring the amount or levels of RNA, mRNA, polypeptides and peptides are well known in the art.
  • mutation refers to any changing of the structure of a gene, resulting in a variant (also called “mutant") form that may be transmitted to subsequent generations. Mutations in a gene may be caused by the alternation of single base in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes.
  • the term "vector” means any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule such as the siRNA molecule of the invention.
  • a "viral vector” is a vector which comprises one or more polynucleotide regions encoding or comprising a molecule of interest, e.g., a transgene, a polynucleotide encoding a polypeptide or multi-polypeptide or a modulatory nucleic acid such as small interfering RNA (siRNA).
  • Viral vectors are commonly used to deliver genetic materials into cells. Viral vectors are often modified for specific applications. Types of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated viral vectors.
  • AAV adeno-associated virus
  • AAV vector refers to any vector which comprises or derives from components of an adeno-associated vector and is suitable to infect mammalian cells, preferably human cells.
  • AAV vector typically designates an AAV type viral particle or virion comprising a nucleic acid molecule encoding a siRNA duplex.
  • the AAV vector may be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary).
  • the AAV vector may be replication defective and/or targeted.
  • the phrase "inhibit expression of a gene” means to cause a reduction in the amount of an expression product of the gene.
  • the expression product can be a RNA molecule transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene.
  • a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom.
  • the level of expression may be determined using standard techniques for measuring mRNA or protein.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
  • in vivo refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).
  • the term “modified” refers to a changed state or structure of a molecule of the invention. Molecules may be modified in many ways including chemically, structurally, and functionally. [00292] As used herein, the term “synthetic” means produced, prepared, and/or
  • Synthesis of polynucleotides or polypeptides or other molecules of the present invention may be chemical or enzymatic.
  • transfection refers to methods to introduce exogenous nucleic acids into a cell. Methods of transfection include, but are not limited to, chemical methods, physical treatments and cationic lipids or mixtures.
  • the list of agents that can be transfected into a cell is large and includes, but is not limited to, siRNA, sense and/or anti- sense sequences, DNA encoding one or more genes and organized into an expression plasmid, proteins, protein fragments, and more.
  • off target refers to any unintended effect on any one or more target, gene, or cellular transcript.
  • the phrase "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • an effective amount of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an "effective amount” depends upon the context in which it is being applied.
  • an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of ALS, as compared to the response obtained without administration of the agent.
  • the term "therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • an agent to be delivered e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.
  • the term "subject” or “patient” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates such as chimpanzees and other apes and monkey species, and humans) and/or plants.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates such as chimpanzees and other apes and monkey species, and humans
  • the term “preventing” or “prevention” refers to delaying or forestalling the onset, development or progression of a condition or disease for a period of time, including weeks, months, or years.
  • treatment refers to the application of one or more specific procedures used for the cure or amelioration of a disease.
  • the specific procedure is the administration of one or more pharmaceutical agents.
  • the specific procedure is the administration of one or more siRNA duplexes or encoded dsRNA targeting SOD1 gene.
  • amelioration or “ameliorating” refers to a lessening of severity of at least one indicator of a condition or disease.
  • amelioration includes the reduction of neuron loss.
  • administering refers to providing a pharmaceutical agent or composition to a subject.
  • neurodegeneration refers to a pathologic state which results in neural cell death. A large number of neurological disorders share
  • ischemia such as stroke
  • trauma such as traumatic brain injury
  • one type of neuron cells are degenerative, for example, motor neuron degeneration in ALS.
  • articles such as "a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
  • any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
  • siRNA design was carried out to identify siRNAs targeting human SOD1 gene.
  • the design used the SOD1 transcripts for human ((Genebank access NO. NM_ 000454.4 (SEQ ID NO: 1)), cynomolgus ((Genebank access NO. XM_005548833.1) from the NCBI Refseq collection (release 63) (SEQ ID NO: 2)) and rhesus (SODl transcript
  • ENSMMUT00000002415 (SEQ ID NO: 3) from the Ensembl project (release 75)) as described in Table 2.
  • siRNA duplexes were designed to have 100% identity to the human SODl transcript for positions 2-18 of the antisense strand, and partial or 100% identity to the non- human primate SODl transcript for positions 2-18 of the antisense strand.
  • position 1 of the antisense strand was engineered to a U and position 19 of the sense strand was engineered to a C, in order to unpair the duplex at this position.
  • D-2884 7700 AUGAUCACUUGGAAG 147 7701 UAAUCUUCCAAGUGAUCA 316
  • Oligoribonucleotides were assembled on an ABI 3900 synthesizer (Applied Biosystems) according to the phosphoramidite oligomerization chemistry.
  • the solid support was polystyrene loaded with 2 '-deoxy -thymidine (purchased from Glen Research, Sterling, Virginia, USA) to give a synthesis scale of 0.2 ⁇ .
  • Ancillary synthesis reagents, DNA and RNA phosphoramidites were obtained from SAFC Proligo (Hamburg, Germany).
  • phosphoramidite monomers of uridine (U), thymidine (dT), 4-N-acetylcytidine (C Ac ), 6-N- benzoyladenosine (A bz ) and 2-N-isobutyrlguanosine (G lBu ) with 2'-0-?-butyldimethylsilyl were used to build the oligomers sequence.
  • Coupling time for all phosphoramidites 70 mM in Acetonitrile was 3 min employing 5-Ethylthio-lH-tetrazole (ETT) as activator (0.5 M in Acetonitrile).
  • oligoribonucleotides were precipitated with 1 M NaOAc and a mixture of acetone and ethanol 4: 1 (v/v). The pellets were dissolved in 1 M aqueous NaCl solution and desalted by size exclusion chromatography. This was accomplished using an AKTA Purifier HPLC System (GE Healthcare, Freiburg, Germany) equipped with a HiTrap 5 mL column (GE Healthcare). Identity of the oligoribonucleotides was confirmed by MALDI mass
  • siRNAs were stored frozen until use.
  • HeLa cells were obtained from ATCC (ATCC in Partnership with LGC Standards, Wesel, Germany) and cultured in HAM's F-12 Medium (Biochrom GmbH, Berlin, Germany) supplemented to contain 10% fetal calf serum (Ultra-low IgG from GIBCO/Life Technologies) and 1% Pen/Strep (Biochrom GmbH, Berlin, Germany) at 37°C in an atmosphere with 5% CO2 in a humidified incubator.
  • HAM's F-12 Medium Biochrom GmbH, Berlin, Germany
  • Pen/Strep Biochrom GmbH, Berlin, Germany
  • Lipofectamine 2000 (Invitrogen/Life Technologies) according to the manufacturer's instructions.
  • SODl siRNA concentrations of 1 nM or 0.1 nM were used.
  • Dose response experiments were done with SODl siRNA concentrations of 10, 2.5, 0.6, 0.16, 0.039, 0.0098, 0.0024, 0.0006, 0.00015, and 0.000038 nM.
  • Control wells were transfected with luciferase siRNA, Aha-1 siRNA, PLGF siRNA, or a control mix of unrelated siRNAs.
  • SODl siRNA or control siRNA For each SODl siRNA or control siRNA, four wells were transfected in parallel, and individual datapoints were collected from each well. For each well, the SODl mRNA level was normalized to the GAPDH mRNA level. The activity of a given SOD 1 siRNA was expressed as percent SODl mRNA concentration (normalized to GAPDH mRNA) in treated cells, relative to the SODl mRNA concentration (normalized to GAPDH mRNA) averaged across control wells.
  • Table 4 provides the results from the in vitro HeLa screen where the SODl siRNAs, the sequences of which are given in Table 3, were tested at either 1 nM or 0.1 nM.
  • a number of SODl siRNAs at 1 nM were effective at reducing SODl mRNA levels by more than 80% in HeLa cells.
  • a number of SOD 1 siRNAs at 0.1 nM were effective at reducing SODl mRNA levels by more than 80% in HeLa cells.
  • duplex 10 2.5 0.6 0.16 0.039 0.0098 0.0024 0.0006 0.00015 0.000038 IC50
  • Example 3 In vitro screen of selected SOD1 siRNAs against endogenous SOD1 mRNA expression in SH-SY5Y cells, U87 cells and primary human astrocytes
  • SH-SY5Y cells were obtained from ATCC (ATCC in Partnership with LGC
  • U87MG cells were obtained from ATCC (ATCC in Partnership with LGC
  • Primary human astrocytes were obtained from LONZA (Lonza Sales Ltd, Basel, Switzerland) and cultured in ABM Basal Medium (Lonza Sales Ltd, Basel, Switzerland) supplemented with AGM SingleQuot Kit (Lonza Sales Ltd, Basel, Switzerland) at 37°C in an atmosphere with 5% CO2 in a humidified incubator.
  • IC50 values are provided in Table 10. In primary human astrocytes, IC50s were higher than in SH-SY5Y and U87MG cells, in general.
  • duplex 10 2.5 0.6 0.16 0.039 0.0098 0.0024 0.0006 0.00015 0.000038 IC50
  • the passenger-guide strand duplexes of the SODl siRNA found to be efficacious are engineered into expression vectors and transfected into cells of the central nervous system or neuronal cell lines.
  • overhang utilized in the siRNA knockdown study is a canonical dTdT for siRNA, the overhang in the constructs may comprise any dinucleotide overhang.
  • the cells used may be primary cells or derived from induced pluripotent stem cells (iPS cells).
  • iPS cells induced pluripotent stem cells
  • a guide to passenger strand ratio is calculated to determine the efficiency of knockdown, e.g., of R A Induced Silencing Complex (RISC) processing.
  • RISC R A Induced Silencing Complex
  • the N-terminus is sequenced to determine the cleavage site and to determine the percent homogeneous cleavage of the target. It is expected that cleavage will be higher than 90 percent.
  • HeLa cells are co-transfected in a parallel study to analyze in vitro knockdown of SOD1.
  • a luciferase construct is used as a control to determine off-target effects.
  • SOD 1 siRNAs were designed. These are given in Tables 1 1A and 1 IB. The passenger and guide strands are described in the tables. In Tables 11A and 1 IB, the "miR" component of the name of the sequence does not necessarily correspond to the sequence numbering of miRNA genes (e.g., VOYmiR-101 is the name of the sequence and does not necessarily mean that miR- 101 is part of the sequence).
  • V0YmiR-115_pre- D-2937 7760 CAAUGUG 350 7753 UUUGU 343 010 Engineered; D- ACUGCUG CAGCA 2806_mir-22-loop; let- ACAACAC GUCAC 7b basal-stem AUUGU instability U

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